Non-Neuronal Cells of the Nervous System: Function and Dysfunction: Structure, Organization, Development and Regeneration: in Molecular and Cell Biology) (Pt. I)

PREFACE

A Danish folk-tale describes how the ‘wise Molboer’ wanted to help a tree hanging over a pond to drink from the water by bending its branches down into the water. Since it was a tall tree and they had no ladder and no rope, they climbed up in the tree, one man hanging on to a branch, the second to his legs and so on (Fig. 1). They started pulling hard, but suddenly the hands of the man closest to the tree started hurting and he wanted to spit in them to alleviate the pain. So, loosening his grip, he shouted to the others ‘stop pulling for a while, so we don’t fall down’, but as illustrated in Fig. 1, that didn’t help. The situation in the nervous system is similar, no cell or cell type, whether neuronal or non-neuronal, can malfunction or abandon its assigned function without serious consequences for other cells with which it interacts. Within the last decades we have begun to realize that the cells that populate the nervous system besides the neurons, and greatly outnumber them, have their specific and important functions, which cannot be abandoned without serious consequences for the nervous system as a whole. The present 3 volumes describe these cells, many of their functions and some of their beneficial or adverse roles in disease. The nonneuronal cells of the nervous system cells include all inhabitants of brain parenchyma with the exception of neurons, e.g., astrocytes with their extensive processes and filopodia (Wolff and Chao; Derouiche) and gap junction-mediated coupling (Scemes and Spray); myelinating and non-myelinating oligodendrocytes (Szuchet and Seeger); microglia, which has both cytoprotective or cytotoxic properties (Schwartz; Kalman);

Fig. 1. When one (cell type) loses its grip, they all fall. From: Fausbøll, V., 1862. Beretning om de Vidtbekjendte Molboers Vise Gerninger og Tappre Bedrifter, Fr. Wøldikes Forlagsboghandel, Copenhagen.

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choroid plexus epithelial cells, which in spite of their very small number are the main regulators of CSF composition (Weaver et al.); ependyma cells and other cells lining the ventricles (Szele and Szuchet), and vascular endothelial cells. Interactions between transport processes across the endothelial cells and across the astrocytic membrane are crucial for regulation of ion and fluid distribution in the extracellular space of the brain (Chen and Spatz). Immune cells, bacteria and virus enter the brain via endothelial cells or via the choroid plexus (Couraud et al.). Recently evidence has been obtained that extraparenchymal macrophages and meningeal cells also are important players both during normal brain function and following injury (Mercier and Hatton; Kalman; Dezawa). Functional interactions between neurons and non-neuronal cells have been especially well studied in the hypothalamic-hypophyseal systems (Salm et al.; Mercier and Hatton; Melcangi et al.) and recently it has been established that endothelial cells and tanycytes also partake in these interactions (Pre´vot et al.). It has long been known that neurons and astrocytes interact closely during development, but only during the last couple of years has information been obtained about the role of radial glial cells as multipotential stem cells, both in the brain (Gomes and Rehen; Szele and Szuchet) and the retina (Bringmann et al.), and about interactions between glial cells (astrocytes and Mu¨ller cells) and endothelial cells during retinal development (Stone and Valter). Regulation of astrocytic cell cycle progression is essential for normal development, and dysregulation may lead to malignant transformation of glial cells (Nakatsuji and Miller). Considerable regeneration of central neurons after trauma has recently been obtained (Dezawa; Schwartz), although this problem is still not solved (Kalman). Enteric glial cells regulate mucosal and vascular integrity, and may be directly involved in inflammatory and permeability disorders of the gastrointestinal tract (Savidge et al.). Astrocytes account for a sizeable fraction of oxidative metabolism in the brain (Gruetter; Hertz, Peng et al.) and both neurons and astrocytes metabolize glucose by glycolysis as well as oxidative metabolism (Roberts and Chih). Astrocytes are essential for supplying glutamatergic neurons with precursors for transmitter glutamate and GABAergic neurons with precursors for GABA (Schousboe and Waagepetersen). They produce cholesterol and generate high-density lipoprotein with apolipoproteins, which are crucial for cholesterol transport between brain cells (Ito and Yokoyama). They express a high density of a ‘peripheral-type’ benzodiazepine receptor, which mediates cholesterol transport from the outer to the inner mitochondrial membrane (Be´langer et al). Cholesterol is used in both neurons and astrocytes for synthesis of membrane constituents (Ito and Yokoyama) and especially in astrocytes and Schwann cells for formation of neurosteroids, which exert neuromodulatory and neurotrophic actions (Melcangi et al.). Astrocytes express a multitude of ionotropic and metabotropic receptors, which affect astrocytic activities and thereby CNS function (Hansson and Ro¨nnba¨ck). They also express neurotrophic factors and cytokines and their receptors (Nakagawa and Schwartz), and a ‘transactivation’ process can take place in which stimulation of a G protein-coupled receptor leads to ‘shedding’ of a growth factor, which in turn stimulates a receptor protein tyrosine kinase by an autocrine/paracrine effect (Peng). Both neurons and astroglial cells express all the molecular components required for NO-cGMP-PKG signaling, and astrocytes can produce large amounts of NO by stimulation of the inducible NO synthase (Garcia and Baltrons).

Preface 2+

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Transmitter effects release Ca bound to intracellular stores of different types (Scapagnini et al.; Hansson and Ro¨nnba¨ck). Neuronally released glutamate triggers Ca2+ waves through the astrocytic syncytium, which in turn may stimulate closeby neurons by astrocytic release of glutamate, modulating neuronal signal transmission (Cornell-Bell et al.; Shuai et al.). A multitude of transporters and metabolic production of CO2 regulate pH in astrocytes and in CSF and brain extracellular fluid (McAlear and Bevensee; Weaver et al.). After increases in extracellular Kþ concentration by release of Kþ from stimulated neurons, excess Kþ is removed from the extracellular space by astrocytes, mainly by active uptake mechanisms (Walz). However, gap junction coupling combined with selective, high Kþ conductance sets the stage for a passive spatial buffering mechanism, which can redistribute locally elevated Kþ concentrations across short distances, e.g., in the retina (Bringmann et al.; Scemes and Spray), and in the toad and fish brain, where they contribute to modulation of neuronal activity and thereby behaviour (Laming). Given these important functions of non-neuronal cells in the nervous system it is exceedingly difficult in most of its diseases to establish whether neurons or one of the nonneuronal cell types were the ones that initially ‘loosened its grip’. However, recently it has been shown beyond doubt that Alexander disease is a primary disorder of astrocytes, caused by mutations in the GFAP gene (Eng and Lee). Hepatic encephalopathy is characterized by a multitude of effects on astrocytes, many of which may be secondary to ammonia toxicity (Be´langer et al.). Astrocytic reactions, combined with microglial activation, also play essential roles in the pathogenesis of HIV-associated dementia (Kovacs et al.; Ghorpade and Gendelman), Prion disease (Brown and Sassoon), and chronic pain (Raghavendra and DeLeo). Proinflammatory cytokines and/or glutamate excitotoxicity are becoming recognized as major pathogenetic factors not only in these conditions, but also in MS (Werner et al.), ischemic brain disease (Ha˚berg and Sonnewald; Schousboe and Waagepetersen), and Alzheimer’s disease (Barger), and may become targets of therapeutic intervention. The reasons for glutamate excitotoxicity are many, and include enhanced release from neurons, astrocytes or microglia, compromised uptake, increased synthesis or decreased degradation in astrocytes or oligodendrocytes and heightened sensitivity of glutamate receptors. Depolarization of retinal Muller cells due to a decreased Kþ conductance in retinopathies contributes to glutamate toxicity by impairing uptake of glutamate in conjunction with Naþ (Bringmann et al.). It is disputed whether astrocytes and microglia carry out a neuroprotective role in Parkinson’s disease or mediate deleterious events related to the production of prooxidant and pro-inflammatory compounds (Przedborski and Goldman). Similarly, heme oxygenase-1 (HO-1), which is upregulated after ischemic and other insults as well as in Alzheimer’s disease may either be cytoprotective or neuroendangering (Schipper). Astrocytes resist anoxia better than oligodendrocytes (Marrif and Juurlink). Oxidative stress, polyol pathway flux, increased advanced glycosylation end product formation and deficient neurotrophic support compromise Schwann cells and the axons they ensheath during diabetic neuropathy (Mizisin). Glial dysfunction appears also to be linked to psychiatric illness, as indicated by a glial cells loss in specific regions of the human brain in depressive disorders (Price) and by the observation that all three conventional antibipolar drugs at therapeutically relevant concentrations exert similar effects on signaling-related parameters in cultured astrocytes (Hertz, Chen et al.). An imbalance between the type-1/type-2 immune systems with preponderance of the type-2

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immune response in schizophrenia may reflect differential activation of astrocytes and microglial cells (Mu¨ller and Schwarz). The description in these three volumes of the complex functions of non-neuronal cells in the nervous system and their interactions with each other and with neurons under normal and pathological conditions will hopefully help in establishing an integrated perception of nervous system function and eventually in the development of rational therapy against many diseases where we now are relatively helpless. This endeavour has been made possible by the dedicated efforts of the authors of each of the 51 chapters. I, as the editor, owe my deepest gratitude to each individual author and thank all authors for contributing their profound knowledge, for their patience with me and for their open-mindedness. I would also like to express my thanks to Alden Books, and especially to Dr. Carol Cooper, for her great helpfulness and effectiveness, to the Series Editor, Dr. E.E. Bittar for encouraging me to produce these volumes and to Ms. Joan Anuels and Mr. Hendrik van Leusen of Elsevier for continued support. LEIF HERTZ Dickey Lake, August 2nd, 2003.

TABLE OF CONTENTS

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Volume I Structure, Organization, Development and Regeneration Chapter 1 Cytoarchitectonics of non-neuronal cells in the central nervous system . . . . . . . . . . . . . . . . . . 1 Joachim R. Wolff and T. Ivo Chao Chapter 2 Oligodendrocyte phenotypical and morphological heterogeneity: a reexamination of old concepts in view of new findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Sara Szuchet and Mark A. Seeger Chapter 3 Regulation of cell cycle progression in astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Yuji Nakatsuji and Robert H. Miller Chapter 4 Role of neuron – glia interactions in nervous system development: highlights on radial glia and astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Fla´via Carvalho Alcantara Gomes and Stevens Kastrup Rehen Chapter 5 Cells lining the ventricular system: evolving concepts underlying developmental events in the embryo and adult . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Francis G. Szele and Sara Szuchet Chapter 6 The perisynaptic astrocyte process as a glial compartment-immunolabeling for glutamine synthetase and other glial markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Derouiche Chapter 7 The astrocytic syncytium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eliana Scemes and David C. Spray Chapter 8 Structural plasticity of nonneuronal cells in the hypothalamoneurohypophyseal system: in the right place at the right time . . . . . . . . . . . . . . . . . . . . . . . . A.K. Salm, A.E. Ayoub and B.E. Lally Chapter 9 Glial – neuronal –endothelial interactions and the neuroendocrine control of GnRH secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vincent Prevot, Sandrine De Seranno and Cecilia Estrella

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147

165

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Chapter 10 Meninges and perivasculature as mediators of CNS plasticity . . . . . . . . . . . . . . . . . . . . . . . . Frederic Mercier and Glenn I. Hatton Chapter 11 Mechanisms of infiltration of immune cells, bacteria and viruses through brain endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P.O. Couraud, X. Nassif and S. Bourdoulous

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Chapter 12 Hydrocephalus disorders: their biophysical and neuroendocrine impact on the choroid plexus epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Charles E. Weaver, Paul N. McMillan, John A. Duncan, Edward G. Stopa and Conrad E. Johanson

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Chapter 13 Roles of retinal macroglia in maintaining the stability of the retina . . . . . . . . . . . . . . . . . . Jonathan Stone and Krisztina Valter

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Chapter 14 Function and dysfunction of enteric glia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tor C. Savidge, Julie Cabarrocas and Roland S. Liblau

315

Chapter 15 Schwann cell interactions with axons and CNS glial cells during optic nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mari Dezawa

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Chapter 16 Control of microglial activity by protective autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . Michal Schwartz

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Part I: Colour Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Volume II Biochemistry, Physiology and Pharmacology Chapter 17 A role for lactate released from astrocytes in energy production during neural activity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eugene L. Roberts Jr. and Ching-Ping Chih Chapter 18 Principles of the measurement of neuro-glial metabolism using in vivo 13 C NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rolf Gruetter Chapter 19 Ion, transmitter and drug effects on energy metabolism in astrocytes . . . . . . . . . . . . . . . . . Leif Hertz, Liang Peng, Christel C. Kjeldsen, Brona S. O’Dowd and Gerald A. Dienel Chapter 20 Role of astrocytes in homeostasis of glutamate and GABA during physiological and pathophysiological conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arne Schousboe and Helle S. Waagepetersen

391

409

435

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Chapter 21 Astrocytic receptors and second messenger systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elisabeth Hansson and Lars Ro¨nnba¨ck

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Chapter 22 Transactivation in astrocytes as a novel mechanism of neuroprotection . . . . . . . . . . . . . . . Liang Peng

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Chapter 23 Roles of glia cells in cholesterol homeostasis in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jin-ichi Ito and Shinji Yokoyama

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Chapter 24 Non-neuronal cells in the nervous system: sources and targets of neuroactive steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roberto C. Melcangi, In˜igo Azcoitia, Mariarita Galbiati, Valerio Magnaghi, Daniel Garcia-Ovejero and Luis M. Garcia-Segura

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Chapter 25 Expression of neurotrophic factors and cytokines and their receptors on astrocytes in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takao Nakagawa and Joan P. Schwartz

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Chapter 26 The nitric oxide/cyclic GMP pathway in CNS glial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agustina Garcı´a and Marı´a Antonia Baltrons

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Chapter 27 Potassium homeostasis in the brain at the organ and cell level . . . . . . . . . . . . . . . . . . . . . . . Wolfgang Walz

595

Chapter 28 Potassium and glia-derived slow potential shifts in relation to behaviour . . . . . . . . . . . . . Peter R. Laming

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Chapter 29 Regulation of Ca2þ stores in glial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Giovanni Scapagnini, Thomas J. Nelson and Daniel L. Alkon

635

Chapter 30 Decoding calcium wave signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.H. Cornell-Bell, P. Jung and V. Trinkaus-Randall

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Chapter 31 Mathematical modeling of intracellular and intercellular calcium signaling . . . . . . . . . . . Jian-Wei Shuai, Suhita Nadkarni, Peter Jung, Ann Cornell-Bell and Vickery Trinkaus-Randall

689

Chapter 32 pH regulation in non-neuronal brain cells and interstitial fluid . . . . . . . . . . . . . . . . . . . . . . . Suzanne D. McAlear and Mark O. Bevensee

707

Chapter 33 AVP effects and water channels in non-neuronal CNS cells . . . . . . . . . . . . . . . . . . . . . . . . . . Ye Chen and Maria Spatz

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TABLE OF CONTENTS Volume III Pathological Conditions Chapter 34 Alexander disease: a primary disease of astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lawrence F. Eng and Yuen Ling Lee

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Chapter 35 Glial reaction and reactive glia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Ka´lma´n

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Chapter 36 Contributions of astrocytes to ischemia-induced neuronal dysfunction in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Asta Ha˚berg and Ursula Sonnewald Chapter 37 Differential vulnerability of oligodendrocytes and astrocytes to hypoxic – ischemic stresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Husnia Marrif and Bernhard H.J. Juurlink

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Chapter 38 Glial heme oxygenase-1 in CNS injury and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hyman M. Schipper

869

Chapter 39 Astrocytes and microglia in Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Steven W. Barger

883

Chapter 40 Non-neuronal interactions in HIV-1-associated dementia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anuja Ghorpade and Howard E. Gendelman

901

Chapter 41 Glycoprotein gp120-mediated astrocytic dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eva Z. Kovacs, Beverly A. Bush and Dale J. Benos

921

Chapter 42 The role of astrocytes and microglia in persistent pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vasudeva Raghavendra and Joyce A. DeLeo

951

Chapter 43 Pathogenic role of glial cells in Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Przedborski and James E. Goldman

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Chapter 44 Upregulation of peripheral-type (mitochondrial) benzodiazepine receptors in hyperammonemic syndromes: consequences for neuronal excitability . . . . . . . . . . . . . . Mireille Be´langer, Samir Ahboucha, Paul Desjardins and Roger F. Butterworth

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Chapter 45 Role of the cytokine network in major psychoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Norbert Mu¨ller and Markus J. Schwarz

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Chapter 46 Shared effects of all three conventional anti-bipolar drugs on the phosphoinositide system in astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1033 Leif Hertz, Ye Chen, Yuly Bersudsky and Marina Wolfson Chapter 47 Glial loss in mood disorders and schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1049 Joseph L. Price Chapter 48 Glutamate excitotoxicity in the immunopathogenesis of multiple sclerosis . . . . . . . . . . . 1059 P. Werner, E. Brand-Schieber and C.S. Raine Chapter 49 Role of glia in prion disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085 David R. Brown and Judyth Sassoon Chapter 50 Schwann cells in diabetic neuropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1105 Andrew P. Mizisin Chapter 51 Mu¨ller cells in retinopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117 A. Bringmann, M. Francke and A. Reichenbach Part III: Colour Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1133 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145

Cytoarchitectonics of non-neuronal cells in the central nervous system Joachim R. Wolff p and T. Ivo Chao Department of Anatomy, Neuroanatomy Unit, University of Go¨ttingen, D-37075 Go¨ttingen, Germany p Correspondence address: Kreuzberging 36, D-37075 Go¨ttingen, Germany E-mail: [email protected](J.R.W.)

Contents 1. 2.

3.

4. 5.

6.

7.

8.

Introduction General considerations 2.1. Relevant features of CNS tissue organization 2.2. The repertoire of cell structuring 2.3. Cytoarchitectonics reveals relations between the structure of cells and tissue 2.4. Morphogenesis couples genetic information to tissue-derived instruction Cytoarchitectonics of endothelia of the terminal vascular bed 3.1. Angioarchitectonic constraints 3.2. Angiogenetic constraints 3.3. Cytoarchitectonics of capillary endothelia 3.4. Conclusions on the cytoarchitectonics of capillary endothelia Diversity of neuroglia Microglia 5.1. Distribution in CNS 5.2. Activation of diverse functions 5.3. Conclusion Oligodendroglia 6.1. The polymorphic cell type 6.2. Composite cell processes and cellular subtypes 6.3. Cell distribution in CNS 6.4. Conclusions Ependymo-astroglia 7.1. Common features of the cell system 7.2. Functional differentiation 7.3. Histoarchitectonic differentiation of cell types 7.4. Common structural components of ependymo-astroglial cell types 7.5. Cooperative tissue monitoring by astrocytes Concluding remarks

Advances in Molecular and Cell Biology, Vol. 31, pages 1–51 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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J.R. Wolff and T.I. Chao

The unique complexity of cellular interactions in central nervous system (CNS) tissue is based on the intra-epithelial association of cells and confrontation of multiple cell types of different origin and contact relations with neurons and/or intracerebral (intra-epithelial) blood vessels. To assess the interactive potential of non-neuronal cell types, their ramified shapes have to be characterized in terms of topography of structural and molecular cell components and their contact relations within metacellular entities and tissue compartments. By utilizing structural, morphogenetic and topographical information including quantitative data, cell-type-specific cytoarchitectonics turns out to be a valuable tool that combines tissue-related cell structures with cell-related tissue compartments. This approach is exemplified by endothelia of intracerebral blood capillaries, microglia, oligodendroglia and ependymo-astroglia, with special reference to astrocytes.

1. Introduction Cytoarchitectonics is commonly used as a descriptor of tissue architectonics. To construct stereotaxic atlases of the central nervous system (CNS), cytoarchitectonics is focused on neurons and distribution patterns of their cell bodies. In this application, neuronal cytoarchitectonics is sometimes supplemented by topographical variations in the density of intracerebral capillaries (angio-architectonics; see Lierse, 1963; Duvernoy, 1983), myelin sheaths (myelo-architectonics; e.g., Zilles et al., 1980; Paxinos and Franklin, 2001) and chemical markers (chemo-architectonics: enzyme histochemistry and immunohistochemistry as explained in the Handbook of Chemical Neuroanatomy; Bjo¨rklund and Ho¨kfelt, 1983 –2003). The term ‘non-neuronal cells’ subsumes all cells that are located in the nervous system without being neurons. At first glance, heterogeneity within this cell group does not make too much sense. Here, we may utilize it as an indicator of important aspects of CNS structure. This organ essentially consists of epithelial tissue, though its organization integrates cell types of (neuro-)ectodermal origin and mesodermal origin. This combination is not only due to intra-epithelial positioning of blood vessels (intracerebral vascularization)—neuroglia also comprise macroglial cell types and microglia. The latter probably stems from hematogenic macrophages, which are of mesodermal origin and invade the mammalian CNS during postnatal development (see Section 5). However, distribution patterns of non-neuronal cell bodies do not vary more, but mostly less than those of neuronal perikarya (for quantitative data see Blinkov and Glezer, 1968). Why should one still study cytoarchitectonics of non-neuronal cells? Cells with non-compact shape are not sufficiently represented by cell bodies suspended in an undefined tissue space. This is the case for neurons and non-neuronal cell types alike, which project processes into the surrounding tissue. By attaining arbor-like (dendritic) or torus-like geometry, such cells penetrate or enclose tissue territories, which they do not fill with their own cell volume (Fig. 1E0 ). These cell territories define tissue spaces for potential interactions with exogeneous signals (diffusible substances, electric fields, contacts with other cells, etc.). In addition, cytoarchitectonics discriminates cell-typespecific structures developing autochtonically from other structures, which vary in different tissue compartments, because their formation is induced by environmental signals.

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For example, presence or absence of surface extensions characterizes astrocytic subtypes as well as location in the gray and white matter, respectively (Privat et al., 1995). Careful observation even extends this criterion to subcellular compartments of ependymoastroglial cells (e.g., Cajal, 1911; Section 7). Statistical analysis of cell territories combined with topographical evaluation of cell structure will prove to be a suitable tool for discrimination of cell types, subtypes and subcellular local differentiation of non-neuronal cells. Cytoarchitectonics characterizes the interactive potential of cell types in accessible compartments of tissue by defining the territorial character of cells and distinguishing between endogenous and tissue-dependent determinants of cell structure. Since cytoarchitectonics is not commonly used as an explicit research method, some general considerations are outlined below (Section 2) to identify the type of information provided by cytoarchitectonic evaluation. This section introduces relevant aspects of nervous tissue organization (Section 2.1) and general cell structure (Section 2.2), the architectonic research strategy (Section 2.3), and points to morphogenetic constraints of cytoarchitectonics (Section 2.4). The sections on non-neuronal cytoarchitectonics classifies cell types subsumed in this category as vascular (Section 3), hematogenic (Section 5), and neural cell types (Sections 6 and 7), and treats selected cell types in more detail: vascular endothelia (Section 3.3), microglia (Section 5), oligodendroglia (Section 6), ependymo-astroglia (Section 7), and astrocytes (Section 7.5). Obvious omissions shall reduce the complexity and sharpen the focus on tissue organization in the CNS. (i) The peripheral nervous system is excluded (but see Fig. 1F –G), as are nervous systems of lower vertebrates and invertebrates. In terms of glial cytoarchitectonics, however, there are similarities with the mammalian CNS. For example, Schwann cells combine characteristics of marginal astroglia and oligodendroglia, and invertebrate glia resemble marginal astrocytes. (ii) Regional variations in tissue architectonics and topographical specialization of glial subtypes are disregarded, except for a few examples selected to elucidate cytoarchitectonic characteristics. (iii) Interactions between more than two tissue components have been excluded because their role in cyto- and histo-architectonics is speculative, at present (for perisynaptic glia in tripartite synapses, see Volterra et al., 2002; Chao et al., 2002). (iv) Contributions of nonneuronal cells to the composition and dynamics of extracellular fluid in the labyrinth of intercellular clefts in the CNS remained beyond the scope of this chapter. (v) The distribution of stem cells and glial precursors in the adult CNS will not be discussed here. (vi) Cells located in the wall of arteries and veins are not treated, while capillary endothelia were selected as interaction partners of astroglia. (vii) The rapidly growing literature on the interactive potential of microglia will not be reviewed here. Rather, microglia will be treated as a cell system. 2. General considerations 2.1. Relevant features of CNS tissue organization As an organ, the nervous system regulates sensory – effectory interactions between the organism and its environment. This function is mediated by physical contacts between neurons and their non-neuronal targets outside the nervous system. To reach targets,

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millions of neuronal cell processes (axons) have to penetrate the outer border of this organ (see Fig. 1E0 ). Inside the CNS, neurons form trillions of synaptic junctions, which are functionally dependent on appropriate composition of the extracellular fluid (see, e.g., the chapter by Walz). Perineuronal homeostasis is achieved by submission of neurons to intra-epithelial organization and association with non-neuronal cells. The nervous system is derived from an ectodermal epithelial anlage (neuroepithelium; Fig. 1E); neurons have (in terms of tissue relation) apolar precursors, which restrict perineuronal spaces to intercellular clefts within the epithelium (‘lateral’ cell surfaces; compare Fig. 1A –G); and non-neuronal cell types originating from different (neuroepithelial, vascular or hematogenic) sources, monitor and modulate the perineuronal milieu. Thus, the nervous system consists of a specialized epithelial tissue that includes non-neuronal cells.

Fig. 1. Cytoarchitectonic characterization of non-neural epithelia (A–D), neuroepithelium (E), its derivatives (central and peripheral nervous system: E0 and F), and the olfactory epithelium as a derivative of the olfactory placode (G). Descriptors are tissue polarity and the polarity, shape and contact relations of epithelial cells. Where epithelial cells form monolayers (A, B, pe in E0 ), every cell has the same polarity, but the size relations may vary between the apical, lateral and basal cell surfaces. Squamous epithelia (A) regulate transepithelial functions with small cell density, large size of apical and basal cell membranes, and minimized lateral cell interactions. These criteria also fit endothelia of blood vessels (bv and oval mesenchymal structures in A–G), including capillaries (c in E0 ). The endothelium continuously lines brain capillaries, except in the choroid plexus (see pe in E0 ), where it is fenestrated. In cuboidal epithelial cells (B; e in E0 ), enlargement of lateral cell surfaces indicates that intraepithelial cell interactions are functionally important. Formation of microvilli (e in E0 ) and infoldings (not shown) may enlarge the apical and basal cell surface, respectively. In pseudostratified epithelia (C), cell polarity varies from complete to incomplete compared with tissue polarity, i.e., all cells adhere to the basal lamina, but not every cell reaches the apical surface of epithelium. In stratified epithelia (D), cells retain compact (polygonal) shapes and either lose contact with the apical or the basal tissue surface, or both. Accordingly, cell polarity is either reduced to apical-to-lateral or basal-to-lateral or lateral-to-lateral (apolar). The neuroepithelium (E) differs from both C and D in that mitoses (mi) are shifted from the epithelial basis to the apical surface (ventricular matrix zone). In addition, E is intermediate between C and D, in that neural cell types, by forming cell processes, lose compact shape and develop various types of intra-epithelial polarities (see E0 ). The choroid plexus is composed of cuboid ependymal cells (compare pe in E0 with B). Corresponding to marginal tanycytes in the adult CNS (mt in E0 ), radial glial cells remain tripolar. Ependymal (e) and astroglial (a) cells are polarized between the lateral cell surface and the apical (ventricular) or basal (meningeal or perivascular) tissue surfaces, respectively. The ‘lateral cell surface’ being enormously enlarged, monitors the complex labyrinth of intercellular clefts in the neuropil. In contrast, oligodendrocytes (o), microglia (m), and neurons (n) completely lose the apico-basal polarity of the epithelium and develop cell-type-specific polarities. The neuron-type-specific polarity spans between afferent and efferent synapses. Also, glia– neuron association depends on expansion of “lateral” cell surfaces, which characterizes the nervous system. Certain neurons project axons into the peripheral nervous system (ax in E0 , F, G). Also in the peripheral nervous system (F), neurons (dark gray) and their processes (e.g., ax) are more or less completely covered by glial cells (light gray), while, in contrast to CNS, the apical tissue surface has been completely lost and basal laminae separate blood vessels from neural tissue. The olfactory epithelium (G) contains neurons and glia-like supporting cells, but otherwise is constructed like a pseudostratified epithelium (compare the position of mitoses in G and C). Abbreviations: a: astrocyte; ax: axon; bv: blood vessel; c: intracerebral capillaries; e: ependyma; m: microglia; ma: marginal astrocyte; mi: mitosis; ms: myelin sheath; mt: marginal tanycyte; n: neuron; o: oligodendrocyte; pe: epithelium of choroid plexus; ra: radial astrocyte; rbv: radial blood vessel; vt: vascular tanycyte.

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2.1.1. Developmental transformations In the adult CNS, the neuroepithelial organization is blurred by developmental transformations. (i)

(ii)

(iii)

(iv)

(v)

Unique growth of neural tissue enormously increases the thickness of the CNS, e.g., by 200:1 in the human forebrain, compared with the epidermis, which is the thickest epithelium outside the CNS (compare Fig. 1D and E0 ). By invasion of blood vessels into the neuroepithelium (intracerebral vascularization, as illustrated at the right edge of Fig. 1E0 ), the ’basal’ tissue surface is extended into the interior of the neuroepithelium and extremely expanded, e.g., in the human neocortex, to about 60:1, compared with the marginal tissue surface covered with pia mater. Extensive formation of branched cell processes and surface extensions (neuronal spines, fine processes of microglia and lamellipodia and filopodia of astrocytes) by neuronal and non-neuronal cells enormously enlarges the internal cell surfaces of neural tissue. For example, the cell surface of astrocytes increases to about 40:1, compared to cuboid epithelial cells with the same volume. Deviation from compact cell shape (see Fig. 1A – D) is genetically controlled in derivatives of neuroepithelium, since neurons in vitro and cells derived from neural crest in vivo form branched processes after translocation into other organs (e.g., melanocytes, Langerhans cells, ‘dendritic cells’; Lotze and Thomson, 1999). Accordingly, the extracellular space of the neuropil largely consists of an extensive labyrinth of intercellular clefts, which have a width of about 15 – 30 nm and an areal extension of about 5000– 7000 mm2/mm3 and sum up to about 0.07 –0.22 mm2/mm3 of neuropil. In addition, perivascular basal laminae occupy about 0.1 –1% of the tissue volume (see Section 3). Taken together, these intercellular spaces make up about 7– 23% of tissue volume. Neuroepithelial stem cells are shifted from the mesenchymal (basal) surface of ectodermal epithelium to the ventricular (apical) surface of neuroepithelium (compare mitotic cells in Fig. 1E with B –D and G). In the postnatal CNS, multipotential stem cells and cell proliferation are progressively restricted to subventricular matrix zones, while glial precursors are often found in perivascular positions (Fig. 2A).

2.1.2. Cell-type-specific polarities Structurally, epithelial tissue types differ in that their cells take different shares of the apical-to-lateral-to-basal tripolarity of each epithelium (compare Fig. 1A –E and G; Simons and Fuller, 1985). In the adult CNS, this criterion discriminates neurons and nonneuronal cell types. Apical-to-lateral-to-basal tripolarity of epithelial sheets is preserved in radial glia, tanycytes and choroid plexus epithelial cells, while ependymal and astroglial cells are confined to the apico-lateral and baso-lateral bipolarity, respectively. Other cell types appear ‘apolar’ in that they are restricted to the intra-epithelial space and their processes are exclusively confronted with ‘lateral’ cell surfaces. Among these,

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oligodendroglia and neurons establish cell-type-specific polarities. The oligodendrocytic polarity is based on attraction to surfaces of thicker axons and formation of myelin sheaths (Section 6). For neurons, it is the polarity between secretory and receptive membrane specializations (presynaptic and postsynaptic elements). Synaptic junctions, in turn, belong to those subcellular complexes, which may attract ependymo-astroglial cells as perisynaptic glia (see ”tripartite” synapses; Chao et al., 2002). As the only type of mobile cells that are consistently present in the CNS, microglia take a special role in that they do not seem to establish stable contacts or lasting associations with other cells in normal CNS tissue. Thus, differential polarities of neuronal and non-neuronal cell types and specific associations between them are important characteristics of neural tissue organization.

2.1.3. Compartmentalization CNS tissue is highly compartmentalized. Even the smallest subdivisions (e.g., spinal segments, brain regions, cortical areas and subareas, cortical layers and columns, nuclei and subnuclei) are larger than the tissue territories occupied by individual non-neuronal cells discussed below. Therefore, differences in regional development may create regionspecific subtypes of glial cells (e.g., Bergmann glia in the molecular layer of the cerebellar cortex and Mu¨ller cells in retina), while region-specific activity in neuronal networks, release of specific transmitters, and trophic agents may modify glial differentiation. Such context-related differences in glial differentiation are lost after several passages of culture in vitro. Thus, the interactive potentials of non-neuronal cell types do not only depend on the tissue territory defined by their own processes, but are also influenced by their position in a specific regional or local context.

2.2. The repertoire of cell structuring 2.2.1. Compact versus stellate shapes In suspension, cells of all types attain more or less a spherical shape. Availability of adhesive substrates, however, results in one of two general forms of cell shaping. (i)

Cells maintaining a compact shape, behave like soap bubbles. Their shape varies from polygonal, if they are interposed between multiple adhesion sites with about equal stability (e.g., epidermis; see Fig. 1D), via cuboid, if cells adhere unilaterally to a stiff substrate and multilaterally to similar cells (Fig. 1B), to disc-like with unilateral adhesion (e.g., squamous epithelia; see Fig. 1A). Cells of all epithelial types belong to this category. Also in the CNS, two types of non-neuronal cells show squamous or cuboid shape (capillary endothelia and ependymal cells, respectively; see Fig. 1E0 ). (ii) Cells of the other category attain uni- or bipolar or stellate shapes by extending processes with or without branching. Processes resemble perikarya, in that both contain mixtures of cytosol, organelles and cytoskeletal components, such as microtubules and intermediate filaments, though aggregated Golgi-lamellae are rare and cytoskeletal elements prevail in processes (Peters et al., 1991).

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Fig. 2. Developmental constraints of ependymo-astroglia. (Note that the pial surface is at the top in this figure, whereas it is at the bottom in Fig. 1.) (A) Radial and non-radial glia have separate precursors and differ in cell polarity. As a direct derivative of ventricular matrix cells (see mitoses ‘mi’ in the ventricular zone ‘VZ’), radial glia maintains the apical-to-lateral-to-basal tripolarity like cells in epithelial monolayers, although epithelial thickness increases enormously. Radial glia combines contacts to the ventricular and marginal (covered by the pial basal lamina, ‘bl’) surfaces of CNS tissue with a large number of intra-epithelial contacts with other glial cells

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2.2.2. Process formation Mechanistically, process formation is not uniform. On one hand, process extension may result from a combination of stretching and focal adhesion of cells. In this way, migrating cell bodies may drag a trail of processes behind or cell elongation passively follows tissuerelated expansion or distortion. Such ‘passive growth’ of processes plays a prominent role in the CNS. In the neocortex of adult rats, for example, about 60% of the collective lengths of axons developed in this manner after they arrived or originated in the tissue. On the other hand, processes may elongate as a result of ‘active growth’ that is guided by growth cones or other programs of morphogenesis. Among non-neuronal cells, we will also have to distinguish between two types of cell processes: One can be regarded as a witness of tissue growth, because length and course are predominantly determined by passive growth (Fig. 2B). Other types of processes show cell-type-specific geometry that is determined by active growth and does not vary in different locations.

2.2.3. Surface specializations Lamellipodia, filopodia and microvilli may appear on cells of all types and all shape categories. These specializations may decorate all parts of surfaces from perikaryon to the tips of processes. Despite differences in shape, surface extensions consist of plasma membranes with attached subsurface gel. The latter is predominantly composed of actin and actin-binding proteins (see the chapter by Derouiche), which enclose soluble proteins (e.g., in astrocytes: S-100 proteins, glutamine synthetase, glycogen phosphorylase; for references see Chao et al., 2002). Since surface extensions in principle differ from perikaryon and processes by the lack of organelles and cytoskeletal elements, the term ‘peripheral processes’ used by Derouiche (this volume), is not adopted here. Among nonneuronal cells, surface extensions play special roles. Like in other epithelia, the huge membrane surface of microvilli is exposed to the luminal (ventricular) tissue surface of ependymocytes. In contrast, astroglial lamellipodia and filopodia, being extremely numerous, are confronted with intra-epithelial cell surfaces. After selective attachment to axonal membranes, oligodendroglial lamellipodia even lose most of their cytosolic

(icc) and neurons (not shown). In the adult CNS, these postmitotic cells may transform into ‘marginal tanycytes’ (see mt in Fig. 1E0 ), which is the prominent type of glia in lower vertebrates and in the spinal cord, brainstem and hypothalamus of mammals, or they may give rise to Mu¨ller cells in the retina. In contrast, precursors of non-radial glioblasts primarily lose contacts with both the outer and inner tissue surfaces. Secondarily, these cells may either establish contacts with newly formed parts of the marginal (pial) surface, developing into marginal or radial astrocytes (including Bergmann glia of the cerebellum), or with intracerebral blood vessels, as is the case for the majority of astrocytes forming vascular endfeet (see also Fig. 1). Note that non-radial glia never regains contact with the ventricular surface. CP: cortical plate; ED: embryonal day; MZ: marginal zone; SVZ: subventricular zone. (Modified from Rickmann and Wolff, 1985.) (B) Ependymo-astroglial cells combine formation of cell processes with focal attachment of endfeet (ef) to marginal (pial) or vascular basal laminae (bl). Depending on positioning, later-forming cell processes (neuropil formation) will displace and distort “older parts” of glial cells. As indicated by arrows, cell bodies (1) and/or branching points of processes (2, 3) may be shifted away from attachment sites into the depth of the tissue. As indicated by two-headed arrows, attachment sites may divide and be moved away from their neighbors as a result of the expansion of the border between mesenchymal and CNS tissues. These aspects of primary processes of ependymo-astroglial cells can serve as witnesses of tissue growth.

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content during myelin formation. Thus, lamellipodia and filopodia are important components of certain non-neuronal cell types. 2.2.4. Structural dynamics In vivo approaches to the study of nervous tissue reveal more and more structural changes in the adult CNS, which are either ongoing spontaneously or induced by exogeneous signals (see the chapters by Salm et al., Mercier and Hatton, and Prevot et al.). Structural dynamics is not restricted to development and in vitro conditions. For example, a cellular molt of glial cells (Korr, 1980), ameboid movements of microglia (Section 5), and formation or retraction of surface extensions and peripheral processes on astrocytes also occur in situ and change intercellular contact relations in the neuropil, at least under pathological conditions (e.g., perisynaptic glia; for details see Chao et al., 2002). Other structures appear stable (e.g., as mentioned above, cell processes which have been passively distorted during development; for marginal and perivascular endfeet of tanycytes and astroglia see Section 7.4). Thus, morphology and contact relations of cells and cellular components have to be analyzed in terms of dynamic changes. 2.3. Cytoarchitectonics reveals relations between the structure of cells and tissue Architects try to match constructive properties of an entity (room or building or landscape) with its potential to interact with surrounding structures. Correspondingly, cytoarchitectonic research does not deal with actual interactions between individual cells. By studying cellular geometry in relation to the texture of surrounding tissue, cytoarchitectonics rather presents an estimate of the interactive potential of specific cells or cell types. Cytoarchitectonics is selective, in that it focuses on properties playing a role in the interactive potential of a cell type. Cytoarchitectonics is janus-faced, in that it enables the separation of cellular features, which are determined by the cell itself (e.g., the typespecific molecular equipment), from those resulting from interactions with the environment (e.g., the lengths of cell processes; see Fig. 2B). Similarly, the cellular environment is divided into exogeneous determinants provided by other cells (e.g., contact partners) and territorial characteristics, which depend on the reach and branching patterns of the cell’s own processes. Thus, cytoarchitectonics views cells as tissue components, for which the interactive potential is cooperatively determined by tissue-related cell architecture (cell territory) and cell-related tissue architecture (potential interaction partners). 2.3.1. Descriptors of cytoarchitectonics These may either be whole cells (tissue territories) or their subcellular representatives in the tissue, such as perikarya (see Introduction), processes, other surface extensions and specialized contacts between plasma membranes. Spatial distribution patterns are quantitatively described by stereological measures, such as the volume fraction occupied by the respective structures (VV), surface densities (SV), numerical densities (NV) of compact structures and collective lengths of elongated structures (LV). All of these measures are related to units of tissue volume (V). In the CNS, however, reference spaces

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need special attention. This organ presents the unique case of an epithelial tissue with an enormously expanded volume (Fig. 1) and heterogeneity. The internal complexity of CNS tissue is based on non-compact cell shapes (cell bodies vs. neuropil), differential distribution of neuronal cell bodies, axons and myelin (white vs. gray matter), and intracerebral vascularization (Section 3). Subcellular structures are usually related to the volume of the surrounding ‘neuropil’. This compartment of the gray matter occupies different volume fractions, depending on the brain region and species studied. For example, neuropil comprises up to 98% of the molecular layers of cerebellar and cerebral cortex, yet its volume fraction may be as small as 3% in layers rich in neuronal cell bodies, as in the cerebellum or hippocampus, while intermediate values are found in other regions of gray matter. Unfortunately, definitions of neuropil are not uniform in the literature. In a strict sense, neuropil is the tissue compartment filled with interdigitating processes of neurons and neuroglia and includes the extensive labyrinth of intercellular clefts, which represent the extracellular space of this epithelial tissue. In morphometric studies, however, tissue components included or excluded from neuropil differ or even remain unclear. Differences and uncertainties may add up to about 8 – 10% of the tissue volume, which is occupied by intracerebral blood capillaries and perikarya of neurons and/or glial cells. Where cell bodies are segregated from zones of neuropil, strategies of evaluation have to be adapted to the task (e.g., distribution of astrocytes in olfactory glomeruli; Chao et al., 1997a; spatial density of Bergmann glia in the cerebellar cortex; see areal vs. volume density in Table 3). Where glial cell bodies associate with neuronal cell bodies or blood vessels or axonal fascicles, measurements can even be restricted to distances between paired structures and relative numbers of pairs (‘satellite glia’ see Section 6). 2.3.2. Complementation of cytoarchitectonic descriptors Cells are usually regarded as elementary entities of biological organization. In CNS tissue, the cellular level is only one of several options of elementary organization. Intercellular cooperation forms multicellular networks, e.g., synaptic coupling between neurons or gap junction coupling between ependymo-astroglial cells (Section 7.5). Cooperation between cell parts establishes subcellular cooperation units, e.g., simple and complex synapses, multilayered stalks of glial lamellae (Chao et al., 2002), and sensory vs. central synaptic compartments of olfactory glomeruli (Chao et al., 1997a). Intercellular and subcellular aspects of cooperation are coupled, where subpopulations of neurons show both, ‘autapses’ and intercellular synapses (e.g., in the suprachiasmatic nucleus; Gu¨ldner and Wolff, 1996). Similarly, astrocytes combine autocellular and heterocellular coupling by gap junctions (Wolff et al., 1998; see also the chapter by Scemes and Spray). Since multilevel organization is not restricted to neurons, but also characterizes non-neuronal cell types, cytoarchitectonic descriptors have to be complemented by networks at the supra- and infracellular levels. (i)

Multicellular networks have to be considered, where cellular functions are governed by intercellular cooperation (e.g., calcium waves in astroglial networks and glia –neuron interactions at ‘tripartite synapses’; Volterra et al., 2002; see chapters by Cornell-Bell et al., and by Jung et al.).

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(ii) Subcellular networks may connect subcellular entities rather than cells (e.g., astroglial gap junctions; Rohlmann et al., 1994). (iii) Even molecular networks have to be added, where cellular functions mechanistically depend on them, e.g., coordinated effects by stimulation of receptors for vasopressin on astrocytes, endothelial cells and choroid plexus epithelial cells (see the chapter by Chen and Spatz). Certainly, these distinctions do not cover all options of cooperation used by neural cells. For example, cytoarchitectonics indicates that cells of one non-neuronal type (microglia; see Section 5) cooperate by parallel actions. This resembles cooperation in dissociated cell systems, e.g., those involved in paracrine functions. 2.4. Morphogenesis couples genetic information to tissue-derived instruction For each cell, morphogenesis determines the site of origin from stem cells, guides the immigration of precursor cells into the area of positioning, provides partners for contact formation, directs individual cell differentiation and develops appropriate tissue architecture. Combined with data on the temporal – spatial course of development, adult cell shape reveals results of active cell growth and passive transformation due to anisometric growth of the surrounding tissue (e.g., ependymo-astroglial cell types; Section 7). Where function is based on interaction between co-localized structures, cytoarchitectonics helps to estimate the capacity of functional adaptation in the respective tissue. For example, the spatial density of blood capillaries in a brain region (angioarchitectonics) sets limits for the regulation of regional blood flow and thresholds for hypoxic cell damage. Thus, morphogenetic constraints determine cell-type-specific differences in adult cell shape. 2.4.1. Genetic versus morphogenetic determination Architects design final products and assure realization by detailed construction plans, while biological development is not completely determined by genetic information. Rather than providing a blueprint of the final product, the genome codes for a potential of morphogenetic options. The inter-individual diversity among organisms of the same species reveals variability that reaches from relatively small differences between identical twins to extreme deviations from statistical means in non-viable malformations (teratology). Among cells of the same class, cytoarchitectonics reveals three types of cellular properties. (i)

Cell-type-specific characteristics are more or less constantly expressed at all locations (e.g., expression of glutamine synthetase and S100 proteins in astrocytes; see Derouiche and Frotscher, 2001; Rickmann and Wolff, 1995). (ii) Optional properties, varying regularly at different locations, may lead to cellular subtypes, e.g., fibrillar vs. protoplasmic astrocytes in white and gray matter, respectively. In other cases, environmental influences only affect subcellular compartments. For example, on its passage through white matter or a glial scar, an astrocytic process may change to smooth surface and fibrillar content, while other

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processes of the same cell located in gray matter, have rough surfaces and contain few intermediate filaments. (iii) Other parameters show inter-individual variations even in co-localized cells and processes (e.g., the positioning of lamellar surface extensions on astrocytic surfaces). Post-mortem tissue does not allow determination of the time, when these variations have been induced and of their durability. In vitro studies, however, suggest that local variations in structure may be transient due to dynamic changes, which differ between non-neuronal cell types. For example, microglia retains migratory activity throughout life (Section 5.2), while the dynamics in cell shape is probably restricted to surface extensions on astrocytes (Section 7.5). Thus, irreversible steps of morphogenesis gradually reduce the potential of cell-type-specific differentiation, while reversible mechanisms of morphogenesis persist and permit adaptive variations in cell structure throughout adult life. 2.4.2. Architectonic consequences of holistic organization Morphogenesis serves a holistic type of organization. (i)

Outer tissue borders appear continuous throughout morphogenesis, though they expand during development and are rapidly restored after injury and during physiological invasion of blood vessels into brain tissue (Section 3). Continuity of the mesenchymal – neural tissue border is based on basal lamina, to which ependymoastroglial cells adhere (Figs. 1E0 and 2A). Depending on the time of development, different subtypes of these glial cells attach to persisting old parts or re-modeled new segments of basal laminae (Fig. 1E0 ; Section 7.5). (ii) Growth is intercalary or interpolated (but not appositional or ‘from basement to roof’). Increase in cell number and active growth of cell processes induce passive growth that elongates pre-existing cell processes (see Fig. 2B). Relations between active and passive growth determine the courses of cell processes of various age and specify the structure of non-neuronal cells (e.g., long and radial short processes of ependymo-astroglial cell types; Section 7.4). (iii) Morphogenesis progressively subdivides cells and tissue. For example, cells taking medial vs. lateral positions in the neural plate, retain or lose apico-basal polarity during neurulation and develop into central and peripheral glial cells, respectively (compare Fig. 1E0 and F). Thus, the structures and contact relationships of cells and cell processes can serve as indicators of developmental interactions with tissue. The earlier structures have been formed, the more they are distorted by passive growth. 2.4.3. The multifunctional potential of morphogenetic entities Morphogenesis provides intermediate products with potentials of functional use. In terms of morphogenetic functions, this potential defines options of the next steps in structural development. As mentioned above, the morphogenetic potential is stepwise reduced to reversible mechanisms, which can be recruited by functional systems and adapt structure to functional use (e.g., conformational changes of macromolecules; recycling of organelles; turnover of cells or molecules; growth and withdrawal of cell processes;

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back-and-forth transport of organelles and molecules). Apparently, the spectrum of such mechanisms is large enough to provide most intermediate products of morphogenesis with the potential of participating in multiple functions. For example, astrocytes seem to act as multifunctional, but spatially fixed units in parallel, while microglial cells may switch from resting to migratory and phagocytic states (Streit, 1995). In addition, there are optional programs of subcellular differentiation. As mentioned above, fibrillar and protoplasmic processes of astrocytes are not bound to determined cell types, but result from local and reversible adaptation to signals that originate from the actual surroundings. Accordingly, the cell-type-specific potential of endocellular determination should be preserved after transplantation and under in vitro conditions, while exogenous or contextual determination is lost after some time in vitro or under pathological conditions. Thus, hypotheses on structures with endogeneous or exogeneous determination are testable by structural adaptation to functional conditions. 3. Cytoarchitectonics of endothelia of the terminal vascular bed Intracerebral vascularization transfers into brain tissue various cell types, which either compose the vascular wall or migrate into perivascular positions. The continuous sheet of endothelial cells lining all vessel segments, will be represented here by the cytoarchitectonics of capillary endothelia, because these cells are well known interaction partners of the ependymo-astroglial cells, which compose the ‘perivascular glial limiting membrane’. Pericytes and smooth muscle cells will be disregarded here, because these cell types do not seem to develop CNS-specific structure. The same criterion can be applied to (peri-)vascular macrophages, which remain separated from brain tissue by basal lamina, at least under normal conditions, and to lymphocytes and other blood cells, which may penetrate the perivascular basal laminae and invade brain tissue, but remain “transient guests” in CNS tissue. Quantitative data derived from rat neocortex (Ba¨r, 1980; for references to other species, including man, see Blinkov and Glezer, 1968), are restricted to vessels with diameters , 8 mm. This includes capillaries (diameter: 5.04 ^ 2.9 ¼ ^ 2 standard deviations; Table 1) and thin parts of capillary-like arterio-venous (a-v) bridges (Fig. 3A and D). Although the endothelial lining of larger vessels is disregarded, essentials of intracerebral angioarchitecture and angiogenesis are introduced to define supracellular or tissue-related constraints of endothelial cytoarchitectonics. 3.1. Angioarchitectonic constraints Brain tissue combines large volume with highly variable metabolic activity. Sufficient blood supply is achieved by transfer of the terminal vascular bed from pia mater, where it is located during early stages of prenatal development, into the intra-neuroepithelial space (Figs. 1E0 and 2A). Depending on the perivascular apposition of astroglial cell processes and fusion of endothelial and glial basal lamina components, endothelia of intracerebral capillaries are ensheathed by thick basal laminae (50 – 100 nm; Ba¨r and Wolff, 1972; see transition from radial blood vessel and capillary in Fig. 1E0 ). Also, in renal glomeruli and

Age [postnatal days]

Diameter of capillaries [mm]

Length of capillaries per tissue volume [mm/mm3]

Number of capillary branches per tissue volume [mm/mm3]

Number of endothelial cells per tissue volume [mm23]

Surface area of endothelial cell [mm2]b

Length of endothelial cells [mm]

Thickness of endothelial cells [mm]

Volume of endothelial cell [mm3]

0–2 7–8 14 20–21 30–35 55 90–120

5.0 5.2 5.4 5.3 5.0 5.0 5.0

220 220 440 660 748 770 806

1,200 3,000 7,800 12,000d 10,800 — 9,600

8,500c 8,500c 12,750 20,400 21,250 20,400 20,400

825 850 1,175 1,080 1,100 1,190 1,260

25.9 25.9 34.5 32.9 34.9 37.7 39.3

— — 0.42 — — 0.28 0.26

— — 247

a

166 163

Mean values; for variances see Ba¨r, 1980. These values are underestimated because irregularities in the luminal cell surfaces and overlapping at interendothelial contacts were not taken into account. c During the first postnatal week, endothelial proliferation is proportional to the increase in tissue volume, while branching increases more. d The overshoot in branching density vanishes proportionally to further tissue growth. b

Cytoarchitectonics of Non-neuronal Cells in the Central Nervous System

Table 1 Developmental cytoarchitectonics of endothelial cells in capillaries of rat neocortexa

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Fig. 3. Angioarchitectonic constraints in the neocortex of adult rats. (A) Intravascular ink injection visualizes radial arteries and veins, which connect intracerebral microvessels with pial vessels. By differences in stem length (a–d) and horizontal reach of terminal branches, radial vessels feed or drain the terminal vascular bed in different layers of cortex. For example, branches of a type-d radial vein terminate at the border between layer 6 and the white matter (wm; compare arrow heads in A and B; bar ¼ 500 mm). (B) According to morphometric analysis, the angioarchitecture of adult neocortex is characterized by four sets of radial vessels, which terminate in different layers as indicated by a –d. (C) As revealed by the angioarchitecture, neocortical tissue is composed of overlapping modules (a –d). The horizontal diameter of the smallest module (a) varies between 180 and 200 micron. As indicated by the gray territories, horizontal diameters of larger modules are multiples of ‘a’. (D) At higher magnification, ink-filled microvessels reveal that the terminal vascular bed comprises two components: (i) precapillaries (a-v) characterized by relatively large diameters (6–10 mm) and straight courses, directly connect arterioles (a) with venules (v), while (ii) capillaries (c) having smaller diameters (5 mm; see Table 1) and winding courses, are highly branched and form a network that is connected to precapillaries (a-v channels; bar ¼ 100 mm). (E) On average, capillary segments between branching points are composed of 2.3 endothelial cells, including approximately one-third of seamless endothelium. The interbranch segment is straightened. By evaluating relative frequencies of capillary sections with one or two intercellular contacts, one can determine the proportion between intercellular overlap (23%) and segments composed of one cell with contact to itself (45%).

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17

pulmonary alveolae, endothelia with thick basal laminae connect with epithelial cells. In these locations, blood interacts with quasi-extracorporeal spaces, such as those containing urine and air, respectively. Brain capillaries are surrounded by the labyrinth of intercellular clefts between neural cells, which can also be regarded as a transcellular space that is protected from unspecific influences of extraneural sources by the blood – brain barrier. This barrier is based on complex interactions between endothelial cells and perivascular glia. The significance of these interactions is underscored by two findings: (i) the mechanistic basis of the blood – brain barrier (i.e., the complete sealing of intercellular clefts between endothelia by tight junctions and probably suppression of vesiculation) is induced by perivascular glia; and (ii) the blood – tissue border is extremely large in the CNS (about 13 mm2/mm3 of neocortical tissue; Table 1), and it amounts to about 20 m2 in an entire human brain. Compared to the pial surface area, intracerebral vascularization enlarges the border between mesenchymal and neural tissues by a factor of 10 – 50, depending on tissue thickness (e.g., in the neocortex of rats and the human CNS: 0.8– 1.5 mm and 2.5 –50 mm, respectively). In addition, intracerebral capillaries are directly apposed to perivascular glia (Fig. 1E0 ), while in most other parenchymal organs capillaries approach epithelia within the mesenchymal tissue, but do not touch their basal surface (Fig. 1A – G). Taken together, intracerebral capillaries enormously increase the potential for interactions between blood constituents, endothelia and neural components, provided the molecules mediating such interactions can pass the blood –brain barrier and the subendothelial basal lamina. In the leptomeninges, arteries and veins branch until they form functional end arteries and venous drainages of demarcated fields of brain tissue, respectively (for details, see Ambach et al., 1986). The next set of branches enters brain tissue, takes more or less radial courses, before they feed or drain the terminal vascular bed in different depths (Fig. 3A and B). The distribution pattern of radial stems and lateral extensions of first and second order branches visualizes the modular organization of brain tissue (e.g., in neocortex; Fig. 3C). The arrangement of vascular modules is region-specific. For example, four sets of modules characterize the neocortex from mouse to man (Duvernoy, 1983). Feeder and drainage modules of different size and position are connected by a common terminal vascular bed. Little attention has been paid to the fact that the fully developed terminal vascular bed consists of two components: (i) precapillary vessels (diameter: 6 –10 mm) directly connect arterioles with venules (a-v channels; but see Ravens, 1984); and (ii) thinner capillaries (diameter: 5 ^ 1.4 mm; Table 1) are highly branched and form a dense network. Spatial relations between these components have not been studied in detail, but some capillary branches certainly fuse with a-v channels (Fig. 3D). Although the functional efficacy of these connections is still unclear, similarities in diameter and arrangement of a-v channels suggest that these may function as thoroughfare channels for the bulk of erythrocytes with

(F) Statistical dimensions of endothelial cells, which line capillaries (after unrolling): 39.3 mm average cell length by including seamless (capillary length divided by cell number). Sections of nuclei appear in about one-quarter of all cases. 15,5 mm corresponds to the circumference of capillaries with an average diameter of 5 mm. Note, that intercellular contact lengths are shared by adjacent cells, while the luminal surface has to be approximately doubled to estimate the cell surface (see Table 1).

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diameters of approximately 7 mm, while net capillaries serve as plasma skimming, like in other organs (see, e.g., Wolff et al., 1975). Once known, their functional dynamics might change our understanding of regional blood flow regulation in brain tissue (see the ongoing discussion on the so-called bold effect in NMR). Roughly estimated, pre-capillary a-v channels make up only 1 – 5% of the terminal vascular bed, but their diameter meets that of erythrocytes. In contrast, the vast majority of terminal vessels are capillaries, and their proportion even increases in brain regions with high densities of terminal vessels, while their diameter is considerably smaller than that of red blood cells. Thus, redistribution of blood plasma and erythrocytes between vessels with smaller and larger diameters might drastically change the rheological (flow-related) resistance of the terminal vascular bed in the CNS. The specific lengths of capillaries (LV ¼ length per unit of tissue volume) differ between brain regions and local tissue compartments (Lierse, 1963; see Blinkov and Glezer, 1968). In white matter, capillary density is relatively low (100 –300 mm/mm3), while LV varies between 300 and 1600 mm/mm3 in gray matter. In the parieto-occipital neocortex of adult rats, for example, LV varies from 400 to 1280 mm/mm3 between layers (average 806 ^ 156 mm/mm3; Ba¨r, 1980; Table 1), though local variations within layers show a similar span (standard deviation about 20% of mean values). In the neocortex of various mammals LV is similar (e.g., man, monkey, dog, cat, rabbit rat and mouse; Blinkov and Glezer, 1968). Depending on capillary length, one can calculate that the average distance between neighboring capillaries varies between 25 and 100 mm. Correspondingly, distances between any point of gray matter and the next neighboring capillary vary between 13 and 50 mm. Conversely, every part of the capillary surface has the chance of making random contacts with astroglial processes (see Section 7.4). Ba¨r (1980) found a mean of 9550 capillary branching points per mm3 of neocortical tissue. The average distance between branching points should then be 47 – 50 mm. Because of irregular meandering, interbranch segments of capillaries are actually longer (87mm; Fig. 3E). The reserve in capillary length of about 50% enables adaptation of the capillary network to changes in tissue volume (during brain edema, cellular swelling, etc.) without elongation or rupture of microvessels. The rigidity of basal lamina material is large enough to allow mechanical isolation of capillaries from brain tissue. Thus, flexibility in capillary bending and mechanical support by basal laminae, rather than changes in the size of endothelial cells, safeguard spatial adaptation of capillary networks to changes in tissue volume and shape.

3.2. Angiogenetic constraints Intracerebral vascularization starts with endothelial sprouting at the epiparenchymal vascular plexus that transiently surrounds the CNS anlage during early stages of neural development (for details, see Ba¨r, 1980). After invading the CNS tissue and branching, the primary sprouts elongate proportionally to the thickening of brain tissue. Intracerebral sprouting fixes the relative positions of branching points. Intracerebral vascularization terminates with capillary elongation that accompanies the last stages of tissue (neuropil) growth, and almost exclusively occurs after postnatal day (PD) 8 (see below). As witnesses

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19

of tissue growth, arteries and veins show preferential radial orientation within the tissue, while capillaries take more or less random courses, at least in gray matter (e.g., in neocortex: radial stems; compare Fig. 3A and B). Elimination of arterio-venous shunts from the epiparenchymal plexus progressively shapes terminal arteries (Ba¨r and Scheck, 1983) and draining veins in the leptomenninges (Duvernoy et al., 1981; Ambach et al., 1986). What will be arterial or venous sides of the terminal vascular bed is finally determined late during the postnatal period. In other words, flow directions are adapted to the functional maturation of brain tissue (Wolff et al., 1992). By witnessing characteristic stages of brain development, intracerebral vascularization can be used as a marker of the modular organization of brain tissue (Fig. 2A – C; rat: Ba¨r, 1980; man: Duvernoy et al., 1981). Interestingly, the late shaping of venous drainage fields in neocortex is influenced by functional use and, therefore, can be permanently distorted by dysfunction during the first and second postnatal week in the rat, e.g. in the visual cortex after enucleation (Toldi et al., 1996). 3.2.1. Four sets of radial vessels During embryonic days (ED) 14 and 15, the first generation of radial sprouts and their terminal branchings establish the subventricular plexus in the neopallium of the rat. This plexus regresses, when the cortical anlage is being vascularized. In adult brains, so-called ‘basal lamina labyrinths’ are regarded as remnants of this regression. However, radial stems and branches in the intermediate zone persist and transform into vessels, which later drain the subcortical white matter. Invasion of radial sprouts proceeds, while migrating neurons populate the cortical plate and differentiate in cortical layers. The second to fifth generation of radial vessels terminates in layers 6, 5 –4, 3, and 2 – 1, respectively (Fig. 3B). Since these vessels persist, their laminar termination pattern in adult neocortex enables reconstruction of the timetable of invasion (layer 6: ED 18 –19; layer 5: ED 21; layer 3: postnatal day, PD 1; layers 1 –2: PD 4). This timetable is confirmed by the intralaminar (horizontal) spacing of radial vessels, and it may be used to reconstruct the growth kinetics of cortex volume (for details, see Ba¨r, 1980; Eins et al., 1983). Establishment of radial vessels between ED 14 and PD 4 suggests that precapillary a-v channels are formed during the same period, whereas the capillary network as previously mentioned is not formed until later. 3.2.2. Network capillaries The vast majority of network capillaries develop during the postnatal period (Table 1). During the first week, endothelial cell proliferation conforms to both elongation of capillaries and increase in cortical volume, though capillary branching increases (Fig. 4B), and double layered capillary walls may elongate by telescope-like sliding of endothelial cells (Ba¨r, 1980). During the second and third week, endothelial proliferation peaks, and it raises the numerical density of endothelial cells to that of the adult cortex (20,400 cells/ mm3; Table 1) and causes a transient overshoot in the capillary branching density. The corresponding undershoot in interbranching length is shown in Fig. 4B. Taken together with the overshoot in capillary width (Table 1), the data suggest that endothelial mitoses do not only cause branching, but also contribute to the elongation (73%; Fig. 4C) and

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Fig. 4. Developmental processes shaping the intracerebral network of capillaries. (A) Endothelial proliferation is accompanied by formation of filopodia (f in 1–4), independently of whether postmitotic cells serve widening (las; lateral separation) or elongation (los; longitudinal separation) of vessels or lateral sprouting (1). Where filopodia contact neighboring vessels, a bridge is formed between pre-existing vessels (2), while filopodia vanish (3, 4). In monocellular bridges, the lumen develops as a transcellular hole, in that intracellular vacuoles fuse and penetrate the tip of the sprout without intercellular contact along the vessel axis (arrow). In the adult brain, these ‘last’ steps of network formation remain labeled by seamless endothelia (see in 5 and 6; modified from Ba¨r, 1980). (B) Capillary sprouting rapidly reduces the average lengths of segments between branching points during the first two weeks after birth (arrow). Number of intercellular contacts is shown in the circles. As indicated by the decreasing frequency of cross-sections with two and three intercellular contacts (white area marked with p ), the formation and elongation of net capillaries reduces the proportion of precapillary a-v shunts (see Fig. 2) to less than 2% in adulthood. Note that the frequency of cross-sections with 0 (‘seamless’) and 1 intercellular contact (net capillaries) increases thereafter. (C) The postnatal increase in capillary length per tissue volume (abscissa scale: capillary density) depends on two mechanisms: cell proliferation (open circles) making the largest contribution (73% of the adult capillary density), was measured as actual endothelial cell numbers per unit of tissue volume (cells/mm3) multiplied by the average cell length at postnatal day 2 (normalization for all ages). The resulting capillary length in the absence of cell elongation rises proportionally to tissue growth during the first postnatal week, increases much more than tissue volume during the second and third postnatal weeks, and produces an

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21

widening of capillaries. Overshoots vanish during the final period of cortex growth that increases tissue volume by 20% between PD 20 and adulthood at about PD 90. Apparently, capillary sprouting is terminated by contact inhibition, where sprouts fuse with preexisting capillaries and stop filopodia formation (Fig. 4A). Even in the adult brain, one can identify ‘last’ sprouts as capillary segments consisting of “seamless endothelia”. Rather than opening of intercellular clefts between adjacent endothelia, in these cases, intracellular vacuoles fuse to form the lumen as a transcellular hole (Figs. 3E and 4A and C; Ba¨r et al., 1984). The finding that about one-third of the total capillary length of adult cortex is lined by seamless endothelia (Fig. 3E), indicates a late and explosive formation of net capillaries. Already at PD 20, astroglial processes cover 97% of the capillary surface, and the subendothelial basal lamina of capillaries has thickened, as described above. The blood – brain barrier seems to mature more rapidly at capillaries, which increase in length by elongation (rather than proliferation) of endothelial cells, as found after PD 20. Where capillary endothelia degenerate under pathological conditions, perivascular basal laminae usually persist in the form of ‘connective tissue strands’ (for references, see Guseo and Gallyas, 1974). These strands consist of basal lamina material, to which astroglial processes remain apposed, though the endothelial cells and vascular lumen are missing. Since this finding is rare in normal brains, the vast majority of capillaries seem to persist during normal development.

3.2.3. Tissue growth-dependent changes in length and shape After angiogenesis has stopped, increases in tissue volume will change the length and shape of vessels in a growth-dependent manner. Indeed, certain angioarchitectonic criteria can be used as descriptors of tissue growth (Ba¨r, 1980). The areal density of radial vessels at the pial surface, their average distance from neighbors in various depths of cortex, and the horizontal reach of their terminal branches, all correlate with the expansion of cortical surface during development (Eins et al., 1983). Similarly, the radial lengths of such vessels correspond with the thickening of respective layers. Little is known about cellular mechanisms, which underlie ‘passive’ vessel growth and adapt capillary length to changes in tissue volume. Primary development of capillaries depends on several different mechanisms such as endothelial cell proliferation, a telescope-like sliding and elongation (by flattening; see Table 1) of endothelial cells. It is still unclear, which of these mechanisms is responsible for length adaptation of capillaries under pathological conditions, e.g., in the vicinity of brain tumors.

overshoot in capillary length ( p , curve indicated by open circles) that is hidden by an overshoot in tissue volume. The overshoot is diluted by further increase in tissue volume after proliferation has stopped. Elongation of endothelial cells having a smaller share (27% contribution to increase in capillary length), was measured as actual endothelial cell length (see Table 1) multiplied by endothelial cell density at postnatal day 2 (cells/mm3; normalization). The resulting capillary length in the absence of endothelial proliferation is proportional to tissue growth until day 8, rapid during the second week, and slowly continues until adulthood (about 90–120 days after birth).

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Conversely, similarities in angioarchitectonics between different brain regions, such as the cerebellar and cerebral cortices (Conradi et al., 1980), indicate a similar coupling between tissue growth and capillary development (Conradi et al., 1979a,b). 3.3. Cytoarchitectonics of capillary endothelia 3.3.1. Cell dimensions Endothelia of intracerebral capillaries represent the smallest cell type in the CNS. Their average cell volume (163 mm3; Table 1) is about 10 times smaller than that of the smallest type of ependymo-astroglia (ependymal cells; Table 2). The cell shape resembles that of extremely flat discs or polygons (see squamous epithelia; Fig. 1A), which are more extended parallel to the axis of vessels than along their circumference (Fig. 3F). Average thickness / length / width are related as 1 / 150 / 60, respectively (0.26 / 39.3 / 16 mm; Table 1; Fig. 3F). Most endothelial cells embrace the cylindrical vessel lumen, make mutual longitudinal contact with themselves and contact two neighbors along the vessel axis. Despite the lack of longitudinal self-contacts, seamless endothelia show similar variations in length and luminal diameters as other endothelial cells (Ba¨r et al., 1984). Three-dimensionally, seamless endothelia have a torus-like shape (Fig. 3E). Where the blood – brain barrier depends on the endothelial lining, as in mammals, tight junctions completely seal the extracellular clefts of endothelia contacts. Without sealing, these would make up approximately 0.1% of the luminal surface area of capillaries (data from Fig. 3F) and less than half of that in seamless endothelia. Tight junctions have to be supplemented by the blocking of vesiculation, which in most capillaries enables random transendothelial passage of extracellular material via vesicles. Only when both passages are prevented, the equipment of endothelial membranes with appropriate transporters and carriers will determine the permeability of the blood –brain barrier. For comparison with glial cell types, it is important to note that despite extreme flattening, capillary endothelia have a relatively small surface-to-volume ratio of 7.7 (1260 mm2/163 mm3; compare Tables 1 and 2). 3.3.2. Segments between branching points The number of segments between branching points is a characteristic element of angioarchitecture, and it determines most aspects of endothelial cytoarchitectonics in network capillaries. Morphometrical analysis revealed that, in the neocortex of adult rats, interbranch segments are statistically composed of 2.3 endothelial cells (Fig. 3E). Since every third endothelial cell is seamless (30%; Ba¨r et al., 1984), about three-quarters of the segments seem to develop from ‘last sprouts’ (2:3 £ 3). One quarter may be derived from terminal vessels, which are perinatally formed and secondarily segmented by capillary sprouts (before PD 14; Fig. 4B). This fraction may indirectly indicate to which degree a-v channels are integrated in to the capillary network. 3.4. Conclusions on the cytoarchitectonics of capillary endothelia (i) Although intracerebral arteries and veins show growth-dependent orientation and topography, capillary endothelial cells are more or less randomly distributed in CNS

Table 2 Quantitative differences in cytoarchitectonics between ependymo-astroglial cell typesa Cell type

Number of cells in tissue volume [£103/mm3] (areal density ¼ NA)b

Average cell Cell volume [mm3] (volume fraction in surface [£103 mm2] tissue ¼ VV)

—

1.4

54 –100–153 18.5 –10.0 –6.5 (NA ¼ 5–20 £ 103/mm2)

68 –125– . 612

5–12

36 –48.6 –65 (NA ¼ 8,300/mm2)

20.6

68 –272– 425

49

Astrocytes in: adult neocortex visual cortex

10 –60c

15 –100

12 –32 16 –29.8 –67

83 –31 61–33.5 –15

parieto-occipital cortex of adult rat

10 –17.6 –28

97–56.6 –35 [D(sphere) ¼ 48 mm]d

Mu¨ller cells (means of rat retina) Bergmann glia

a

3.4

248.5 [D(sphere) 70– 96–120 ¼ 78 mm]d

3,400 (VV ¼ 100%)

Surface-tovolume ratio

References

,0.5

Estimates based on cubes with diameter of 15 mm 3400– 5000– . 6250 12.1 Chao et al., 1997a; (VV ¼ 5– 20%) 5.6 –19.8 Reichenbach and Wolburg, 2003 3,600 13.5 Reichenbach (VV ¼ 15 –18%) et al., 1995; Reichenbach and Wolburg, 2003 Blinkov and Glezer, 1969b Distler et al., 1991 Gabbott and Stewart, 1987 3,500–5,700– 9,700 12–17 –21 Chao et al., 2002e (VV ¼ 10%)

Minimal (left) and maximal values (right) flank mean values (bold), where data were available. In tissue sheets, like the ependymal layer, the retina and the Purkinje-cell layer, cell densities have been determined as cell numbers per area. By multiplication with the thickness of the sheet, volume densities have been calculated. c Similar variations (10,000 and 60,000 astrocytes/mm3 of tissue) can be obtained by measurements and calculation from data of Glees, Brownson, Schroeder, and others (cited in Blinkov and Glezer, 1968), when the percentage of astrocytes among all glial cell types (40–45%) is combined with the overall glial cell density [16–40.5 £ 103 and 30–59 £ 103 astrocytes/mm3 of tissue occur in the neocortex and facial nucleus, respectively; adult rat neocortex]. d Based on diameters of HRP-filled astrocytes including all cell processes and surface extensions. Data represented by the diameters of spheres with equal volume (D(sphere); Rollmann, 1992). e Layers 5–6 of the parieto-occipital cortex of adult albino rats (90–120 days after birth; Sprague–Dawley strain; data of Rollmann, 1992). b

Cytoarchitectonics of Non-neuronal Cells in the Central Nervous System

Ependymal cells 300 (NA ¼ 4,500/mm2)

Tissue volume monitored Interdigitating by one cell [103 mm3] cell territory [£103 mm3]

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tissue. (ii) Capillary endothelia monitor tube or torus-like cell territories. Their lumen provides the passing blood with a high surface-to-volume ratio of 0.8 m2/cm3. The outer half of the cell surface adheres to the perivascular basal lamina and mediates interactions with ependymo-astroglial cells. (iii) Between endothelia, interactions are minimized by cell flattening (thickness: 0.26 mm; Table 1), small overlap at intercellular contacts and “seamless” endothelia, which make up one-third of the length of intracerebral capillaries. 4. Diversity of neuroglia The concept of neuroglia subsumes non-neuronal cell types, which appear to be permanent and integrated residents of nervous tissue. In the CNS, these criteria combine macroglia (ependymo-astroglia and oligodendroglia) and microglia, though they probably originate from different (neural and non-neural) stem cells. The criterion of ‘tissue integration’ excludes cells that are separated from neural cells by basal laminae (cells in vascular walls, meningeal cells and macrophages in perivascular or pial positions), while ‘permanent presence’ (residential status) excludes hematogenic cells, which may transiently invade the CNS under pathological conditions (lymphocytes, granulocytes and monocytic macrophages; see Streit, 1995). By playing different roles, neuroglial cell types cooperate in the maintenance and restitution of tissue integrity, even when neurons are absent as during scar formation and reorganization of peritumoral tissue (see the chapter by Kalman). In contrast to neurons, which are confined to gray matter, neuroglia is ubiquitously distributed within the CNS and makes up about 90% of all cells. Despite numerical superiority, neuroglial cells occupy only 15– 20% of gray matter volume and about 60% of the white matter, though including myelin membranes the proportion of glial plasma membranes should be considerably higher. The diversity of neuroglial types serves different CNS functions. Apart from differences in molecular equipment between microglia, oligodendroglia and ependymoastroglial cell types (Streit, 1995; Reichenbach and Wolburg, 2003), glial cell types differ in cytoarchitectonics (e.g., contact relations to neurons and tissue-related polarity). Only few glial cell types maintain the triple polarity of neuroepithelium (epithelial cells of the choroid plexus, radial glia, tanycytes; compare Fig. 1E and E0 ). Some maintain contact with one of the tissue surfaces (apical: ependymal cells; basal: tanycytes and astrocytes), while others keep all of their membranes within the CNS and are unrelated to tissue polarity (oligodendroglia, resting microglia). Among neuroglia, macroglial cells make up the largest fraction (about 40– 45% for each, ependymo-astroglial cells and oligodendrocytes; see Blinkov and Glezer, 1968). Like neurons, all types of neuroglia develop ramified shapes, which enormously expand the lateral cell surfaces in CNS tissue. 5. Microglia Cytoarchitectonically, microglia appear intermediate between neural cell types and hematogenic cells, which transiently invade the CNS via blood vessels under pathological conditions. On one hand, microglia resemble neural cells (macroglia and neurons), in that these cells ubiquitously populate the whole CNS, attain more or less stellate cell shapes by

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forming branched processes, and adhere to neural cells without intervening basal laminae. On the other hand, microglia cells appear as derivatives of macrophage precursors, which invade CNS tissue via intracerebral blood vessels during development (for ongoing discussions of cellular subtypes and descent see Fedoroff, 1995; Streit, 1995; Gehrmann and Kreutzberg, 1995). In addition, microglial cells remain highly mobile throughout life and do not seem to establish durable contacts with any other cell, including other microglial cells. Focally, they can even displace astrocytic processes from perivascular basal lamina. Unlike vascular endothelia forming multicellular sprouts (Section 3.2), microglial cells seem to invade neural tissue as individuals and form a system of cells that navigates separately in the adult CNS. By forming a system of independently acting cells, microglia contribute to tissue architectonics in a different manner than the multicellular networks consisting of neurons or ependymo-astroglia connected by intercellular junctions (Section 7.5). 5.1. Distribution in CNS 5.1.1. Numerical density Under normal conditions, the numerical density of microglial cells is on average much smaller than that of macroglial cell types (5 – 20% of all glial cells; see Gehrmann and Kreutzberg, 1995). In addition, variations are relatively small between and within brain regions (e.g., neocortical layers: 4200 –6600 cells/mm3 of tissue volume; 3500– 6600 cells/mm3 in various parts of the hippocampal formation; Dewulf, 1937). Using silver impregnation to visualize microglia, Dewulf found variations between 3500 and 8600 cells/mm3 in gray and white matters between brain stem and frontal cortex of man and monkey. Similar cell densities were found in the CNS of other mammalian species (mouse, rat, cat, marmoset monkey), when detection of microglia was based on binding of Griffonia-simplicifolia agglutinin; e.g., 3300– 9400 microglial cells per mm3 of cortical tissue (mouse: Lawson et al., 1990; Saftig et al., 1997; other species: Boettcher and Wolff, unpublished study). Taken together, these data confirm that microglial cells are more or less evenly distributed with low density in the CNS of healthy adult mammals. Cell density suggests that each microglial cell monitors a mean tissue volume of 100,000 to 300,000 mm3. Spheres with equal volumes would have diameters of 60– 80 mm. These estimations are comparable with the characteristic structure and size of microglial cells (Fig. 1E0 ). ‘Resting’ microglial cells have cell bodies with small perikaryon that extend highly branched processes forming more or less stellate or bipolar cell shapes. Microglial processes penetrate elliptoid tissue territories with small and large diameters of 20 –40 mm and 50– 100 mm, respectively. 5.1.2. Spacing In the resting state, microglial cells show a characteristic spacing that largely avoids overlapping of processes. In the vast majority of cases observed in chemically fixed material, contact formation between processes of neighboring cells is either avoided or restricted to what appears as random events (see Streit, 1995). However, positions of

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microglia might not be static. It is widely accepted that conditions existing in brains of healthy adult mammals keep the majority of microglial cells in a ‘resting state’, which maintains the distribution pattern described above. Since microglia can hardly be observed in undisturbed tissue, it remained unclear whether microglia in situ is immobile in the resting state. The enormous mobility of these cells seen under in vitro conditions, e.g., in cell cultures or in sliced preparations, raises the question of whether or not microglial cell positioning is dynamically adjusted by random movements and some sort of contact inhibition. 5.2. Activation of diverse functions One of the intriguing properties of microglia is functional plasticity (Streit et al., 1988), which includes a large number of options for reactions to activating stimuli. 5.2.1. Migration With and without local injury, the migratory potential can be activated by exogeneous signals, which chemotaxically guide microglia toward targets, such as neuronal perikarya in unusual metabolic states. In motor neurons, such states can either be induced as the retrograde reaction to peripheral axotomy (see Gehrmann and Kreutzberg, 1995) or result from prolonged motor activity (swim training; Kulenkampff, 1952). Latencies varied between 24 h in the first case and 0.5 h in the latter, though variations were probably due to differences in neuronal reaction time, rather than latencies of microglial response (but reaction latencies differ between astroglia and microglia around axotomized motor neurons; see Rohlmann et al., 1994). In both cases, microglial migration transiently increases the number of microglial cell bodies located near those of motor neurons (‘glial satellites’ of neurons). Release stop of unknown attractants might explain the removal of microglia from positions near neuronal perikarya. Return to previous distribution patterns, however, indicates that the normal distribution pattern also depends on active (chemotaxic?) regulation. Migratory activity and hypertrophied microglia have also been seen in the supraoptic nucleus in response to dehydration (see the chapter by Salm et al.) and a number of other conditions, not discussed here. Thus, migration induced under various conditions is an option for microglia that does not exist in other mature glial cells. 5.2.2. Membrane incorporation or release of synthesis products Microglia can modify its receptive properties by changing the equipment with transmembrane proteins (receptors, ion channels etc.). These cells can also express immunophenotype by expressing and exposing complement CD3, MHC complexes of types I and II. This type of activation may or may not be accompanied by synthesis and release of cytokines and other agents, such as oxygen peroxide (for details, see Streit, 1995). In this way, microglia can dramatically modify its potential of interactions with adjacent cells and cell parts in its vicinity. It is tempting to speculate, that combination with migratory activation increases the reach of membrane-bound and secreted effectors within brain tissue.

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5.2.3. Phagocytosis When reaching sites of tissue injury or cellular degeneration, microglial cells turn into phagocytes. In this state, microglia appears hypertrophic and is morphologically indistinguishable from hematogenic macrophages (Gehrmann and Kreutzberg, 1995). In contrast to the latter, however, microglia accumulates residual bodies and seems to migrate towards pre- or postcapillary blood vessels, which contain perivascular macrophages. 5.2.4. Mobile waste management After phagocytosis, microglia filled with residual bodies of phagosomes migrate towards specific parts of mesenchymal borders of CNS tissue. For unknown reasons, microglia prefer to approach pial margins and intracerebral veins or arteries, but not capillaries and a-v channels (Section 3.2). After detecting an appropriate target, microglia may penetrate perivascular or pial basal laminae and, for refuse disposal, either leave the CNS via blood flow or hand their lysosomal content over to hematogenic macrophages. Interestingly, this mobile waste management is apparently restricted to phagosomes, while in metabolic storage diseases (e.g., after knockout of lysosomal acid phosphatase; Saftig et al., 1997) storing microglial cells maintain their positions like astrocytes. 5.2.5. Proliferation Microglial cell numbers can be regulated not only by immigration from and emigration into intracerebral blood vessels. Labeling with thymidine and bromouridine suggests that microglial cells are capable of proliferation within the CNS tissue (see Gehrmann and Kreutzberg, 1995). Among parenchymal microglial cells, the spontaneous turnover rate seems to be much lower than is the case with perivascular and leptomeningeal phagocytes (1:30:60%; Hickey et al., 1992). However, this proliferative potential can be activated, though it is not clear whether activation is restricted to persisting precursor cells or may recruit all microglia in a resting state. Thus, activated microglia are extremely versatile, because activation is variable in that one to several of these potentials may be switched on at the same time. Even the distinction between activations with and without phagocytosis (see Gehrmann and Kreutzberg, 1995) is a considerable simplification, which does not cover the morphological variability of microglia in normal brain regions (Lawson et al., 1990). 5.3. Conclusion Among neuroglial cell types in the CNS, microglia is unique in that it forms a system of individualized cells, which act in parallel and are cytologically janus-faced. Similarity with neural cells is based on the resident status of microglia in all CNS regions, ramified cell shapes, occupation of non-overlapping tissue territories in the resting state and integration into neuropil by direct adherence to neural plasma membranes. At the same time, microglia preserves properties of hematogenic precursors: in the activated state it changes morphology, expresses migratory, proliferative and phagocytotic capabilities,

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shows synthesis and exposure on membranes or release of synthesis products, which are important in the regulation of immunophenotype (local), cell death and tissue repair after injury. Thus, diversity among microglial cells reveals different states of transient activation, rather than stable subtypes of cells. 6. Oligodendroglia As macroglial cell types, both oligodendroglia and ependymo-astroglia are derived from neuroepithelial stem cells (ectodermal origin). Details of cytogenesis, molecular specification and the myelination process are beyond the scope of this chapter (but see Baumann and Pam-Dinh, 2001). Taken together, these criteria suggest that the oligodendroglia is a uniform cell type in the vertebrate CNS, though individual oligodendrocytes show a characteristic polymorphism, which was confirmed in many CNS regions and vertebrate species, and led to the distinction of subtypes (for details, see Szuchet, 1995; see also the chapter by Szuchet and Seeger). Here, we discuss cytoarchitectonic constraints as the possible source of cellular polymorphism that even includes structural differences between neighboring cells. 6.1. The polymorphic cell type 6.1.1. Common tissue relations In terms of tissue relations oligodendroglia may be defined as follows: (i) Loss of apicobasal polarity of neuroepithelium separates these cells (like neurons) by interposition of ependymo-astroglia from both cerebro-spinal fluid (CSF) and mesenchymal tissue components (leptomeninges and blood vessels). This may be an important restriction for oligodendrocytes, because these cells and their precursors, for example, react to proinflammatory mediators circulating in the CSF (Kong et al., 2002). Tissue-related apolarity is not a criterion for all myelinating cells. Schwann cells would rather myelinate axons in spite of regularly making direct contact with the mesenchymal environment. (ii) In the adult CNS, the vast majority of oligodendroglial cells are involved in the formation and maintenance of one-to-many myelin sheaths, which surround specific subpopulations of axons. (iii) Most oligodendroglial cells form cell processes that enable them to reach distant axons and myelinate more than one axonal internodium. For each cell, these processes define a tissue territory that overlaps more or less completely with that of neighboring oligodendrocytes. Overlap is obvious, especially where cell bodies are tightly packed or form long rows in white matter (Hortega’s type I– IV; interfascicular oligodendrocytes, see, e.g., Ogawa et al., 1985). 6.1.2. Polymorphism Hortega (1928) distinguished four types of oligodendrocytes, which do not depend on topographical separation. Although multipolar type-I cells projecting processes in different directions are more frequently found in the gray matter, all four types are mixed in the white matter (e.g., in the feline medulla; Hortega, 1928) and in the pons, where gray and white matter are interwoven (Ogawa et al., 1985). Type-II differ from type-III cells by unipolar vs. bipolar orientation of trunk processes which originate from

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the perikaryon. These trunks branch at variable distances and form thin processes, which preferentially run parallel to each other. Type-IV cells differ from all the others by focusing on one axon and forming only one myelin sheath, while in all the other cell types each oligodendrocyte forms and maintains multiple (20 – 30) myelin sheaths on different axons. 6.2. Composite cell processes and cellular subtypes 6.2.1. Components Variability among cell processes is based on differences in the combination of three components: leaf-like extensions, long thin processes and short stout processes. Leaf-like extensions wrap around internodal parts of axons and form myelin sheaths (see Fig. 5C). Formation of such surface extensions is based on a morphogenetic program that is common to oligodendrocytes and Schwann cells. Growth of such extensions and myelin formation occurs near potentially internodal surfaces of axons. Since morphological differentiation may follow biochemical specification (Remahl and Hildebrand, 1990), contact formation may be initiated by a sensing of specialized axon membranes. Whatever surface markers attract lamellar extensions, these do not seem to exist on unmyelinated axon types and on nodal segments of the axon surface, to which oligodendrocytic processes do not attach more than randomly (for attraction of ependymo-astroglia to nodal regions, see Chao et al., 1994; Rosenblut, 1995). During myelination, plasma membranes develop molecular specification, lose the cytoplasmic matrix between most, but not all, parts of leaf-like extensions (see Fig. 5C) and undergo compaction. In the adult CNS, the number of leaf-like extensions equals that of myelin sheaths formed and maintained by one cell. It remains to be investigated, whether the complex (‘mossy’) periphery of processes in young oligodendrocytes includes lamellar extensions, which have not yet found appropriate axon segments (see Szuchet, 1995). Long and thin processes (diameter: 0.1– 0.4 mm) largely define the tissue territory, which is penetrated by one oligodendrocyte. In gray and white matter, the majority of cells give rise to several (up to 30) of these processes. They vary in length (up to 200 mm) and are often unbranched, while others show a few dichotomous branches. Thin processes may head into all possible directions. Many run closely parallel, others take opposite or divergent directions (a – d in Fig. 5B). Although some contain one or a few microtubules and filaments, the ultrastructure of these processes mostly resembles that of microvilli or filopodia (Fig. 5A0 ). It is not clear how these thin processes relate to the verges and Schmidt – Lanterman’s incisures of myelin sheaths. Both are continuous at the transition zone between process and myelin sheath, and are filled with organelle-free and filopodialike cytomatrix (Fig. 5A0 and C). As the third component, 1 –5 stout processes connect several thin processes with the perikaryon in a subpopulation of cells. This is the only type of process that contains cell organelles (Fig. 5A and B: part d). 6.2.2. Cell shaping The four types of oligodendrocytes described by Hortega apparently result from variations in cell shaping. Differences at the cellular level can be described by differential

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composition and arrangement of cell processes. Cell shaping of oligodendrocytes depends on two types of fixation in tissue and one type of connecting structure. (i) By formation and/or maintenance of myelin sheaths, sites of contact between any part of the cell surface and internodal parts of axon surfaces are fixed in tissue. (ii) As oligodendrocytes do not migrate, the position of the cell body largely corresponds to the site of last mitosis (last location of the precursor cell). The perikaryon then provides another type of fixation within the tissue. (iii) In most cases, stout and thin processes connect the cell body with multiple myelin sheaths made by the same cell (1 in Fig. 5A; see also Fig. 5A0 ). Exceptions are type-IV oligodendrocytes, which maintain contact between cell body and a single myelin sheath (similar to Schwann cells; a in Fig. 5B), and perisomatic origin of myelin sheaths (2 in Fig. 5A and Fig. 5A00 ). 6.2.3. Immature oligodendrocytes In the early stages of development, the distance between cell body and myelination sites is probably small and may be bridged by processes. Indeed, young oligodendrocytes have a ‘mossy’ form, in which stout trunks connect the cell body with a complex periphery that encloses branched thin processes and numerous lamellar extensions. Disappearance of these mossy cells during further development (for details, see Szuchet, 1995) might be due to transformation into one of the adult cell shapes. In this case, stout processes would largely preserve their structure (see Fig. 5B: d), while expansion of surrounding tissue (arrows in Fig. 5B) by elongating the thin processes might determine their length and orientation. Continuation of myelination into early adulthood, would make adaptive prolongation of early-formed processes an important shaping factor. Many authors agree that oligodendroglial processes are maximally about 200 mm long. This interpretation asks

Fig. 5. Cytoarchitectonic characteristics of oligodendrocytes. (A) Oligodendrocyte (o) in the neocortex of adult rat forms both myelin sheaths (‘1’ and ‘2’) and direct contact with the cell body of a neuron (n; ‘neuronal satellite’; for higher magnification see A000 ). Stout processes (1– 2 mm thick), arising from the perikaryon, contain organelles. Myelin sheaths are connected to the cell by thin processes (0.1–0.4 mm thick; arrowhead in A0 ), which are in most parts devoid of organelles (except microtubuli and filaments; not shown). Thin processes originate from both perikaryon (2 in A and open arrowhead in A00 ) and stout processes (1 in A). Bars: 1 mm (A), 0.5 mm (A0 ), 0.7 mm (A00 , A000 ). (B) Cartoons of variations in cell shape. a: Similar to Hortega’s type-IV, the cell body is adapted to the only myelin sheath formed (note similarity with Schwann cell, except for the lack of a basal lamina). b: The cell body moved away from the one myelin sheath (arrow; compare with a), forms additional processes, which connect with additional myelin sheaths, similar to Hortega’s type-II and type-III. c: The cell body gives rise to several thin processes, which rarely branch and connect to different myelin sheaths, similar to type-III. The arrow indicates spatial separation of cell body from myelin sheath. d: More complex connections between several thin processes and perikaryon are mediated by stout and branched trunks, though thin processes directly originate from the cell body. Such a cell would be intermediate between type-I and type-II. (m. and unm. represent bundles of myelinated and unmyelinated axons, respectively.) (C) Unrolled myelin sheath of an ‘a‘-cell visualizes thin-process-like verges (outer and inner pockets, incisures). (D) Comparison between numerical densities of neuroglial cell types in neocortical layers 1–6: data of Schlote (1959), on human macroglia (þ, W, p ); Dewulf (1937), on monkey microglia (points); Ogawa et al. (1985), on oligodendroglia in cat pons (mean: square). Note that oligodendroglia increases ‘neuroglia-density’ by a factor of four in layers and regions that are rich in myelinated axons, while that of astrocytes decreases (by a factor of two) and microglial cell density does not change significantly.

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for studies, which clarify whether process length indicates limits of passive ‘growth’ or the maximum dissociation by intercalary growth, separating primarily adjacent tissue points. Alternatively, oligodendrocytes might send out filopodia which, used as sensors of potential internodal segments of newly formed axons or vacated by degeneration of myelin, could transform into processes (like in endothelial cells; see Fig. 4) and thus initiate myelination in later stages of development. 6.3. Cell distribution in CNS 6.3.1. Cell density Since oligodendrocytes outnumber ependymo-astroglial cells in the white matter by a factor of two to four (see Blinkov and Glezer, 1968; Ogawa et al., 1985), they are on average the most frequent type of glial cell in the CNS of adult mammals. Also, in gray matter, the density of oligodendrocytes correlates with that of myelinated axons (compare, e.g., numerical densities of about 10,000 cells/mm3 of layer 1 with five times higher densities in the myelin-rich layers 4 – 6 of human neocortex; Fig. 5D). Similarly, averages of 55,000 and 56,000 cells/mm3 have been found in the pons (mixture of gray and white matter) and cerebellar peduncle (white matter), respectively (Ogawa et al., 1985; square in Fig. 5D). On average, one oligodendrocyte monitors a tissue territory that can be described by a sphere with equal volume and a diameter of about 30 mm. As defined by the length of thin cell processes, much larger cell territories (diameters . 100 mm) have been documented in various CNS regions of different species (for details, see Szuchet, 1995). This clearly shows that oligodendroglial cell territories deeply overlap and that this is not restricted to dove-tailing between parallel processes but includes longitudinal interdigitation. 6.3.2. Positioning in tissue Apart from correlated increases in average densities of oligodendrocytes and myelinated axons (Fig. 5D), the distribution of cells varies locally (for quantitative assessment in the pons, see Ogawa et al., 1985). Rather than being diffusely distributed in the white matter, oligodendrocytes tend to accumulate near the surfaces of myelinated axon fascicles. The concentration of cell bodies is up to ten times higher in inter- or perifascicular spaces than intrafascicularly. It is unclear whether these differences are due to the stopping of immigrating precursor cells at borders of myelinated tissue compartments (e.g., Chari and Blakemore, 2002) or progressive mitotic activity of local precursors, or both. The second CNS structure, around which oligodendrocytes may accumulate, are neuronal perikarya. In the cerebral cortex, oligodendrocytes make up about 50% of all perineuronal satellites (astrocytes: , 40%; microglia: 6– 10%; Brownson, 1956). In the feline pons, more than half of the oligodendrocytes are in satellite position to neuronal cell bodies, though perineuronal spaces collectively occupy only about 30% of the tissue volume. The function of this accumulation in satellite positions remains obscure. As shown in Figure 5A, oligodendrocytes directly contacting neuronal perikarya may be involved in myelination, like other cells located in the neuropil or near axon fascicles. In contrast to

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microglia and astrocytes in this position, it is unknown, whether their number is fixed or changes under special conditions, which might induce movements of oligodendrocytes toward or away from neuronal cell bodies. 6.4. Conclusions Cytogenesis, molecular differentiation and the myelination process suggest that oligodendroglia is a uniform cell type of the CNS. The polymorphism of oligodendrocytes does not depend on topographical variations but even occurs between neighbors. Here, the idea is advocated that differences in cell shaping of oligodendrocytes are due to developmental changes in spatial relations between two types of fixed points, the positions of the cell body and myelinated axon segments, which remain connected to each other. 7. Ependymo-astroglia 7.1. Common features of the cell system In the vertebrate CNS, ependymo-astroglia was the first cell system identified as ‘neuroglia’. The term suggested that a special class of cells fill tissue spaces like glue which had been poured between the neurons. By staining individual neuroglial cells, the Golgi method certified the cellular nature of neuroglia (see Cajal, 1911), though the complicated course of glial plasma membranes remained obscure until electron microscopy became available (see Peters et al., 1991). Three-dimensional reconstructions of submicroscopic cell parts reveal more and more of the complex structure and contact relationships of lamellipodia and filopodia. Characteristic surface extensions decorate not only protoplasmic astrocytes (Wolff, 1965; Spacek and Harris, 1998; Chao et al., 2002), but also astroglia-like cells (Bergmann glia in the cerebellar cortex; Grosche et al., 1999) and parts of tanycytes (see Reichenbach and Robinson, 1995) and Mu¨ller cells in the retina (Reichenbach, 1989; Chao et al., 1997a). Other structural criteria of ependymo-astroglial cells are interglial contacts (see Rohlmann, 1994) and the characteristic adaptivity of their cell surfaces to adjacent tissue components, especially neurons and synapses (see Chao et al., 2002). Finally, increasing numbers of molecular markers confirmed the concept that cell types sharing these properties at least under certain conditions (e.g., in certain developmental stages or reactive states; for details, see Reichenbach and Wolburg, 2003) may be subsumed as ‘ependymo-astroglia’. Both, structural criteria and molecular tools also define differences between ependymo-astroglia, on the one hand, and oligodendroglia or microglial cells, on the other. Thus, positive and negative criteria can be used to define ependymo-astroglial cell types as a common cell system. 7.1.1. Multiple functional potentials Ependymo-astroglial cell types may be identified by common marker molecules. Expression levels, however, may vary in different cell types, locations and states of reactivity (see table in Reichenbach and Wolburg, 2003). The functional diversity of markers is impressive: cytoskeletal components and regulators of their relation to

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membranes, cytosolic components involved in different metabolic pathways or calcium buffering, and a variety of glia-specific transmembrane proteins involved in gap junction formation, transport, or detection of ions or transmitters. Taken together, diversity and optional expression of markers indicate that ependymo-astroglial cell subtypes serve multiple functions, which partly depend on certain conditions in brain tissue. For example, glial fibrillary acidic protein (GFAP) composes intermediate filaments, which increase in number during reactive cell states and scar formation (see the chapter by Kalman) and may cyclically increase or decrease during development (Missler et al., 1994). Recent findings suggest that intermediate filaments may influence the capacity for changes in cell shape and intracellular transport. Ezrin is an actin-binding protein that modulates association between the cytoplasmic matrix and the plasma membrane (Derouiche and Frotscher, 2001). Such dynamics is important during formation and withdrawal of lamellipodia and filopodia, which differentially expand the cell surfaces of ependymocytes, tanycytes, protoplasmic and fibrillar astrocytes and various types of astroglia-like cells (see Table 2). Connexin-43 is the major component of gap junctions (see chapter by Scemes and Spray), which knit together ependymo-astroglial cell types in a common network. Transfer of signal molecules through gap junctions enables the characteristic spread of calcium waves within this network. These waves may be related to S100b protein, which is a Ca/Znbinding molecule typically expressed in these cell types (see Rickmann and Wolff, 1995). A set of transmembrane proteins enable interactions with neuronal function. These include receptors for synaptic and non-synaptic transmitters (see the chapter by Hansson and Ro¨nnba¨ck), specific potassium channels and carriers (see the chapter by Walz), gliaspecific transporters of amino acid or monoamine transmitters enable these cell types to be important players in the homeostasis of intercellular fluid, especially in perineuronal and perisynaptic spaces (see Chao et al., 2002). In addition, metabolic cooperation with neurons are indicated by expression of specific glucose transporters, glycogen phosphorylase, glutamine synthetase and pyruvate carboxylase (see the chapters by Schousboe and Waagepetersen and by Ha˚berg and Sonnewald). Depending on the developmental stage studied and the location or reactive state of the cell, expression of such markers may vary quantitatively, e.g., GFAP, glutamine synthetase (for details see the table in Reichenbach and Wolburg, 2003). 7.1.2. Subcellular differentiation Some marker molecules are restricted in subcellular distribution (‘subcellular architectonics’), accumulate in characteristic cell parts (e.g., in perisynaptic lamellipodia; Table 4) or focus on subpopulations of filopodia involved in gap junction formation (connexin-43; Wolff et al., 1998). Marker molecules, like those forming orthogonal arrays of particles, may even be focused on ependymo-astroglial membranes contacting perivascular and marginal basal laminae, while these arrays are scarce or missing in other parts of the cell surface (Wolburg, 1995). Similarly, ependymo-astroglia of higher vertebrates perivascularly releases factors that induce the formation of the tight junctions in vascular endothelium, which form an essential part of the blood – brain barrier (Wolburg and Lippodt, 2002). Thus, although location and polarity of ependymoastroglial cell types influence their differentiation to a certain extent, they do not

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eliminate common characteristics of subcellular structure, molecular equipment and cellular functions. 7.2. Functional differentiation 7.2.1. Interaction with neuronal functions This cell system is organized in a way that enables coordination of various functions in CNS tissue. Ependymo-astroglia interacts with neuronal functions. These cells focally adhere to the ‘receptive surfaces’ of neurons (e.g., dendrites and synapses; for a recent review see Chao et al., 2002) and are equipped with molecular tools to clear from perineuronal spaces neurotransmitters, potassium ions, certain metabolites etc. (see the chapters by Schousboe and Waagepetersen and by Walz). Where Mu¨ller cells are complemented with astrocytes, as in vascularized retinae, both cell types apparently cooperate in monitoring and responding to neuronal activity (see Chao et al., 1997b and the chapter by Stone and Valter). Enzymatic equipment of ependymo-astroglia additionally suggests metabolic interactions with neurons (see the chapters by Schousboe and Waagepetersen and by Ha˚berg and Sonnewald). 7.2.2. Monitoring of inner and/or outer CNS surfaces Cooperation between cell types apparently serves monitoring of the inner and/or the outer surfaces of nervous tissue (Fig. 1). In adult vertebrates, the inner CNS surface enclosing the ventricular system is covered by a monolayer of ependymal cells together with ventricular processes of radial glia or its mature derivatives, e.g., tanycytes in spinal cord and brain, Mu¨ller cells of retina (for further cell types, see Reichenbach and Robinson, 1995; Reichenbach and Wolburg, 2003). As mentioned above (Section 3.1) the outer surface or mesenchymal border of the CNS is much more complex than the basal surface of epithelia, in that the marginal surface covered by the pia mater is supplemented by the huge surface of intracerebral blood vessels (Fig. 1). Both marginal and vascular surfaces are selectively covered by endfeet and en-passage contacts of astroglial cell processes or processes of astroglia-like cells (Bergmann glia, tanycytes) including Mu¨ller cells of the retina. Within the ependymo-astroglial cell system, functional cooperation is activated by electrical and metabolic coupling via gap junctions (see the chapter by Scemes and Spray). However, appropriate techniques are still missing to visualize the complexity and dynamics of intercellular contact relations in CNS tissue. 7.3. Histoarchitectonic differentiation of cell types 7.3.1. Contact with the ventricular tissue surface Ependymo-astroglial cell types differ in cellular origin and tissue-related polarity. Cell types lining the ventricular surface, i.e., radial glia, tanycytes and ependyma, share not only their contact with the cerebrospinal fluid, but also their direct descent from stem cells located in the ventricular zone (Fig. 2A). This origin is independent of whether contacts with mesenchymal tissue surfaces are missing (as in most ependymal cells; e in Fig. 1E0 ), restricted to the pial brain surface (as in radial glia and partly in marginal tanycytes; mt in

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Fig. 1E0 ; or Mu¨ller cells in avascular retinae), confined to perivascular basal laminae (vascular tanycytes; vt in Fig. 1E0 ), or include both marginal and vascular contacts (as Mu¨ller cells in vascularized retinae). 7.3.2. Contact with mesenchymal tissue surfaces Astrocytes and astroglia-like cell types (e.g., Bergmann glia of the cerebellar cortex) originate from glioblasts in extraventricular positions (‘non-radial’ glioblasts in Fig. 2A). During migration from the ventricular zone, these glioblasts are transiently apolar (i.e., without contact to either ventricular and marginal tissue surfaces). Before the next mitosis, however, these glioblasts establish contact with the mesenchymal tissue border (intracerebral vessels or pia mater; see Fig. 2A), while re-establishment of ventricular contact remains impossible. Depending on the position of the cell body or the termination of endfeet, mature astrocytes have been subdivided into marginal, radial or perivascular varieties (ma, ra, and a in Fig. 1E0 ). Ependymo-astroglia then comprises four cell classes: One maintains the apico-basal polarity of the neuroepithelium (radial glia, marginal tanycytes; choroid plexus epithelial cells are a special case; see pe in Fig. 1E0 ). The others either give up the basal (marginal) pole (ependyma) or lose the apical (ventricular) pole and selectively re-establish contact with mesenchymal (marginal or vascular) tissue surfaces (astrocyte, marginal astrocyte, and radial astrocyte). 7.3.3. Interdigitating (lateral) cell surfaces Only ependymal and the epithelial cells of choroid plexus acquire the shape of cubed epithelia, which show little interdigitation of lateral cell surfaces (compare Fig. 1B with E0 ). All other ependymo-astroglial cell types enormously expand their ”lateral cell surfaces” (Table 2) by formation of processes and surface extensions (lamellipodia and filopodia), which interdigitate with other neural cells within the intra-epithelial space (neuropil; see Section 2.3). Processes and surface extensions apparently play different roles in cellular interactions because they differ in dynamics from permanent (primary processes and trunks of secondary ones; Section 7.4) to highly dynamic (surface extensions; Safavi-Abbasi et al., 2001; Section 7.4). 7.3.4. Morphogenetic programming of cell types among ependymo-astroglia Ependymo-astroglial cell types differentiate due to local variations in tissue-related influences. They have different precursors (ventricular stem cells vs. subventricular glioblasts; see Fig. 2A), undergo different morphogenesis, and maintain different tissuerelated polarity, which provides cell types with different probabilities of cellular interactions throughout life. For example, protoplasmic astrocytes and Bergmann glia interact with thousands of synapses (e.g. Chao et al., 2002), while fibrillar astrocytes either restrict interactions with neurons to signals arising from nodal segments of axons in the white matter (e.g., Chao et al., 1994) or exclude neurons as local interaction partners, as many ependymal cells, choroid plexus epithelial cells and astrocytes in scars. Therefore, the genetic program for common properties is established as a potential during early stages of cytogenesis, while realization of subroutines (e.g., expression of GFAP, formation of

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intermediate filaments, and lamellipodia) requires activation by exogeneous signals. By determining position and polarity within tissue architecture, morphogenetic programming differentiates the availability of such signals to members of different cell types. Thus, differences between ependymo-astroglial cell types are determined cytoarchitectonically. 7.4. Common structural components of ependymo-astroglial cell types 7.4.1. Primary processes Radial glia or tanycytes and most astrocytes initially form one or a few processes, by which they establish primary contacts with mesenchymal surfaces (see Fig. 2A). At least one of these attachments to marginal or pial and vascular basal laminae, which is mediated by terminal widenings (so-called endfeet), persists into adulthood and responds with elongation and distortion to dislocations of the cell body from the site of origin (‘1’ in Fig. 2B). Primary processes may branch anywhere between the cell body (Bergmann glia) and two endfeet (compare ‘2’ and ‘3’ in Fig. 2B). Degrees of branching are usually low in mammalian CNS (but see lower vertebrates; Reichenbach and Wolburg, 2003). There are a few exceptions from these rules: ependyma and plexus epithelia do not extend processes at all, while marginal and perivascular astrocytes do not dislocate cell bodies from the primary position. Dislocation may vary between none, in latter cases, and much more than the length of secondary processes (compare BG with fA and pA in Fig. 6A and cells in B –D). The reason is unknown as to why some primary processes in gray matter are devoid of surface extensions (e.g., the smooth surface of radial glia and astrocytes in Fig. 6A), while others are studded with them (e.g. many tanycytes, retinal Mu¨ller cells, astrocytes in Fig. 6B). 7.4.2. Secondary processes Secondarily formed processes project more or less radially into the surrounding neuropil or white matter (see all astrocytes in Fig. 6A –D). Secondary processes branch to varying degrees (within neuropil more than in white matter) and terminate freely in tissue. It is still unclear why certain astrocytes develop subpopulations of secondary processes belonging to different length classes (e.g., Fig. 6D). Long secondary processes tend to associate or intermingle with other glial processes in marginal plexuses or perivascular plexuses (compare Fig. 6A with D). Short secondary processes tend to be more radially oriented, carry most of the branches, branch with blunt angles and take fine winding courses. 7.4.3. Surface extensions Lamellipodia and filopodia or finger-like protrusions may decorate all parts of the cell surface from the cell body to the tips of processes of all ependymo-astroglial cell types. Surface extensions have submicroscopic dimensions (thickness: 0.02 –0.3 mm, length: 1– 10 mm; radial reach: 1.9mm; Fig. 7F) but by overlap and aggregation they cause the light microscopical ‘roughness’ of cell surfaces at protoplasmic astrocytes, Bergmann glial cells and tanyctes (Fig. 6; for retinal Mu¨ller cells, see Reichenbach and Wolburg, 2003).

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Fig. 6. Subtypes of astrocytes and astroglia-like cells tend to be spatially separated, but may interdigitate with their processes. (A) In the cerebellar cortex, Bergmann glia (BG), protoplasmic astrocytes (pA; or ‘velate astrocytes’; see Palay and Chan-Palay, 1974) and fibrillar astrocytes (fA) are separately located with cell bodies and most of their processes in the molecular layer (ML), the granular layer (GL) and the white matter (WM), respectively. Bergmann glia project all processes into the molecular layer, where these cells are studded with lateral protrusions (‘domains’; see Grosche et al., 1999), and they mostly terminate with endfeet at the pial surface. In contrast, protoplasmic and fibrillar astrocytes carry at least two different types of processes. Short processes with radial orientation penetrate the tissue territory that surrounds the cell body. Depending on positioning in gray or white matter, these processes vary in the degree of branching and decoration with lamellar surface extensions. In addition, long processes pass through neighboring layers ( p ), even in gray matter they have smooth surfaces, and they intermingle with other long processes to form the marginal plexus (arrowheads). (B) Marginal astrocytes (mA) also show two types of processes: the relatively short processes are radially oriented, while the longer processes are smaller in number, carry fewer surface extensions and terminate with endfeet at the pial basal lamina (bl). (C) Depending on the distance between cell body and blood vessel surface (bv), perivascular astrocytes (pvA) in the gray matter may or may not show a process with endfoot (arrows; the top one has its cell body adjacent to bv). (D) Short and long processes as well as variations in the length, branching pattern and endfeet of long processes also occur on perivascular astrocytes of the white matter (arrows). Note that long processes without visible endfeet contribute to perivascular glia plexuses, which are restricted to

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Since lamellipodia and filopodia have complicated shapes (Fig. 7G and H), they have been given different names: e.g., lamellipodia and filopodia (Chao et al., 2002, and here); lamellae and finger-like extensions (Wolff, 1965); peripheral astroglial processes (Derouiche and Frotscher, 2001). Indeed, lamellae may be difficult to distinguish from peripheral segments of processes because they may ‘branch’ and form complex structures like the glial domains observed on Bergmann glial cells (Grosche et al., 1999). However, lamellar and finger-like extensions differ from processes in that they are devoid of organelles. By large numbers they constitute about half of the astroglial cell volume (Wolff, 1970; Fig. 2 in the chapter by Scemes and Spray), but by their enormous surfaceto-volume ratio determine 80% of the cell surface (see Table 3). 7.5. Cooperative tissue monitoring by astrocytes Astrocytes are most suitable to display class-specific relations between cell and tissue structure. Apart from covering mesenchymal tissue borders, this cell type combines the smallest cell density with largest cell surface, and subcellular cooperation in interactions with neurons (see Tables 2 and 3; for interactions with synapses see Chao et al., 2002). 7.5.1. The composite perivascular glial sheath Most authors seem to believe that all intracerebral blood vessels including capillaries are covered by glial endfeet. The idea of a homogeneous composition of perivascular glial sheaths (perivascular glia limitans) is probably based on uniform descriptions given for intracerebral ‘vessels‘ in most older papers (e.g., Cajal, 1911). However, if one compares diameters of the blood vessel segments and astrocytes (about 80 mm; see Figs. 6C and D and 7; Table 2) shown in those papers, the vascular segments drawn have diameters much larger than 10 mm and, therefore, do not represent capillaries (see Section 3.2; Fig. 3D). Visualization of cell borders between perivascular glial processes either by silver impregnation (vascular perfusion with silver nitrite; Fig. 7B and C) or gap-junction staining (connexin-43 immunohistochemistry; Fig. 7D) enables comparison between the glial sheaths of large and small vessels. Both techniques confirmed that large plates formed by astroglial endfeet are restricted to arterial and venous vessels, though these are intermingled with smaller plates. In contrast, capillaries are exclusively covered by small plates, such as those formed by by-passing processes (Wolff, 1970). Absence of glial endfeet from capillary surfaces is in agreement with the fact that most astrocytes have undergone last mitosis before capillaries are formed (Section 7.4). The presence of small plates derived from en-passage contacts with astroglial processes and lamellipodia, corresponds to the late increase in vascular surface due to widening that follows capillary

larger pre- and postcapillary vessels (open arrows; Cajal did not distinguish between capillaries and vessels with larger diameters). (E) Although so-called ‘cellules inde´pendants’ (iA) do not show processes terminating with endfeet, these cells will at least have en-passage contacts with blood vessels (see text). These drawings are meant to display cell shapes, and they do accordingly not show the high degree of interdigitation that exists between neighboring glial cell processes (see text; drawings derived from Cajal, 1911, modified).

40

J.R. Wolff and T.I. Chao

Fig. 7. Two types of processes and surface extensions characterize astroglial cell structure. (A) Astrocyte filled with horseradish peroxidase, is completely filled with histochemical reaction product. Note that in spite of the perivascular position of the whole cell and extended contact with the surface of the blood vessel (bv), one process is longer than the others and terminates in a vascular endfoot (EF). Intercellular contacts are visualized either with vascular perfusion of silver nitrate (B, C) or immunohistochemistry of connexin-43 labeling astroglial gap

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formation. Thus, perivascular glial sheaths differ in composition: astrocytic endfeet are restricted to vessels already existing during astroglial proliferation (peak between postnatal days 4 and 10; Wolff, 1987): these are arteries and veins (see Section 3.2); while capillaries are mainly covered by en-passage contacts of secondary processes (Section 7.4).

7.5.2. Enlargement of territorial borders by interdigitation of processes Morphometry of rat neocortex suggests that, based on cell density, astrocytes on average monitor a five times smaller tissue volume than that determined by measuring process lengths (57,000 vs. 249,000 mm3; Table 2). Accordingly, cellular monitoring territories of neighboring astrocytes interdigitate. This interdigitation varies proportionally to process lengths and nearness of next neighbors (Fig. 8C –F). Interstingly, statistical variations in territorial diameter found in different studies (see Table 2) resemble those variations, which can locally be observed in tissue (compare Fig. 8A – C with D – F). It is unclear, whether ‘small territories’ belong to recently formed astrocytes (cellular moult in adult CNS; Korr, 1980), i.e., they will grow further, or under which conditions territorial sizes might change in persisting cells. In both cases, interdigitation of territories enlarges borders between neighboring cells and make intercellular interactions easier.

7.5.3. Tissue monitoring by surface extensions GFAP-immunohistochemistry visualizes relatively few contacts between neighboring cell processes (Fig. 8A and B). Correspondingly, stereological analysis revealed that contacts between astroglial processes (primary and secondary ones; see above) are less frequent than randomly expectable (Wolff, 1970). Spacing between astroglial processes is due to the fact that staining of intermediate filaments marks the cytoskeletal cores of astroglial processes, while lamellar and filopodial surface extensions remain invisible (compare Figs. 7A and 8C). When the latter are included, as in Golgi-preparations (Fig. 6) or three-dimensional reconstructions (Fig. 7G and H), every process is surrounded by a cylindrical tissue space that is penetrated by surface extensions (Fig. 7E). The cylindrical form of such process-related monitoring spaces (shown in Fig. 7E) is a simplification. Statistically, however, this tissue volume (about 20 mm3 per micron process length;

junctions (D). (B) The pattern of astroglial contacts with the outer surface of a small artery is shown in a tangential section. Note larger plates formed by endfeet (EF) are combined with pleomorphic small plates corresponding to en-passage contacts with astroglial processes (arrows). (C) On capillaries, small contact plates completely cover the vascular surface, while large plates are missing. (D) Gap junctions discontinuously line astroglial contact zones. (E) Based on a three-dimensional reconstruction of a segment of astroglial process (AP) including all its surface extensions and the vascular endfoot (EF; note its smooth surface). This drawing wraps up the distribution space of surface extensions in neuropil. Note the variability of radial reach along the axis of the astroglial process territory. (F) Statistical representation of a one-micron segment of an average process territory including AP and lamellar and filiform surface extensions (AL). (G, H) Three-dimensional reconstructions of AP, including AL arising from these segments. The cut surface is outlined. Note the similarity of views in (H) and (F). Bars: 20 mm (A), 10 mm (B–D), 5 mm (E), 1 mm (G, H).

42 Table 3 Cytoarchitectonic components of an average type-1 astrocyte in the adult rat occipital cortex (based on Fischer von Weikersthal, 1992; Rollmann, 1992; Rohlmann et al., 1994) Cell parts

Volume (V)

Surface area (S)

Radial dimensions

Diameter: D(CB) ¼ 5.6 mm Mean radial reach: R(P) ¼ 36.2 mm; Collective length: SL(P) ¼ 3,000 mm Radial reach from cell surface: R(Se) ¼ 2.8 mm; Thickness: T(Se) ¼ #0.2 mm Average monitoring space: D(sphere) ¼ 48 mm; D(CB)z þ 2R(P) ¼ 78.0 mm

per cell [mm3] (volume fraction ¼ VV)

volume fraction of space monitored by one cella

per cell [mm2] (surface fraction ¼ SS)

fraction of all membranes in space monitored by one cella

Cell body (CB) Branched processes (P)

100 (VV ¼ 2%) 3,400 (VV ¼ 59%)

0.2% 6%

100 (SS ¼ 0.1%) 19,000 (SS ¼ 20%)

,0.01% 2.4– 3.2%

1 7

Surface extensions (Se): lamellipodia and filopodia

2,200 (VV ¼ 39%)

4%

77,000 (SS ¼ 80%)

9.6– 13%

35

Whole cell

5,700 (VV ¼ 100%)

10%

96, 000 (SS ¼ 100%)

12–16%

17

a The tissue volume on average monitored by one astrocyte amounts to V ¼ 57,000 mm2 (see Table 2). In practice astroglial volume and surface fractions are determined by stereological methods in ultrathin sections of whole tissue and related to all astrocytes contained in the tissue volume used as reference.

J.R. Wolff and T.I. Chao

S/V (surface-tovolume ratio)

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Fig. 7F) encloses all contacts established between astroglial surface extensions of the process segment under investigation and surrounding tissue structures. Contact partners may be astroglial structures, whether these originate from a neighboring cell, belong to another process of the same cell or is a self-contact with another segment of the same surface extension. Morphometric analysis in cerebral and cerebellar cortex of adult rats revealed that interastroglial contacts are formed somewhat less than randomly (Wolff, 1970). In the meantime, detection of astroglial components has been improved by using S100 proteins as a marker (Rickmann and Wolff, 1995). This led to somewhat smaller absolute values for the fractions of volume and surface area of cortical astrocytes (both about 10% of total tissue volume and all neuropil membranes, respectively; Fischer von Weikersthal, 1992). Nevertheless, histochemical identification confirmed, that interastroglial contacts occupying about 9% of astrocytic cell surfaces, are formed somewhat less frequently than expected by chance (see Table 3; Rohlmann and Wolff, 1996). The overall probability of forming interastroglial contacts is heterogenously composed of relatively rare direct contacts between cell bodies and processes (primary and secondary ones) and more frequent contacts between surface extensions, which locally accumulate in perivascular glial sheaths (Section 7.5), near subpopulations of simple synapses and in multilamellar sheaths surrounding certain types of complex synapses (for details, see Chao et al., 2002). Probably because of scarcity, contact relations between astrocytes and other glial cells have never been quantified, to our knowledge. Contact relations with neurons are highly variable. Topographically, these relations vary from complete ensheathment of certain neurons and synapses to avoidance of astroglia-free neuropil compartments. Astroglia is consistently missing, for example, in the sensory synaptic neuropil compartment in olfactory glomeruli (Chao et al., 1997b), bundles of thin unmyelinated neurites (Wolff, 1970), and within the synaptic cleft of simple synapses and in the interior of certain complex synapses, though these may be completely wrapped by astroglia, and at some places even by multilamellar sheaths. Perisynaptic attachment of astroglial lamellae shows considerable dynamics that can be modified within minutes (Rohlmann et al., 1994; Landgrebe et al., 2000). This dynamics includes formation and attachment of astroglial surface extensions to subpopulations of synapses and withdrawal from others. Such changes can be induced by transneuronal signaling. For example, the peripheral transection of the 7th cranial nerve reduces the perisynaptic glial covering to about onehalf in the motor cortex within 1– 4 h (Landgrebe unpublished results; for details, see Chao et al., 2002). In addition, the molecular equipment of astroglial surface extensions is apparently adapted to the chemical specificity of transmission in synaptic subpopulations (Table 4). This table presents a selection of molecules which have been preferentially found in peripheral astroglial structures often surrounding synapses. Although the list is incomplete, e.g., numerous reports on glutamate transporters are missing, it demonstrates that the specificity of astroglial surface extensions is not confined to structure and attachment to specific contact partners, but includes the specificity of molecular equipment that must somehow depend on exogenous signals. Mechanisms of aim-directed trafficking and focal accumulation of molecules in lamellipodia and filopopdia are poorly understood, but these must be able to enrich connexin-43 in subpopulations of filopodia as a prerequisite of gap-junction formation (Wolff et al., 1998).

44

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Fig. 8. Distribution of astrocytes in the neocortex of adult rats. (A) In layers 5 and 6 of the parieto-occipital region, variations in the spatial density of astrocytes are relatively small (mean cell density: 17,600 cells/mm3; immunohistochemical identification with antibodies against GFAP; see E). Accordingly, distances between neighboring cell bodies vary around 50 mm (bar). (B) In the same preparation, however, there are cell clusters in which distances between neighboring cell bodies are reduced to about 30 mm (bar ¼ 50 mm). (C) Because astroglial cell processes interdigitate, their reach is larger than these distances. If expressed as the sphere that statistically includes one-half of the process tips, the mean diameter of the monitored tissue sphere is 78 mm. (D–F) Interestingly, astroglial cell densities found in different parts of rat cortex vary to the same extent as local variations. The minimum density of about 10,000 cells/mm3 corresponds to a territory of 100,000 mm3 per cell and a sphere diameter of 57 mm (D). Corresponding values are presented for intermediate (E; see also A)

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7.5.4. Gap junctions couple subcellular entities Despite randomness of contact relations between processes and surface extensions, astroglial cells form multicellular networks (see the chapter by Scemes and Spray) which enable functional interactions between cells as indicated by calcium waves spreading from one cell to its neighbors (see the chapters by Cornell-Bell et al. and by Shuai et al.). Connectivity of astroglial networks is based on gap junctions, which connect all cell parts with each other but accumulate near synapses. Morphometric analysis in rat neocortex suggests that the number of gap junctions is extremely high, i.e., an average astrocyte carries 30,000 gap junctions, which occupy about 1% of the cell surface and about 15% of the interastrocytic contact area (Rohlmann and Wolff, 1996). Interestingly, gap junctions are not restricted to intercellular contacts (heterocellular coupling; see e.g., Fig. 7D). A considerable proportion of them (at least 4000 gap junctions per cell) connect parts of the same neocortical astrocyte and may even couple different segments of the same lamellipodium (autocellular coupling). Gap junctions connecting parts of the same astrocyte, are also found in purified cultures, i.e., in the absence of other cell types (Wolff et al., 1998). At least in astrocytes, gap junction formation does not select membranes of different cells for coupling, but combines half channels in membranes of subcellular compartments, independently of whether these belong to the same cell or another one. In cell types with complex structure, such as astrocytes, gap junctions enable equilibration of cytosolic components between subcellular compartments, rather than cells. Within astroglial networks, formation of such entities indicate a certain degree of functional autonomy of surface extension and peripheral processes. Cellular signal transfer may then indicate cooperation between many of these subcellular entities, which combine adjacent astrocytes. This is similar to synapses (even synaptosomes) between neurons, and may attract further attention in the future.

8. Concluding remarks Cytoarchitectonics is utilized as a method to detect cell-related tissue compartments (cellular territories) and separate tissue-related cell structures (topographical variables) from endogenously determined ones (topographical invariables). Both the organization of CNS tissue as an enormously expanded epithelium and the non-compact cell shapes of neurons and most non-neuronal cell types make cytoarchitectonic views profitable, provided not only cell bodies, but the full repertoire of cellular structuring (processes and surface extensions) is included as parameters. Intracerebral (intra-epithelial) vascularization is a CNS-specific factor that deeply influences the organization of tissue and the diversity of cell types. It does not only

and maximum densities of 67,000 cells/mm3 (F) reported for the neocortex (see Table 2). Note that the radial overlap between neighboring astrocytes increases from 10.5 mm (D) and 15 mm (E) to 24.5 mm (F), if the length of cell processes (cell size) is kept constant. Thus, the possibility for intercellular interactions increases dramatically when cell density is increased.

46

Table 4 Labeling of peripheral and perisynaptic astroglia processes and lamellar extensions with a selection of transmitter-related markers Regions

Species

Reference

mu and delta opioid receptors alpha(2A)-adrenergic receptor beta-adrenergic receptor

Cervical spinal cord Hippocampus Dentate gyrus Visual cortex Various Cortex Thalamus Peri-aqueductal gray Cortex Peri-aqueductal gray Cortex Thalamus Cerebellum Peri-aqueductal gray Various Dorsal cochlear nucleus Locus coeruleus Dorsal cochlear nucleus Dorsal cochlear nucleus Locus coeruleus Dorsal cochlear nucleus Dorsal cochlear nucleus Ventro-basal nucleus Cerebellum Median eminence Hippocampus

Rat Rat Rat Cat, rat Rat Human, monkey Rat Cat Rat Cat Rat Rat Rat Cat Rat Rat Rat

Cheng et al., 1997 Milner et al., 1998 Milner et al., 2000 Aoki, 1992 Aoki and Pickel, 1992 Conti et al., 1998 DeBiasi et al., 1998 Barbaresi et al., 2001 Conti et al., 1999 Barbaresi et al., 2001 Minelli et al., 1996 DeBiasi et al., 1998 Itouji et al., 1996 Barbaresi et al., 2001 Norenberg and Martinez-Hernandez, 1979 Petralia et al., 1996 Van Bockstaele et al., 2000 Petralia et al., 1996 Petralia et al., 1996 Van Bockstaele et al., 2000 Petralia et al., 1996 Petralia et al., 1996 Mineff and Valtschanoff, 1999 Loesch and Burnstock, 1998 Kawakami et al., 1998 Snyder and Kim, 2000

GABA-transporter 1

GABA-transporter 2 GABA-transporter 3

Glutamine synthetase GluR1-4 GluR5-7 KA2 NR1 mGluR2/3 P2X1 receptor NOS D-serine

Rat Rat Rats Rat Rat Various

J.R. Wolff and T.I. Chao

Marker

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determine the structural diversity of ependymo-astroglial cell types, but also provides the CNS with microglia as an intra-epithelial resident with mesodermal origin, and it forces neurons and oligodendrocytes to deviate from tissue polarity by developing cell-type-specific polarities. Based on morphogenetic and tissue-related constraints, which can be quantitatively estimated by morphometric and stereological analyses, the adult potential for induction of reversible morphogenesis actually determines the functional potential of cell types and individual cells. This includes structural reorganization of limited extent, changes in cooperation between cellular and subcellular entities as well as quantitative and topographical changes in molecular equipment. This article demonstrates that differences in structural –functional potentials can be utilized to define the differential organization of non-neuronal cell types in the CNS. According to cytoarchitectonic criteria, capillary endothelia appear as a system of cells, which maximize apico-basal cell surfaces but minimize lateral cellular interactions within the system, to cooperate in parallel to monitor the border between blood and CNS. Microglia use territorial spacing to monitor the extracellular fluid of CNS tissue with a minimum number of multifunctional cells, which act as individuals. Oligodendroglia also forms a system of individual cells, which adapt to and isolate internodal segments of axonal membranes. In contrast, ependymo-astroglia is polarized between ventricular and mesenchymal tissue surfaces (including all perivascular surfaces) and the intra-epithelial, mainly perineuronal intercellular spaces. Quantitative and microtopographical assessment revealed that the specific connectivity of astroglia, via gap junctions, couple subcellular units rather than cells and include autocellular and heterocellular coupling. Data suggest that in a manner similar to synapses, gap-junction-coupled subcellular entities have to cooperate intracellularly to achieve coupling between neighboring astrocytes as visualized by spreading calcium waves.

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Oligodendrocyte phenotypical and morphological heterogeneity: a reexamination of old concepts in view of new findings Sara Szucheta,* and Mark A. Seegerb a

Department of Neurology and the Brain Research Institute, Pritzker School of Medicine, The University of Chicago, Chicago, IL 60637, USA p Correspondence address: Tel.: þ 1-773-702-6396; fax: þ1-773-702-4066 E-mail: [email protected] b Department of Neurology, Pritzker School of Medicine, The University of Chicago, Chicago, IL 60637, USA

Contents 1. 2.

3. 4. 5. 6.

Introduction Oligodendrocyte phenotypes 2.1. The myelinating phenotype 2.2. The nonmyelinating phenotype Morphological heterogeneity: how does it arise? Oligodendrocyte –neuron cross talk: role on oligodendrocyte differentiation? Lessons from postmyelinating oligodendrocytes: an in vitro model for regeneration Concluding remarks

The morphological and phenotypical heterogeneity of oligodendrocytes (OLGs) were already captured by del Rio-Hortega in his 1928 historic monograph. His keen observations have withstood the passage of time (75 years!) but are only now being recognized. In this chapter, OLG complexity is addressed in the context of its possible determinants. Factors such as territories of OLG origin and OLG – neuron interaction are considered as possible modulators of OLG final destination and fate of acquiring a given phenotype. Experimental evidence that supports a hypothetical link between these events is presented and discussed. Two OLG phenotypes—myelinating and nonmyelinating—are examined in detail. The contributions of transcription factors and signals from growth factors in directing OLGs to adopt a given phenotype are also described. A novel gene, otmp, and its protein product, Oligodendrocyte TransMembrane Protein (OTMP), are portrayed and shown to be expressed in nonmyelinating OLGs of gray matter, such as Advances in Molecular and Cell Biology, Vol. 31, pages 53–73 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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perineuronal OLGs, and in OLGs of white matter tracts that are not connected to axons. OTMP is shown to be instrumental in revealing these latter cells for the first time. Finally, the lessons that can be derived from cultures of postmyelination OLGs as an in vitro model for regeneration are discussed.

1. Introduction In his monograph on oligodendrocytes (OLGs), del Rio-Hortega (1928) called attention to the morphological heterogeneity not only of OLGs associated with myelin but also among the neuronal and vascular satellites, whose processes form the rich plexus that envelops neurons and blood vessels. Thus, implicitly, del Rio-Hortega (1928) illustrated, at a minimum, three phenotypes. In the gray matter, satellite cells often abut a neuronal soma and are referred to as perineuronal OLG (Polak et al., 1982; Raine, 1997). In recent years, the notion of OLG structural heterogeneity has taken hold (Szuchet, 1995 and references therein; Spassky et al., 1998; Miller, 2002), but the issue of OLG phenotypical heterogeneity is still not being considered. Nearly every paper on OLGs in the last two decades, with the sole exception of some textbooks, begins by defining OLGs as the central nervous system (CNS) cells that make and maintain myelin. While this statement is correct, it does not suffice as a definition of OLGs because it fails to encompass all cell types. Furthermore, it should be stated that the switch from ‘making’ to ‘maintaining’ myelin might in itself represent a phenotypic change (Yim et al., 1986). It may be argued that, in certain circumstances (e.g., perineuronal OLGs in a pathological situation; Ludwin, 1984), every OLG has the potential to gear up its myelin genes and make myelin, but this should not detract from acknowledging the existence of physiological phenotypes other than the myelinating ones. To complicate matters even further, the thought should be entertained that each of the structural entities recognized by del Rio-Hortega (1928) may well represent the expression of a specific phenotype. Whereas the field of OLG heterogeneity has seen relatively little progress (but see below), there have been significant advances in pinpointing OLG origins. The discovery of early fate-determining genes such as olig1 (Lu et al., 2002), olig2 (Zhou et al., 2001) and sox10 (Sun et al., 2001) has forced a review of previously held notions on neurogenesis, gliogenesis and lineage relationships (Kintner, 2002). Thus, while the final verdict is still a subject of controversy, and issues such as one territory vs several sites of origin of OLG progenitors are being disputed (Miller et al., 1997; Spassky et al., 2000; Richardson et al., 2000, 2001; Qi et al., 2002), the existence of a lineal relationship between motor neurons and OLGs seems to stand on firm ground (Zhou and Anderson, 2002). Nevertheless, disagreement still persists as to whether OLGs are also lineally linked to astrocytes (Levison and Goldman, 1997; Lee et al., 2003). There are those who support the existence of a glial restricted precursor (GRP), distinct from the O2A bipotential cell, which is now believed to be an in vitro artifact (Luskin and McDermott, 1994; Miller, 2002). The GRP is supposed to be a tripotential cell that gives rise to the two types of astrocytes (i.e., fibrous and protoplasmatic) as well as to OLGs. Figure 1 illustrates these two diverging viewpoints. Experimental evidence seems to tilt the balance toward a single motor neuron/ OLG lineage (Zhou and Anderson, 2002; but see Miller, 2002). The discovery of new

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Fig. 1. Two possible models for OLG lineage progression in the developing spinal cord. (A) Neuroepithelial cells (NE) in the ventral region of the spinal cord differentiate into motor neuron/oligodendrocyte precursors (MNP/OLP). Expression of Olig2 and neurogenins (Ngn) in these cells induces motor neuron (MN) differentiation. Down-regulation of neurogenins and up-regulation of Nkx2.2 with time induces oligodendrocyte (OLG) differentiation in these cells. (B) Neuroepithelial cells along the dorso-ventral axis of the spinal cord differentiate into either neuronal or glial restricted precursors (NP or GRP, respectively). GRPs differentiate primarily into the two types, fibrous and protoplasmatic, astrocytes (Ast1 and Ast2) in the dorsal and intermediate regions, and OLGs in the ventral region. OLG differentiation is induced by sonic (hedgehog) Shh at the expense of astrocytes. See text for details (adapted from Miller, 2002).

genes should shed light on this and other related issues. To dwell further on these topics would be outside the main theme of this chapter; excellent reviews are available (Wegner, 2000a,b, 2001; Woodruff et al., 2001; Miller, 2002). In this chapter, we discuss OLG morphological and phenotypical heterogeneity in the context of their possible determinants. Thus, we examine whether a link can be established between multiple territories of OLG origin and their subsequent differentiation into distinct subtypes. We also consider the contribution of cell –cell interaction (e.g., OLG – neuron interaction) to generating a given class of OLGs. In like vein, we ask

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how the currently accepted OLG phenotypes can be derived from the founder cell(s)? Additionally, we describe a novel gene, otmp, and present experimental evidence that its protein product, Oligodendrocyte TransMembrane Protein (OTMP), is uniquely expressed in nonmyelinating OLGs (Szuchet et al., 2001, 2002). Moreover, with the use of antiOTMP antibodies (Abs) we document, for the first time, the presence of nonmyelinating OLGs in white matter tracts (Szuchet et al., 2002; Szuchet, Hudson and co-workers, unpublished). Among the challenging issues that lie ahead is the discovery of the stage of development at which there is a bifurcation of the myelinating vs the nonmyelinating phenotypes.

2. Oligodendrocyte phenotypes As documented above, OLGs are a highly complex and heterogeneous population of cells. In broad terms, they can be classified as myelinating and nonmyelinating. Interfascicular OLGs belong to the former class and perineuronal and perivascular to the latter. Additionally, interfascicular OLGs can be grouped into four subtypes that differ, among other ways, in their association with axons and in their metabolism (Hartman et al., 1982; Szuchet, 1995; Butt and Berry, 2000). Among the nonmyelinating OLGs, apart from those already cited, a new type that resides in white matter tracts has recently been revealed by the expression of a novel gene, otmp (Szuchet et al., 2001, 2002; see below). If we accept that each of these structural and biochemical entities is endowed with a specific function, we have indeed, a large number of phenotypes to consider. For the sake of simplicity and because we lack solid data on function for most of these cell types, we will only entertain two phenotypes: myelinating (recognizing that it is not a single species) and nonmyelinating (similarly being aware of the existence of more than one entity).

2.1. The myelinating phenotype Myelination is a unique cellular process in which immature OLGs migrate from their site of origin to presumptive white matter tracts, proliferate, exit the cell cycle and begin the massive synthesis of lipids and proteins, which are incorporated into the cellular membrane as it extends and enwraps neighboring axons. The magnitude and complexity of myelination is illustrated by the fact that during its active phase, OLGs must produce as much as 5000 mm2/cell of myelin surface area a day and on the order of 105/cell/min of myelin-associated proteins (Morell and Toews, 1984; Pfeiffer et al., 1993). This OLG task is vital because the insulation conferred by myelin enables the rapid and efficient saltatory propagation of action potentials along axons and loss of myelin leads to crippling diseases such as multiple sclerosis. We will succinctly review the current understanding of the extrinsic and intrinsic signals that may be operative in OLGs acquiring a myelinating phenotype. In doing so, we will strive to differentiate between in vivo and in vitro models because results from culture tend to measure the cell potential rather than its in situ fate. This is particularly true for undifferentiated, plastic cells.

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2.1.1. Axonal signals and their receptors The developmental step addressed here is the transition between an oligodendrocyte progenitor (OLP), characterized by proliferation and differentiation events, and a mature myelinating OLG defined by its association with axons and expression of myelin-specific genes. It is difficult to define the exact point at which this shift takes place since there appears to be no clear genetic switch that induces the myelinating phenotype. Moreover, fate determination and differentiation are separated in both space and time (Miller, 2002). Experimental evidence implicates platelet-derived growth factor AA (PDGF) and fibroblast growth factor 2 (FGF-2) in the development of OLGs; however, results are not always consistent and depend on whether an in vivo or an in vitro model is used. PDGF is secreted by neurons and astrocytes in the CNS and, in conjunction with axonal electrical activity, appears to be essential for the survival and differentiation of mature OLGs in the rat optic nerve in vivo (Demerens et al., 1996; Barres and Raff, 1993). Conversely, addition of PDGF retards the rate of proliferation and differentiation of myelinating OLGs in vitro (Bogler et al., 1990) and in vivo in the rat anterior medullary velum (AMV) (Butt et al., 1997). FGF-2 induces the up-regulation of PDGF-a receptors and stimulates the proliferation of OLPs in vitro and in vivo (McKinnon et al., 1990; Goddard et al., 1999); it also blocks terminal differentiation of O4þ/galactocerebroside (GC)2 OLGs in culture and in vivo (Gard and Pfeiffer, 1993; Bansal and Pfeiffer, 1997). FGF receptor-2 (FGFR-2) is expressed primarily by cultured terminally differentiated OLGs (Bansal et al., 1996), and it is down-regulated by FGF-2 (Yim et al., 2001). Thus FGF-2 appears to maintain the undifferentiated and proliferative state of the OLP. Insulin-like growth factor 1 (IGF-1) significantly increases the population of myelinating OLGs in the rat AMV, most likely by promoting the survival and differentiation of OLP (Barres and Raff, 1993; McMorris et al., 1990). IGF-1 knock-out mice exhibited a decrease in myelinating OLGs, which was attributed to reduced numbers of neurons in regions of hypomyelination (Cheng et al., 1998). Thus, IGF-1 functions as a positive regulator for the survival and differentiation of both OLGs and neurons, thereby adjusting the axon/OLG ratio. Triiodothyronine (T3) has been implicated at several stages throughout OLG development. OLGs carry two thyroid hormone receptors (THRa and b), each of which generates two isoforms (THRa1 and 2, b1 and 2) through alternative splicing (Hudson et al., 1996). THRb2 is expressed by OLPs (Barres et al., 1994) and T3 potentiates the responsiveness of these cells to FGF-2 (Ben-Hur et al., 1998). T3 induces OLPs to upregulate THRb1 as they exit the cell cycle in vitro (Barres et al., 1994) or to initiate active myelination in vivo (Carre et al., 1998). T3 also has a role in the transcription of myelin specific genes such as myelin basic protein (MBP) (Tosic et al., 1992) and proteolipid protein (PLP) (Granneman et al., 1998). Hence, the interaction between T3 and its receptors appears to be intimately involved in the development and maintenance of the OLG myelinating phenotype in vitro and in vivo. Ciliary Neurotrophic Factor (CNTF), in contrast to the aforementioned growth factors, seems to directly enhance the process of myelination (Stankoff et al., 2002). CNTF exerts its effect via the CNTF receptor (CNTFR) associated with the LIFRb/gp130 complex, which in turn activates the 130 kDa glycoprotein Janus kinase (gp 130-JAK) pathway

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(see Section 2.1.2). CNTF also acts, in conjunction with PDGF, to promote the survival of OLPs in vitro through a JAK/STAT pathway (Dell’Albani et al., 1998). In summary, it is clear that our understanding of the role of growth factors in OLG myelination is still very rudimentary. Complicating matters further are the large number of parameters that influence the outcome of in vitro studies and the difficulty of establishing experimentally controlled models in vivo. Notwithstanding these obstacles, affirming the notion that there is interplay among the various growth factors and elucidating its nature are essential for deciphering the molecular events that lead to myelin formation.

2.1.2. Intrinsic signaling pathways Following the model proposed by Edlund and Jessell (1999) for CNS neuronal differentiation, we speculate that the development of the OLG myelinating phenotype is a process initially regulated by extrinsic factors, but that as the cells get closer to terminal differentiation the regulatory signals become increasingly intrinsic. This seems to be a reasonable proposition since OLG differentiation appears to be largely regulated by molecules and mechanisms similar to those active in neurons. From this, it follows that it is the intrinsic signal transduction pathways of developing OLGs that transmit the extrinsic signals (see section above) to the nucleus, which in turn induce transcription factors (see section below) to initiate the expression of myelin specific genes. A comprehensive review of all the signaling pathways implicated in OLG development (Wegner, 2000a,b) is beyond the scope of this chapter. Here we will only consider those most relevant to the theme of this article (Fig. 2). Protein kinase cascades such as the extracellular-signal regulated protein kinase (ERK) and protein kinase C (PKC) pathways have been shown to modulate the proliferation (Bhat et al., 1992) and differentiation (Heinrich et al., 1999) of OLPs through the binding of growth factors such as PDGF and FGF-2. The PKC and ERK pathways have also been shown to interact with each other (Stariha et al., 1997), stimulate OLG process extension (Althaus et al., 1997; Yong et al., 1988), and modify myelin-specific genes and their products in cultured OLGs (Vartanian et al., 1986, 1988; Yong et al., 1994; see also Casaccia-Bonnefil, 2000; Stariha and Kim, 2001). Fyn is a member of the Src family of cytoplasmic nonreceptor tyrosine kinases, and is one of the early regulatory signals for the initiation of myelination (Umemori et al., 1994). It is not known how Fyn is activated, but Fyn has been implicated in the expression of myelin-specific proteins, cytoskeletal rearrangements and membrane synthesis by myelinating OLGs (Osterhout et al., 1999; Umemori et al., 1999; Wolf et al., 2001). The binding of CNTF to CNTFR induces activation of intermediate kinase proteins followed by various STAT proteins (Davis et al., 1991, 1993; Dell’Albani et al., 1998; Heinrich et al., 1999). STATs can also be activated via receptor tyrosine kinases such as PDGFRAA and FGFR-2 (Novak et al., 1996; Yamamoto et al., 1996). It is postulated that activated STAT proteins dimerize and translocate into the nucleus where they are presumed to activate as yet unidentified OLP genes. Further investigation of these pathways is therefore essential to understanding how the differentiation of OLPs progresses from an extrinsically to an intrinsically regulated event.

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Fig. 2. Extrinsic to intrinsic signal transduction pathways in differentiating OLGs. Pictured are the PDGF/FGF-2-activated ERK and STAT pathways, the CNTF-activated JAK/STAT pathway, the T3-mediated activation of the nuclear receptor TR, and the Fyn-mediated activation of p190 RhoGAP and possibly myelin associated glycoprotein (MAG) and STAT. See text for details (adapted from Wegner, 2000a,b).

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2.1.3. Transcription factors relevant to myelinogenesis Significant progress has been made in defining the expression and establishing the function of families of transcription factors whose presence in specific domains determines the types of cells that emerge from such domains (Kessaris et al., 2001). For example, the orderly expression of transcription factors olig1, olig2 and nkx2.2 and their interplay underlie OLG fate determination (see reviews by Miller, 2002; also Wegner, 2000a,b, 2001; Hudson, 2001). Herein we are strictly concerned with transcription factors that regulate the transition from an immature OLG phenotype to a myelinating one. Although a number of putative transcription factors have been identified, the precise mechanism of their action remains to be elucidated. Myelin transcription factor 1 (MyT1) is a C2HC zinc finger protein that binds specifically, in vitro and in vivo, to the PLP gene promoter; its expression decreases inversely to that of PLP, indicating that MyT1 may be involved in inducing the proper conformation of the PLP promoter so that other transcription factors can bind properly (Kim and Hudson, 1992; Armstrong et al., 1995). Using a dominantnegative approach, Nielsen, Hudson and Armstrong (personal communication) showed that MyT1 modulates OLG proliferation and differentiation. Sry-box 10 (Sox10) is a member of the high-mobility group of proteins; and its expression is restricted to OLGs and persists from embryonic to adulthood. It is also present in cultured cells. Sox10 acts synergistically with the POU-domain protein Tst1/Oct6/SCIP to induce MBP promoter activity (Kuhlbrodt et al., 1998b; Stolt et al., 2002). Independently, Tst1/Oct6/SCIP appears to be involved in the activation of genes controlling the proliferation, rather than the differentiation, of immature OLGs in vitro (Collarini et al., 1992; Jensen et al., 1998; Monuki et al., 1989). Two other members of the Sry-box family, Sox4 and Sox11, are highly upregulated in immature OLGs but are downregulated as OLGs begin to myelinate. Sox4 and Sox11 act synergistically and indiscriminately with the POU-domain proteins Brain1 and 2 (Brn1 and Brn2) to induce promoter activity, suggesting that Sox4 and Sox11 may be functionally redundant (Luhlbrodt et al., 1998a). Brn1, Brn2 and Tst-1/Oct6/SCIP also appear to be largely redundant in the transcriptional events leading up to myelination (Schreiber et al., 1997). Krox-24 is a homeodomain zinc finger protein that appears to regulate the genes that maintain OLPs in a proliferative and nondifferentiated state (Sock et al., 1997). Rat Kruppel-type 1 and 2 proteins (rKr-1 or -2) are C2H2 zinc finger transcriptional regulators, which seem to have opposite effects on cultured OLGs. rKr-2 behaves as a repressor by keeping OLPs in a proliferative and nondifferentiated state (Pott et al., 1995), whereas rKr-1, detected in MBPþ/PLPþ OLGs, appears to sustain their myelinating phenotype (Pott et al., 1996). Gtx is a homeobox domain protein that is found specifically in differentiated OLGs in the adult CNS (Komuro et al., 1993). There are four Gtx binding sites in the MBP and PLP promoters. Gtx and MBP appear to be coordinately regulated (Awatramani et al., 1997). We have presented an overview of the current knowledge of factors that influence the transition from an OLG progenitor to a mature cell. Some of these results originated from in vitro models that may or may not have their in situ counterparts. What is clear is that this is the very beginning. Future research should be able to integrate all of this information into a consistent model that explains, in molecular terms, the steps required to progress

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from a cell with a specified destination, to a migratory entity, to a proliferating cell and, finally, to a cell poised for its ultimate accomplishment, i.e., the production of myelin.

2.2. The nonmyelinating phenotype Knowledge of the existence of OLGs that are not connected to a myelin sheath, but instead extend their network of processes, not unlike the arms of an octopus, to touch, cover, or envelop neurons and blood vessels dates back to the early description of OLGs (del Rio-Hortega, 1928). Beyond this morphological characterization, few advances have been made in our understanding of their physiological role. The situation is not as bleak for the gray matter cells that abut the neuronal soma, the so-called perineuronal OLGs, but even here our information concerning what these cells do is still in it its infancy. 2.2.1. Perineuronal oligodenrocytes The morphological and structural characterization of perineuronal OLGs can be found in any textbook dealing with the nervous system (Polak et al., 1982; Raine, 1997; Peters et al., 1991). Overall, there is nothing remarkable about them. They are regarded as analogous to capsule cells of the dorsal root ganglia. Perineuronal OLGs come in different flavors that include two or more OLGs attaching to large neurons or a single OLG ‘overseeing’ several small neurons. Although the distance between abutting cells can be very narrow, no junctional complexes have been detected. Sometimes an astrocytic process intercalates in between the neuron and the OLG. The functional significance of these structural units is still an open question. On the one hand, the suggestion has been made that perineuronal OLGs may specialize in tending a specific type of neuron, e.g., Purkinje cells (Monteiro, 1983). On the other hand, there is some experimental evidence indicating that this type of OLG may partake in remyelination under pathological circumstances (Ludwin, 1984). There are no inherent contradictions between these findings since our current understanding of how cells change their phenotype in response to internal or external cues can easily account for such situations. There is a general belief that perineuronal OLGs express all the necessary myelin proteins and are competent to make myelin. However, to the best of our knowledge, this belief is not supported by experimental evidence. In fact, attempts to demonstrate the presence of MBP in these cells were unsuccessful (Polak et al., 1982; Sternberger, 1984). An interesting hint for the functional relevance of perineuronal OLGs to the neurons they adjoin came from the work of Taniike et al. (2002). These authors have shown that under conditions of demyelination, perineuronal OLGs protect neurons from apoptosis by upregulating a specific prostaglandin. The discovery of a novel gene (Szuchet et al., 2001), uniquely expressed by nonmyelinating OLGs, may pave the way to unravel the mysteries of this significant population of cells. 2.2.2. otmp—a novel gene expressed solely by OLGs of the nonmyelinating phenotype The identification and characterization of otmp and its protein product, OTMP, have been described (Szuchet et al., 2001). Otmp was isolated from an ovine OLG cDNA

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library as a 3500 bp cDNA. The cDNA encodes a protein of 511 amino acids with a predicted molecular mass of 55.7 kD and a pI of , 9.3. OTMP belongs to the emerging subfamily of ‘glytamate-binding proteins’ with highly conserved representatives in all species. However, OTMP has a topology that distinguishes it from the other family members: it is modeled as a four-pass transmembrane protein with both termini anchored inside the cell and two external loops of 92 and 10 amino acids each. Polyclonal Abs directed against a 20 amino acid segment of the external (92 amino acid) loop were utilized to establish the cellular localization of OTMP in rat/mouse/human brain sections. Interestingly, these Abs selectively recognized OLGs of the nonmyelinating phenotype in gray and white matter. In gray matter, OLGs and their processes abutting neuronal soma were intensely decorated by anti-OTMP Abs (Fig. 3) (Szuchet et al., 2002). Significantly, OTMPþ cells are also interspersed among interfascicular OLGs in white matter tracts (Fig. 4) but do not colocalize with GFAPþ cells, which demonstrates that they are not astrocytes. OTMPþ cells are postulated to be OLGs that are not connected to an axon via a myelin sheath (Szuchet et al., 2002). The existence of such cells can be inferred from the work of Suzuki and Raizman (1994) and have now been revealed for the first time. Suzuki and Raizman (1994) performed a detailed examination of the development of a white matter tract such as the fimbria. Two of their observations are directly pertinent to

Fig. 3. OTMP recognizes perineuronal OLGs. Triple staining of P29 mouse cerebrum with anti-OTMP, NeuN (marks specifically neuronal nuclei) and DAPI (stains nuclei). Note that an OTMPþ cell abuts a NeuNþ cell. Epifluorescence image.

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Fig. 4. OTMP is not colocalized with markers that identify myeline-making OLGs. Immunostaining of the corpus callosum of a 15-day old GFP/CNP transgenic mouse (the green fluorescence protein, GFP, was driven by the CNP promoter) with anti-OTMP and DAPI. Notice that OTMPþ cells are interspersed among green OLG in a row of interfascicular cells. There is no overlap between the markers. Confocal image.

the issue under consideration: (1) cells within a given row may be clonally related; and (2) myelination in a given row is initiated by one OLG at a time (Fig. 5). Since it is not possible to discern whether each of the OLGs in a given row has invested an axon with myelin or whether some remain in a supporting role (‘cells in-waiting?’) once myelination is complete, it was tacitly assumed that they all make myelin. The data of Szuchet et al. (2002) challenge this concept and raise important questions as to the function of the OTMPþ cells.

3. Morphological heterogeneity: how does it arise? It is not our intention to review the morphological heterogeneity of OLGs. First, the initial description of del Rio-Hortega (1928) rests on firm ground and all recent work on this topic only reaffirms his observations. No changes in 75 years! This stands as a strong tribute to del Rio-Hortega’s intellect and sagacity. Second, this subject is well covered in textbooks (Haymaker and Adams, 1982; Peters et al., 1991) and reviews (Szuchet, 1995 and references therein). It is also described in the chapter by Wolff and Chao. Here, we will examine whether a link—however tenuous—can be established between the various morphological subtypes and the territories from whence OLG

64 S. Szuchet and M.A. Seeger Fig. 5. A schematic representation of the multifocal mode of differentiation of immature OLGs in the fimbria. In each row of interfascicular immature OLGs, a single OLG (red), referred to as the ‘precocious cell’ gives rise simultaneously to a complement of varicose myelinating processes. Immature OLG (pink) and astrocytes (green) are also picture (adapted from Suzuki and Raizman, 1994).

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progenitors originate. It should be stated at the start that the origin(s) of OLGs is still a hotly debated topic. del Rio-Hortega (1928) classified OLGs into three classes: (1) interfascicular (associated with myelinated axons); (2) perineuronal (abutting neuronal soma); and (3) perivascular (related to blood vessels). The interfascicular OLGs were further subdivided into four subtypes (I – IV) based on cellular size, morphology, number of processes and number of internodes they subserved. While the size of the soma increased from type I to IV, the number of myelinated internodes decreased from many to one. Thus, formally, the type IV OLG resembles a Schwann cell in its association with the axon. It is generally accepted that OLG types I/II reside mostly in brain while types III/IV are found in the spinal cord. The key question is whether the fate of a cell to become a given OLG subtype is predetermined by genetic programming (e.g., originating from a specified progenitor) and/or by its microenvironment (e.g., resulting from an exchange of signals with the neuron). What is the evidence? It is generally accepted that OLG precursors originate at restricted territories and then migrate widely toward presumptive white matter tracts. However, the notion that there might be more than one progenitor type, recognized by distinct markers and populating different regions, is highly contested. Thus, Spassky et al. (1998; 2000; 2001) argue for the existence of at least two, and possibly more, biochemically defined OLG precursors that are distinguished by expressing either the plp/dm-20þ gene or the pdgf-aRþ gene (or neither) and populating the brain and the spinal cord, respectively. In stark contrast, Richardson et al. (2000) (see also Woodruff et al., 2001) vehemently oppose this view by advocating a single pdgf-aRþ founder for all OLGs. If confirmed, the hypothesis of Spassky et al. (1998, 2000, 2001) could, in principle, account for the segregation of type I/II and type III/IV OLGs to the brain and spinal cord, respectively. And with a stretch of the imagination, one might suggest that the population that expresses neither of these two genes (Spassky et al., 2000) is the founder cell of nonmyelinating OLGs. While this is very tempting as a mechanism to explain OLG structural diversity, a sobering warning comes from transplantation experiments, which have shown that type I/II OLGs are capable of myelinating large axons (Fanarraga et al., 1998). Clearly we are not there yet, but are moving in the right direction. It is hoped that future research will solve the ‘mystery’ of OLG structural and functional complexity.

4. Oligodendrocyte – neuron cross talk: role on oligodendrocyte differentiation? We will only discuss the most pertinent evidence (when available) in support of the concept that OLG – neuron association constitutes an inseparable partnership, both at the level of the myelin– axon connection and soma– soma interaction (as in the OLG perineuronal unit). A priori, there is no reason to presuppose that the nature of OLG – neuron interaction is the same, or even similar, in the two units under discussion. In considering the myelin– axon unit, the dogma which holds that the sole function of myelin is that of a static insulator that facilitates fast nerve conduction is no longer tenable. Physiologists were long perplexed by the lack of correlation between the actual thickness of the myelin sheath and their awareness that only a few lamella would suffice to sustain

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fast conduction. This puzzle has now been solved! Myelin has emerged as a necessary modulator of axonal differentiation. Not only does myelin affect the cytoskeletal composition and chemistry, and thereby the final thickness of the axon it enwraps, but it also sends retrograde signals to the soma to control gene expression and axonal transport (Brady et al., 1999; Witt and Brady, 2000). The employ of animal models such as the shiverer mouse, a demyelinating mutant, and an MBP transgene on a shiverer background (Readhead et al., 1987; Readhead and Hood, 1990) was instrumental in unraveling some of these myelin– neuron interactions. Hence, the findings of Ledeen and co-workers (Chakraborty and Ledeen, 1993; Larocca and Ledeen, 1993) that myelin had the biochemical machinery to transmit signals—dismissed at first as purification artifacts— now have a role to play. Additionally, there are membrane –membrane interaction at the so-called paranodal loops that are crucial for the proper organization of the nodes of Ranvier and the focal alignment of sodium channels (Popko, 2000). In conclusion, the road is now wide open for searching deeper into the codes utilized by OLGs and neurons to communicate with one another. Turning to OLGs, it is pertinent to ask: what do they receive from neurons in return? A coherent picture is still missing. Evidence that neurons provide growth factors that influence OLG proliferation and differentiation was given in Section 2.1. Others observations can be added to this list (e.g., Fruttiger et al., 2000; Stankoff et al., 2002). It seems that neurons may even control OLG numbers (Burne et al., 1996). But among the major challenges that still remain is discovering the molecules and events that direct OLGs to become a specific cell type. Is it a signal from the neuron? Is it the microenvironment of the particular white matter tract that the cells populate? Or is it a bit of both? The discovery of nonmyelinating OLGs within rows of myelinating ones may provide a new approach to addressing the questions outlined here. As for the OLG-perineuronal unit, as pointed out above, it remains essentially a tabula rasa. Again, the novel gene otmp and its expressed protein, OTMP, may become powerful tools for initiating exploration into the forces/signals driving this association. How different are they from those fostering OLG –axon association?

5. Lessons from postmyelinating oligodendrocytes: an in vitro model for regeneration Our laboratory has developed a model, albeit idealized, consisting of pure cultures of ovine OLGs isolated from young, myelinated brains. Using this model, we have shown that OLGs recover from the trauma of separation from myelin and reassemble multilamellar membrane in vitro, in the absence of axons (a process we have named myelin palingenesis) (Arvanitis et al., 1992a,b), and in vivo, in the shiverer mouse (Ludwin and Szuchet, 1993). These findings demonstrate the suitability of employing such OLG cultures to investigate the mechanism of remyelination (i.e., regeneration). Moreover, as remyelination by OLGs recapitulates the developmental steps of myelination, this in vitro model can also be utilized to study the mechanism of myelination (Yim et al., 1986; Vartanian et al., 1986, 1988). In fact, we have discovered that the state of OLG differentiation (or regeneration) can be modulated in our model by

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facilitating or interfering with their attachment to a special substratum consisting of a heparin-binding glycoprotein, named GRASP, that we purified from horse serum (Schirmer et al., 1994). Not only can this model be used to study the morphological and biochemical changes that occur in OLGs upon adherence to a substratum which, we postulate, mimics an axonal surface, but also to identify alterations in the expression of genes that are important for (re)myelination. Indeed, taking advantage of such a strategy and using techniques such as differential display and cDNA library screening we have identified several genes which are up-regulated upon OLG adhesion to GRASP—an event that marks the commencement of their myelinogenic phenotype (Yim et al., 1993; Sanyal et al., 1996). We have characterized several of the genes implicated in establishing this phenotype (Sanyal et al., 1996; Szuchet et al., 2001, 2002). We have also demonstrated that adhesion induces OLGs to assemble a cell-associated matrix and have characterized the major matrix constituents as heparan sulfate proteoglycans (Szuchet et al., 2000). Our working hypothesis, based on these findings, is schematically illustrated in Fig. 6. To further characterize the importance of proteoglycans to the function of OLGs, we screened a cDNA library generated from adhered OLGs with oligonucleotide probes complementary to conserved segments of genes that encode proteoglycans important in other cell types. Using this approach, we isolated a number of cDNAs representing novel

Fig. 6. Hypothetical events leading to the regeneration of postmyelination OLGs. It is postulated that upon binding to the substratum GRASP, an OLG surface receptor connects to the cytoskeleton, forming an adhesion signaling complex. This complex then transduces a number of biochemical events (including the assembly of a cell-associated matrix, polarization of the plasmalemma and activation of stress-related genes) that culminate in the cells acquiring the myelinogenic phenotype.

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genes unrelated to the targeted proteoglycans and sharing similarity only in the region of the probe. One of them, a 3300 bp cDNA, is a novel proteoglycan that we named novocan and its protein product NOVOcan; ‘novo’ to stress the fact that it is novel and ‘can’ because the predicted protein sequence contains glycosaminoglycan binding sites (Szuchet et al., 2001; see also chapter by Szele and Szuchet). Another cDNA (3500 bp) is otmp. It is instructive to examine the properties of the genes identified thus far, for they provide insight into the events that transpire in regeneration. For example, while the modulation of ferritins by iron is posttranslational, under conditions of differentiation or growth, heavy-chain ferritin is transcribed independently of iron. Thus, the observed up-regulation of heavy-chain ferritin in adhered OLGs can be taken as a measure of their attempts at recovery (Sanyal et al., 1996). In like vein, the appearance of genes such as novocan and otmp, associated with early events of development, provides a glimpse of pathways that might be activated. In summary, this model is providing useful clues of the processes that an OLG may have to undergo when trying to regenerate.

6. Concluding remarks The last two decades have seen a great impetus in unraveling the biology of OLGs. Major players in these advances were: (1) development of methods to isolate OLGs from mature and neonate brains and to maintain them in culture; (2) discovery of surface markers that specifically recognize cell types both in vitro and in vivo; (3) identification of adhesion molecules and the manner in which they foster cell – cell interactions; (4) progress in the use of retroviral vectors to follow cell fate; and (5) improvement in imaging techniques. This increased technological sophistication marched hand in hand with the depth of scientific inquiries. Thus, questions dealing with how cells talk to one another, signals that define the site(s) of a cell’s origin, migration, proliferation, terminal differentiation and lineage relationship are amenable to experimentation. Concerning OLG progenitors, their territories of origin can be assigned in space and time and their migration followed. Progress has been made in deciphering some of the interactions between OLGs and neurons and the role of myelin above and beyond that of an insulating tape. Yet the road ahead is long and hard for many issues remain to be solved. Among other items, the controversy regarding whether a single or multiple founder cells give rise to the different OLG subtypes needs resolution. Which signals determine that a given progenitor lands in a specific white matter tract to become an OLG of a given subtype? Then there exist the nonmyelinating OLGs, some of which, e.g., perineuronal OLGs, are mere textbook oddities. Moreover, the finding that interspersed among interfascicular OLGs are cells that do not seem to be involved in the making of myelin sends many of us back to the drawing board. How are these cells programmed? If neurons tell OLGs what phenotype to adopt, how do they select which ones to address? The discovery of a gene, otmp, uniquely restricted to a (novel?) phenotype opens up new questions, but should also help to formulate experimental paradigms to answer them. Of prime importance is the need to

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understand at what stage of development these different phenotypes segregate and what event marks this critical point? As it is, and as it should be, answering one question is just a way of opening the door for many more to come in. Acknowledgements Some of the work reported here was done in the Laboratory of Developmental Neurogenetics, NINDS, NIH, where one of us (S.S.) was on a one-year sabbatical leave. Sara Szuchet expresses her heartfelt thanks to Dr Lynn Hudson, laboratory chief, for her unconditional support. We are grateful to Drs Gabor Lovas and Javier Martinez de Velasco for the photographs on OTMP and Ms. Gretchen Hendrie for help with chapter preparation. This work was supported in part with NIH grant RO3-HD40832 and pilot project PP0843 from the National Multiple Sclerosis Society. Sara Szuchet was the recipient of a National Research Service Award.

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Regulation of cell cycle progression in astrocytes Yuji Nakatsujia,* and Robert H. Millerb a

Department of Neurology (D-4), Osaka University Graduate School of Medicine, Yamada-oka 2-2, Suita, Osaka 565-0871, Japan p Correspondence address: E-mail: [email protected] b Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA

Contents 1. 2.

3.

4.

Introduction Control of the cell cycle in mammalian cells 2.1. Overview of the cell cycle 2.2. Cyclin dependent kinase inhibitor p27Kip1 Cell-cycle regulation in astrocytes 3.1. Regulation of astrocyte proliferation 3.2. Density-dependent inhibition of astrocyte proliferation 3.3. Cell cycle in pathological conditions 3.4. Brain tumors and cell cycle Concluding remarks

Many of the regulatory effector mechanisms of the astrocytic cell cycle are shared with other cell types, although the control of those effectors through growth factor stimulation and contact inhibition of cell proliferation appear to be specific to CNS glia. Many of the molecules involved in astrocyte cell cycle progression are discussed, especially the role of the cyclin-dependent kinase inhibitor p27 –pRb pathway in contact inhibition and reactive gliosis under various conditions. 1. Introduction Understanding of the regulation of cell-cycle progression in astrocytes is important for the interpretation of events occurring in the course of development, during recovery from central nervous system (CNS) injury, in neurodegenerative diseases, and in the development of tumors. Since much more information is available about regulation of Advances in Molecular and Cell Biology, Vol. 31, pages 75–95 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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the cell cycle in other eucaryotic cells, we will begin this chapter by a general discussion of cell-cycle regulation in mammalian cells and then proceed to cell-cycle regulation in astrocytes under normal and pathological conditions.

2. Control of the cell cycle in mammalian cells 2.1. Overview of the cell cycle The proliferation of all eucaryotic cells proceeds through a carefully controlled sequence termed the cell cycle. The cell cycle can be considered as four distinct phases termed G1, synthesis (S), G2 and mitosis (M). Completion of mitosis is usually followed by cytokinesis and under appropriate stimulation both daughter cells may re-enter the cycle at G1. Progression through the cell cycle is regulated at a number of distinct steps by the sequential expression of cyclins and activation of cyclin-dependent kinases (CDKs) (cdk1, cdk2, etc. in Fig. 1). After the completion of mitosis, a cell may enter a quiescent state, called G0 phase that precludes further progression through the cycle. Upon suitable stimuli, however, such as the activation of growth factor receptors, a cell may re-enter the G1 phase. This phase is characterized by the preparation of components necessary for further progression through the cell cycle. There is a critical stage or restriction point in the late phase of G1, where entry into the rest of the cell cycle is controlled. After progression through this restriction point, a cell may traverse the remaining cell cycle without further mitogenic stimulation. There are a variety of molecular signals that contribute to cell-cycle progression, including the cyclins, CDKs, specific CDK inhibitors (CKIs) and selective phosphatases. Central in this control cascade are the CDKs. The major regulatory mechanisms controlling progression through the cell cycle appear not to be absolute levels of CDK

Fig. 1. The cell cycle is governed by the interaction of cyclins, CDKs and CKIs. Dotted lines indicate inhibitory effects.

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expression, since they are relatively constant, but rather their activity that is controlled by the expression of cyclins, CKIs, CDK activating kinase (CAK), and phosphatases. The cyclins are divided into two major classes: the G1 cyclins (e.g., cyclins D1-3, and E1-2) and the G2/M cyclins (cyclins A and B). Each cyclin interacts with specific CDKs. For example, cyclin D interacts with cdks4 and 6; cyclin E with cdk2; cyclin A with cdks2 and 1, and cyclin B with cdk1 (Fig. 1). Expression levels of the cyclins are regulated at both the transcriptional level and by protein degradation mediated by the ubiquitin – proteasome pathway. The pattern of expression of the different cyclins is independently regulated. D-type cyclins (D1-3) are expressed relatively constantly throughout the cell cycle, although they are down-regulated in G0. Their expression may be induced by external stimuli including growth factors, and the specific D-type cyclins induced is dependent on the responding cell type. In contrast, the expression of other cyclins is periodic throughout the cell cycle, and their expression is less dependent on external stimulation (Sherr and Roberts, 1995; Harper and Elledge, 1996; Sherr and Roberts, 1999). The activation of CDKs is regulated by binding to their appropriate cyclins (left part of Fig. 2), and the activity of the resultant cyclin –CDK complexes is further controlled by phosphorylation –dephosphorylation events. For example, the phosphorylation of a threonine (T) residue on CDK by CAK activates a cyclin – CDK complex, in which cyclin

Fig. 2. The activity of CDK is regulated by cyclin and the phosphorylations of threonine (T) and tyrosine (Y) residues of the CDK by a series of kinases and phosphatases. The active CDK– cyclin complex in the upper righthand corner is inactivated both by dephosphorylation mediated by KAP, and by hyperphosphorylation at T14 and Y15, mediated by Wee1/Myt1. The latter inactivation is counteracted by the phosphatase Cdc25. CKI (CDK inhibitor) also inhibits the activity of CDK.

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H –cdk7 complex is a major component (Matsuoka et al., 1994). This activity is suppressed by the CDK-interacting protein phosphatases (Fig. 2) such as kinase-associated phosphatase (KAP) (Song et al., 2001). By contrast, phosphorylation of other threonine (T14) and tyrosine (Y15) residues by Wee1/Myt1 inactivates the complex, and this inactivation is counteracted by Cdc25 (Fig. 2), a phosphatase, present as Cdc25A, Cdc25B or Cdc25C. While Cdc25A dephosphorylates cdks2/4/6 and is required for G1/S progression, both Cdc25B and Cdc25C induce mitosis by dephosphorylating and activating cdk1 (Borgne and Meijer, 1996; Bartek and Lukas, 2001; Bollen and Beullens, 2002). The retinoblastoma tumor suppressor protein pRb is an additional important cell-cycle protein that acts as a negative regulator for progression through the cell cycle. The activity of pRb is regulated by its level of phosphorylation, and its overall expression level is relatively constant throughout the cell cycle. During early G1 the active, hypophosphorylated form of pRb predominates (left part of Fig. 3), and this directly binds the transcription factor E2F. During progression through the cell-cycle, pRb is progressively hyperphosphorylated by cyclin D – cdk4/6 and cyclin E – cdk2 complexes and becomes inactivated (Weinberg, 1995). The hyperphosphorylated pRb releases E2F, and the released E2F, in association with another transcription factor DP, activates the transcription of cell-cycle regulated genes such as cyclin A, cyclin E, cdk1, DNA polymerase-a, thymidylate synthase, c-myc, and E2F itself (Fig. 3) (Lam and La Thangue, 1994; Lania et al., 1999). In addition to the inhibitory mechanism by sequestering E2F, pRb can actively suppress transcription of some proteins by binding to the promoter in a complex with E2F. Under these conditions, pRb binds simultaneously to E2F and histone deacetylase (HDAC), which induces a tight association between the DNA and nucleosomes leading to transcriptional inhibition (Harbour and Dean, 2000). There are

Fig. 3. The hypophosphorylated form of pRb directly binds the transcription factor E2F. Following active cyclin–CDK complex-mediated hyperphosphorylation of pRb, it releases E2F. Free E2F, in association with the transcription factor DP, activates the transcription of cell cycle regulated genes important for further cell cycle progression.

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a number of pRb related proteins including pRb/p105, p107 and pRb2/p130, all of which share substantial homology in a central motif known as the pocket region. These pocket proteins bind specifically to distinct E2F family members and repress transcription (Paggi et al., 1996; Ferreira et al., 1998). pRb preferentially interacts with E2F1-3, p107 with E2F4 and pRb2/p130 with E2F4-5. pRb2/E2F complexes are formed primarily in G0 and early G1 phases, pRb/E2F complexes in G1 to S phases, and p107/E2F complexes in late G1 to S phases (Claudio et al., 1996). pRb-deficient mice die in the early embryonic stage before complete genesis of glial cells. Neural precursor cells derived from pRb-deficient mice exhibit delayed cell-cycle exit but undergo neurogenesis, and up-regulated p107 may compensate for the lack of pRb (Lee et al., 1992). Mice deficient in p107 show little or no alteration in neural cell development, but pRb2/p130-deficient mice exhibit disorganization in neural structures along with a reduction of developing neurons (Yoshikawa, 2000). A recent study showed that overexpression of pRb2/p130 in astrocytes resulted in the inhibition of proliferation and induction of astrocyte differentiation (Galderisi et al., 2001). An additional and critical regulation of cyclin – CDK complexes is mediated through CKIs. There are two major classes of CKIs termed INK4 family and the Cip/Kip family. The INK4 family consists of p16INK4a, p15INK4b, p18INK4c and p19INK4d, which are specific inhibitors of cyclin D – cdk4/6 complexes (Figs. 1 and 2). These proteins interact directly with the subunit of cyclin D-dependent kinases, and this results in inactivation of cdk4/6-INK4 dimers (Sherr and Roberts, 1995; Ruas et al., 1999). The importance of p16INK4a regulation of the cell cycle is indicated by relative abundance of mutations in this molecule that accompany tumorigenesis and by the observation that while p16INK4a deficient mice show no gross abnormalities, they have relatively high rates of spontaneous tumorigenesis (Serrano et al., 1996). This phenomenon may reflect a role for p16INK4a in senescence related cell-cycle arrest (Alcorta et al., 1996). The specific functions of p18INK4c and p19INK4d are less well understood. The expression of both molecules is consistent with a role in proliferative control, since in many tissues including the CNS the expression of p18INK4c and p19INK4d are high during mouse embryogenesis, and fall to low levels in the adult (Zindy et al., 1997). The second major family of CKIs is the Cip/Kip family that consists of p21Cip1, p27Kip1 and p57Kip2. Members of this protein family possess binding sites for both cyclins and CDKs, and they are potent inhibitors of cyclin E– cdk2 and cyclin A –cdk2 complexes (Harper and Elledge, 1996; Sherr and Roberts, 1999). Though the Cip/Kip family proteins had been considered general inhibitors of G1 and S phase CDKs, they stabilize the cyclin D – cdk4/6 complex and contribute to its nuclear localization (Blain et al., 1997; Cheng et al., 1999). A number of distinct signals induce the expression of p21Cip1 that inhibits proliferating-cell nuclear antigen (PCNA)-dependent DNA replication as well as cyclin – CDK complexes (Waga et al., 1994). For example, DNA damage activates protein kinase ATM (ataxia telangiectasia mutated) and checkpoint kinases (Chks), which stabilize p53 protein leading to the expression of p21Cip1 (Pearce and Humphrey, 2001). In addition, differentiation signals such as a myogenic factor (MyoD) and nerve growth factor (NGF) also increase p21Cip1 levels (Parker et al., 1995; Yan and Ziff, 1997). Other members of the family such as p27Kip1 are induced in cultured cells by serum starvation, contact inhibition, and elevations in cAMP levels (Kato et al., 1994a; Polyak et al., 1994), and p57Kip2 is

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primarily expressed in terminally differentiated cells. Not surprisingly, antiproliferative signal such as transforming growth factor (TGF) b induces p15INK4b and p27Kip1 (Reynisdottir et al., 1995), although the activity of TGFb is not restricted to its effects on CKIs, since it also decreases the G1/S phosphatase Cdc25A (Fig. 2), required for activation of cdk4/6 (Iavarone and Massague, 1997). As with several other components of the cell-cycle regulator mechanisms, lack of p21Cip1 or p27Kip1 does not cause gross developmental defects. By contrast, p57Kip2-deficient mice show severe developmental defects and most die after birth (Yan et al., 1997). The effects of p27Kip1 will be discussed in more detail below. 2.2. Cyclin dependent kinase inhibitor p27Kip1 A major regulator of cell-cycle progression is the cyclin-dependent kinase inhibitor p27Kip1 that belongs to Cip/Kip family of CKIs. Cell-cycle arrest may be induced by p27Kip1 in a number of ways. This inhibitor interacts with a variety of cyclin –CDK complexes, including cyclin D –cdk4/6 and leads to cell-cycle arrest in G1 (Blain et al., 1997; Cheng et al., 1999). Furthermore p27Kip1 is both a substrate for and an inhibitor of the cyclin E – cdk2 complex. In addition, p27Kip1 inhibits CAK and causes cell-cycle arrest (Kato et al., 1994b). Several signals including TGFb, serum deprivation, ganglioside GM3, cAMP and contact inhibition are known to induce p27Kip1 expression and subsequently negatively regulate cell proliferation (Polyak et al., 1994; Coats et al., 1996; Nakatsuji and Miller, 2001a). The expression of p27Kip1 in the vertebrate CNS is developmentally regulated. In the developing brain it is abundantly expressed and this expression is maintained in postmitotic neurons into adulthood (Lee et al., 1996; Nakayama et al., 1996). The expression of p27Kip1 in the developing CNS appears to have important consequences for glial cell development. For example, the proliferation and differentiation of oligodendrocyte precursors is regulated in part through a p27Kip1 dependent mechanism, and the differentiation of the CG4 cell line into astrocytes is dependent on p27Kip1 (Casaccia Bonnefil et al., 1997; Durand et al., 1997; Tikoo et al., 1997; Nakatsuji and Miller, 2001c). In p27Kip1 knock out mice, like other Cip/Kip gene family knock out mice, there is not a dramatic phenotype, or obvious CNS defects although they exhibit multiple organ hyperplasia and predisposition to tumors (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). The expression of p27Kip1 is primarily regulated at the posttranslational level, by protein degradation via the ubiquitin – proteasome pathway (Pagano et al., 1995), although under some conditions p27Kip1 may also be under transcriptional control (Hengst and Reed, 1996). CDK dependent phosphorylation of threonine residue 187 is required for its degradation by the ubiquitin mediated pathway (Vlach et al., 1997; Nguyen et al., 1999). Ubiquitin mediated protein degradation cascade involves ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3) (Pickart, 2001). The SCF complexes are E3s consisting of Skp1, Cul1, Rbx1 and a member of F-box proteins. The F-box protein Skp2 specifically recognizes p27Kip1 in a phosphorylation dependent manner. A role for Skp2 in control of p27Kip1 levels is evident by the increased p27Kip1 levels in Skp2 knock out mice (Carrano et al., 1999; Sutterluty et al., 1999;

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Nakayama et al., 2000). Accelerated proteolysis of p27Kip1 is frequently seen in cancers (Slingerland and Pagano, 2000). The roles of p27Kip1 in cancer are discussed in detail later in this chapter. 3. Cell-cycle regulation in astrocytes 3.1. Regulation of astrocyte proliferation The vertebrate CNS is composed of neurons, macroglia (astrocytes and oligodendrocytes) and microglia. Neurons arise from precursor cells that actively proliferate during early embryonic periods, while the differentiated progeny is basically nonproliferative in the adult CNS. Recent studies suggest that specific regions of the CNS retain the capacity for active neurogenesis, and certain neuronal precursors retain the capacity to proliferate in the adult (Alvarez Buylla et al., 2000; Gage, 2000). In general, glial cells arise after the majority of neurons are born. While it is unlikely that mature oligodendrocytes can reenter the cell cycle, the adult CNS retains significant populations of proliferative glial precursors capable of giving rise to both astrocytes and oligodendrocytes (Miller et al., 1986; Noble et al., 1990). Much of our understanding of the mechanism of cell-cycle machinery (outlined above) has been elucidated from studies using fibroblastic cells and cell lines. By comparison, information on the role of the cell cycle in regulating CNS development and pathology is poorly understood. During development, classical studies suggest that in any specific region of the rodent CNS, the majority of astrocyte proliferation occurs either during late embryogenesis or early postnatal periods. In rat spinal cord, for example, most neurogenesis is completed by E18, while most astrocyte proliferation occurs in the first postnatal week (Altman and Bayer, 1984). Thus, the majority of astrocyte precursors actively proliferate during a defined developmental period ending during early postnatal development. There is also clearly defined spatial separation in the timing of glial precursor cell proliferation, even within a single region. For example, in the ventral rat spinal cord gliogenesis occurs several days or even weeks earlier than in selected dorsal regions. This temporal and spatial control of glial cell proliferation suggest that a variety of environmental cues regulate local cell proliferation, and these environmental cues guide the activity of the cell-cycle machinery. During the cell cycle, particularly during G1 phase, regulators of the cell cycle are ideal candidates to respond to the extracellular cues. Indeed, the cyclin Ds serve as growth factor sensors, and once a cell has passed the restriction point in G1 it can complete the cell cycle without further mitogenic stimuli (Sherr, 1994). Receptor tyrosine kinases (RTKs) and integrins are important as transducers of stimuli from the extracellular environment for growth factors and extracellular matrix (ECM) proteins, respectively (Huang and Ingber, 1999; Assoian and Schwartz, 2001; Miranti and Brugge, 2002). Several different growth factors regulate astrocyte proliferation. These include epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) (Besnard et al., 1987; Hunter et al., 1993). These ligands drive robust proliferation of cultured astrocytes and are present in the developing and adult CNS. In addition, there are several other potential mitogenic activators for astrocytes such as cytokines and

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chemokines (Giulian and Lachman, 1985; Barna et al., 1990; Yong et al., 1991; Bakhiet et al., 2001). Growth factors, such as EGF and bFGF, bind to RTKs, which lead to the activation downstream of the Ras –Raf– MAPK pathway. These signaling pathways may link directly to the cell cycle. The expression of cyclin D1 mRNA is up-regulated by p21Ras, Raf, and p44/p42 MAPK which are downstream of RTKs (Lavoie et al., 1996; Aktas et al., 1997; Bottazzi et al., 1999). The functional significance of this signaling is demonstrated by the finding that the protein level of cyclin D1 is dramatically increased without significant effect on cyclin E and cyclin A levels in EGF treated astrocytes. EGF treatment concomitantly leads to an increased ratio of hyper-to-hypophosphorylated pRb (Fig. 3) and transiently overrides contact inhibition of proliferation (Nakatsuji and Miller, 2001b). Serum stimulation of rat astrocytes in primary cultures induces the expression of cyclin E followed by the activation of cdk2, whereas the expression of cyclin D1, p27Kip1 and the activities of cdk4, CAK are not affected (Tanaka et al., 1998). In addition to growth factors, cell adhesion to the ECM is required for the transcription of cyclin D1 mRNA. The requirement for anchorage dependent expression of cyclin D1 (Zhu et al., 1996) suggests that growth factors and ECM jointly stimulate the phosphorylation signals that lead to the activation of cyclin D – CDKs –pRb pathway and drive the G1 phase of cell cycle. This combination of signals is probably multifactorial. For example, cross-talk between RTKs and integrins induce activation of the MAPK, extracellular signal-regulated kinase (ERK), and of Ras, leading to activation of the cyclin D pathway. Simultaneously, the same signals promote cyclin E – cdk2 activity by suppressing the inhibitors p21Cip1 and p27Kip1 (Huang and Ingber, 1999; Assoian and Schwartz, 2001; Miranti and Brugge, 2002). The regulation of the astrocyte generation in the CNS may be a reflection of the balance between mitotic and anti-mitotic signals. Growth factors such as EGF and other positive environmental cues promote progression of cell-cycle machinery, whereas anti-mitotic signals such as cell contact or negative environmental cues inhibit cell-cycle progression by regulating the expression of cell-cycle proteins. Less is known about how the proliferation of progenitor cells, including astrocytes, is suppressed during CNS maturation than what actively drives their proliferation during development. The inhibition of cell proliferation can be induced in several ways, including serum deprivation and contact inhibition although the underlying mechanisms in each case appear to be significantly different (Moreton et al., 1995). Density-dependent inhibition of cell proliferation has been widely studied as a model to understand the mechanisms that might actively inhibit cell proliferation.

3.2. Density-dependent inhibition of astrocyte proliferation Many nonneural cell types are actively inhibited from cell proliferation in a densitydependent manner, such that in vitro the formation of a confluent cell monolayer frequently leads to a cessation of cell proliferation. This phenomenon has been termed contact inhibition or density-dependent inhibition of cell growth (Stoker and Rubin, 1967; Blat et al., 1994). Several distinct mechanisms may mediate density-dependent inhibition of cell proliferation. Increased cell number may deplete essential growth factors leading to cell quiescence. Alternatively, cells may release soluble molecules that inhibit

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proliferation. Several candidate molecules have been identified. For example, Swiss 3T3 cells secrete a molecule termed fibroblast growth regulator (FGR-s) (Hsu and Wang, 1986), embryonic mouse fibroblasts secrete an insulin-like growth factor binding protein (IGFBP-3) (Blat et al., 1994), while cultured human mammary cells secrete an inhibitor termed mammastatin (Ervin et al., 1989), all of which are thought to inhibit proliferation of the appropriate cell type. Likewise, astrocyte conditioned medium contains a soluble activity that appears to inhibit astrocyte proliferation (Aloisi et al., 1987). An inhibitor of the EGF receptor (ERI), which mediates the major proliferative pathway of astrocytes, was suggested to be secreted and block cell proliferation. Isolation of this factor indicated that it belonged to the glycosphingolipid family, and it was renamed neurostatin (Nieto and Broderick, 1989; Abad Rodriguez et al., 1998). Not all the inhibitors of astrocyte proliferation are synthesized and secreted by astrocytes. Astrostatine, a small secreted molecule found in neuronal conditioned medium also inhibits astrocyte proliferation (Rogister et al., 1990). Some potential inhibitors of cell proliferation are not particularly cell specific. TGFb is secreted by several cell types and has been proposed to inhibit proliferation of many cell types (Roberts et al., 1985; Hunter et al., 1993). The proliferation inhibitory mechanisms of TGFb have been well studied and include the induction of CKIs p15INK4b and p27Kip1 as a major mechanism to inhibit the cell cycle. This operates in conjunction with the down-regulation of phosphatase Cdc25A that is required for activation of cdks2/4/6 (Fig. 2), and together these two events contribute to the G1/S arrest (Iavarone and Massague, 1997; Bollen and Beullens, 2002). The third mechanism that could account for the density-dependent inhibition of cell proliferation is the direct interaction between membrane associated molecules on adjacent cells resulting in cessation of proliferation. The proliferation of type-1 astrocytes (the conventional type of astrocyte cultures) is inhibited in a density-dependent manner. This inhibition of proliferation is cell type specific. It can be induced by contact inhibited fibroblasts, but not by smooth muscle cells (Nakatsuji and Miller, 1998). In addition, O-2A progenitor cells (a bipotential progenitor cell in vitro for oligodendrocytes and the so-called type-2 astrocytes) continue to proliferate extensively on a contact inhibited monolayer of astrocytes (Noble and Murray, 1984). Thus the density-dependent inhibition of astrocyte proliferation is cell type specific and maybe dependent on homotypic cell – cell interaction. This density-dependent inhibition of proliferation is also induced by contact with fixed contact-inhibited astrocytes, suggesting it is mediated by membrane-associated molecules (Nakatsuji and Miller, 1998). While the signals that mediate this inhibition of proliferation are unknown, it clearly signals to the regulators of the cell cycle. The expression of CKI p27Kip1 is dramatically increased in contact-inhibited confluent astrocytes, compared with cultures in their growth-phase. In contrast, the level of cyclin A is down-regulated, although the levels of cyclin D1 and cyclin E are not significantly changed. The increased p27Kip1 and decreased cyclin A levels are correlated with the decreased ratio of hyper-tohypophosphrylated pRb (ppRb/pRb), shown in Fig. 4 (Nakatsuji and Miller, 2001b). The significance of p27Kip1 and cyclin A in the mechanism of astrocytic contact inhibition was confirmed by utilizing a p27Kip1 expression vector and p27Kip1-deficient mice. Astrocytes in the growing phase stop proliferation, accompanied by down-regulation of cyclin A, when p27Kip1 is overexpressed. Astrocytes derived from p27Kip1-deficient mice

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Fig. 4. Expression levels of p27Kip1 and cyclin A, and the concomitant change of hyper-to-hypophosphorylated pRb (ppRb/pRb ratio) in contact-inhibited astrocytes. Type-1 astrocytes were plated at an initial density that generated confluent cultures between day 0 and day 1 in vitro. The intensity of the bands of each protein by Western blot analysis was quantitated densitometrically. The Y-axis of the left figure is indicated in arbitrary units where expression levels on day 0 was set at 1.0 for p27Kip1 and 10.0 for cyclin A.

show weaker contact inhibition of cell proliferation than wild-type astrocytes (Fig. 5). Reactive astrocyte proliferation following cerebral cortical injury lasts longer in p27Kip1deficient mice than in wild-type mice (Koguchi et al., 2002). Effectors other than p27Kip1 may contribute to the density-dependent inhibition of cell proliferation. An increase of CKI p21Cip1 in confluent astrocytes, resulting in the inhibition of cyclin D-associated phosphorylation of pRb and cyclin A expression has been reported (Teixeira et al., 2000). Further, cyclin D1 and D3 are also expressed in astrocytes and endothelin-1 stimulation induces cyclin D1 through an ERK-dependent and cyclin D3 through a cytoskeletondependent pathways. Taken together, the following model is proposed to explain contact inhibition of astrocytes: certain cell-type specific inhibitory molecule(s) expressed on the surface of contact-inhibited astrocytes can lead to the induction of CKIs p27Kip1 and p21Cip1, resulting in pRb hypophosphorylation, accompanied by the reduction of cyclin A level, and together these cause cell-cycle arrest. The molecules, which are expressed on the cell membrane of astrocytes and cause inhibition of proliferation through modulation of cell-cycle proteins, are currently unknown. Several cell surface molecules have been implicated in contact inhibition including E-cadherin, contactinhibin (Fagotto and Gumbiner, 1996) and connexin43 (Chen et al., 1995). Contactinhibin, a plasma membrane glycoprotein, appears to inhibit the proliferation of some fibroblast cell lines (Oesch et al., 1987; Wieser and Oesch, 1988). The induction of CKI p16INK4a and inactivation of cdk4 appears to be the underlying mechanism of contact inhibition of human diploid lung fibroblasts (Wieser et al., 1999). Recently, contactinhibin was suggested to be involved in contact inhibition of proliferation of human Schwann cells (Casella et al., 2000). A number of previously defined cell adhesion molecules have been proposed to play a role in contact inhibition of a variety of different cell types. The neural cell adhesion molecule (NCAM) has also been proposed to inhibit astroglial proliferation (Sporns et al., 1995). Homophilic binding of

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Fig. 5. An increased number of proliferating astrocytes in high-density cultures of p27Kip1-null cells. Astrocytes derived from wild-type (A, B) and p27Kip1-deficient (C, D) mice were plated at high-density and BrdU was added for the final 24 h. Four days after plating, cells were fixed and labeled with anti-BrdU antibody (B, D). Many cells are still proliferating in high-density cultures of p27Kip1-null cells (D) indicating weaker contact inhibition. (A), (C) are corresponding phase contrast micrographs.

NCAM leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor (Krushel et al., 1998). Similarly, N-cadherin induces contact inhibition of cell growth of chinese hamster ovary (CHO) cells and causes G1 arrest, in which only CKI p27Kip1, but not p21Cip1 expression is elevated (Levenberg et al., 1999). E-cadherin exerts an inhibitory effect on cell growth of fibroblasts at confluence by regulating b-catenin (Soler et al., 1999). Integrin is an important sensor for ECM molecules such as collagen and laminin. In melanoma cells, fibrillar collagen inhibits cellcycle progression, and this inhibition involves adhesion through the a2b1 integrin and upregulation of CKI p27Kip1 (Henriet et al., 2000). In a less well-defined system, binding of the monoclonal antibody AMP1, which recognizes an antigen similar to Target of the Anti-Proliferative Antibody (TAPA-1), has been shown to reduce proliferation of astrocytes (Geisert et al., 1996). This antigen is present on the surface of astrocytes; as it is

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an integral membrane protein, it is unlikely to be sensitive to trypsin treatment, and it is thus a strong candidate for mediation of contact inhibition in this cell type. How cell – cell contact regulates cell proliferation is not clear. Cell– cell contact has been proposed to increase the tyrosine phosphatase activity (Pallen and Tong, 1991; Mansbridge et al., 1992), and decrease EGF and PDGF receptor tyrosine phosphorylation (Sorby and Ostman, 1996). Since EGF and PDGF are mitogens for type-1 astrocytes, down-regulation of the signal pathways utilized by these receptors may contribute to the inhibition of proliferation. Indeed, in contact-inhibited human umbilical vascular endothelial cells (HUVECs), increased phosphatase activity suppressed ERK and phosphatidylinositol 3kinase (PI3-K)/Akt pathways, resulting in cell-cycle arrest by down-regulation of cyclin D1, cyclin E, and cyclin A and up-regulation of p27Kip1 (Suzuki et al., 2000). We propose that during development the initial proliferation of astrocyte precursors in a specific region of the CNS is driven in large part through the release of mitogens from local neuronal cells. As the number and density of astrocytes increase, homotypic contact inhibition between adjacent cells tends to suppress further proliferation. The local release of soluble proliferation inhibiting factors or loss of growth factors may then contribute to limit further division and together totally suppress astrocyte proliferation, leading to a stable balance of astrocyte numbers. In this model, the number of astrocytes would be directly proportional to the number of neurons and other neural cells, which would tend to dilute the effect of homotypic contact inhibition. Such a model is consistent with the finding that penetrating injuries to the CNS frequently promote local astrocyte proliferation (Miller et al., 1986). Astrocytes in the region of the injury are released from contact inhibition, while such inhibition is maintained further from the injury site. Defects in signaling or receptors for contact inhibition of astrocyte proliferation may also underlie formation of astrocytomas that are one of the more common forms of CNS tumors. Other than homotypic interaction between astrocytes, heterotypic neuron – astrocyte interactions may also affect astrocyte proliferation and determine appropriate neuron– astrocyte ratio in the CNS. Under certain conditions, neuronal membrane associated molecule(s) inhibit the proliferation of astrocyte (Hatten, 1987; Hatten and Shelanski, 1988). In contrast, in other conditions, neurons do not inhibit astrocyte proliferation, rather they appear to promote it (Nakatsuji and Miller, 1998). Thus, it seems likely that neurons may regulate astrocyte proliferation both negatively and positively, depending on the conditions. Recently, CD81, a molecule expressed on astrocytes, was reported to be involved in neuron-induced astrocyte cell-cycle exit (Kelic et al., 2001). This neuron– astrocyte interaction is discussed later in more detail.

3.3. Cell cycle in pathological conditions In the mature CNS of vertebrates, glial cells reside ubiquitously in a relative quiescent state, retaining the capacity to proliferate. In response to a variety of stimuli such as trauma, inflammation and neurodegenerative diseases, they can become activated, and reactive gliosis occurs. Reactive gliosis is characterized by hyperplasia, hypertrophy and an increase in the immunostaining for glial fibrillary acidic protein (GFAP) (Yu et al.,

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1993; Wu and Schwartz, 1998). Regulation of the cell cycle seems to be important in some types of reactive gliosis. In in vitro models of gliosis, where confluent monolayers of astrocytes were scratched with pipette tips, p27Kip1 immunoreactivity disappears from the cells lying along the scar after injury and the cells re-enter the cell cycle. Later, an increase in the expression of p27Kip1 is observed, concomitant with the refilling of astrocytes in the denuded area, suggesting that p27Kip1 participates in the cessation of proliferation by contact-dependent inhibition of growth. In in vivo models of gliosis induced by a cortical stab wound, the reactive proliferation of astrocytes of p27Kip1-deficient mice lasts longer than that of wild-type mice (Koguchi et al., 2002). In Mu¨ller glial cells of the adult mouse retina, p27Kip1 interacts with cyclin D3, and retinal injury down-regulates p27Kip1 protein level, allowing Mu¨ller glial cells to re-enter the cell cycle and up-regulate GFAP and subsequently down-regulate cyclin D3 (Dyer and Cepko, 2000). These lines of evidence suggest that p27Kip1 participates in the mechanism of reactive gliosis. Activation of cell cycle-associated proteins in neurodegenerative diseases such as Alzheimer’s disease (AD) is known to occur (Giovanni et al., 1999; Copani et al., 2001). However, most studies have focused on neuronal responses and much less is known in terms of regulation of the glial cell cycle. Amyloid b (Ab) peptide induces c-jun and c-fos expression in astrocytes (Ferrer et al., 1996), and nuclear expression of Ki-67 (a protein involved in cell proliferation) of astrocytes in hippocampus of AD patients has been demonstrated (Nagy and Esiri, 1997). Activated astrocytes surrounding Ab-containing plaques are immunoreactive for hyperphosphorylated pRb and E2F1 (Jordan-Sciutto et al., 2002), suggesting activation of the astrocyte cell cycle in AD. In prion disease such as Creutzfeldt– Jacob disease and Gerstmann – Stra¨ussler – Scheinker syndrome, astrogliosis is one of the pathogenic hallmarks (Prusiner, 1991—see also chapter by Brown and Sassoon). Prion protein (PrP) induces increased progression through the cell cycle to late G1, and it enhanced the level of p53 and phosphorylated ERKs in astrocytes, in which cyclin E is greatly enhanced, while cyclin D and cdk4 are unchanged (Hafiz and Brown, 2000). Astrocytes and oligodendrocytes in progressive multifocal leukoencephalopathy patients express elevated levels of Ki-67, cyclin A and cyclin B (Ariza et al., 1998), suggesting a proliferative response. Thus, the involvement of activation of the astrocyte cell cycle in various pathological conditions is gradually being revealed. The regulatory mechanisms underlying this activation are unclear. The cyclin D1 promoter has been shown to be regulated by NF-kB, c-Fos/c-Jun, SP1 and E2F-1 (Herber et al., 1994; Albanese et al., 1995; Lukas et al., 1996). NF-kB is elevated in several pathological conditions, and it belongs to the Rel family of transcription factors that regulate genes involved in immune and inflammatory responses (Baeuerle and Henkel, 1994; Baldwin, 1996). It seems therefore likely that neural cell proliferation, including astrocytosis, seen in various pathological conditions such as AD, multiple sclerosis, brain injury, in which inflammatory process are involved, is in part related to the NF-kB –cyclin D pathway.

3.4. Brain tumors and cell cycle Tumorigenesis, in terms of cell cycle, may reflect either activation of cell cycle controlling molecules such as cyclins and CDKs that positively regulate proliferation or

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inactivation of molecules such as CKIs, pRb and p53 that negatively regulate proliferation. Gliomas like many other tumors are characterized by a number of molecular changes affecting two critical tumor suppressor pathways: the pRb and p53 pathways. In human malignant gliomas, combined inactivation of p16INK4a, p15INK4b and p14ARF (human, p19ARF in mouse), which are coded on chromosome 9p21, is one of the most consistent genetic alteration (Jen et al., 1994; Kamb et al., 1994; Moulton et al., 1995; Simon et al., 1999; Simon et al., 2001). These inactivations or deletions result in disruption of the pRb and p53 pathways. Both p16INK4a and p15INK4b belong to INK4 family CKIs and inhibit cyclin D – cdk4/6 mediated pRb phosphorylation. Further, overlapping reading frames in the INK4a/ARF gene encode p16INK4a and a distinct tumor suppressor protein p14ARF (p19ARF), such that p16INK4a and p14ARF (p19ARF) share exons 2 and 3 (Quelle et al., 1995). The tumor suppressor p14ARF (p19ARF) promotes degradation of mouse double minute-2 (Mdm2) and thereby stabilizes p53 (Pomerantz et al., 1998; Zhang et al., 1998). Hence the deletion of p14ARF results in the degradation of p53, which leads to the inactivation of both the p21Cip1 – pRb pathway and the p53– apoptosis pathway. Thus, p16INK4a, along with p14ARF, function as a tumor suppressor (Sharpless et al., 2001). There is a correlation between malignancy and homozygous deletion of the p16INK4a gene, since low-grade astrocytomas do not exhibit homozygous deletion (Walker et al., 1995). Not only the deletion of the p16INK4a locus, but also the lack of expression of p16INK4a protein due to hypermethylation of the p16INK4a promoter result in tumorigenicity (Li et al., 1998). Although the cell-cycle inhibitor p27Kip1 plays an important role in the astrocytic cell cycle, mutations in this gene appear less critical in tumor formation. In contrast to the loss of the activities of INK4 family members, alteration of Cip/Kip family genes are rare events in human cancers (Shiohara et al., 1994; Kawamata et al., 1995; Pietenpol et al., 1995; Ponce Castaneda et al., 1995). Loss of p27Kip1 protein expression is, however, frequently seen in many cancers, as reviewed by Slingerland and Pagano (2000). The reduction of p27Kip1 expression correlates with many cancers and can be a prognostic marker of tumor progression (Catzavelos et al., 1997; Newcomb et al., 1999; Sgambato et al., 1999). In astrocytomas, p27Kip1 modulates cell-cycle progression (Naumann et al., 1999), and there is a direct correlation between survival of the patients and the expression level of p27Kip1 (Mizumatsu et al., 1999; Piva et al., 1999; Fuse et al., 2000). Changes in the expression levels of p27Kip1 in tumors, including gliomas, may result from the accelerated proteolysis of the protein (Piva et al., 1999; Slingerland and Pagano, 2000; Strohmaier et al., 2001). Consistent with this hypothesis, p27Kip1 is destabilized in many types of human cancers, and this destabilization correlates with tumor aggressiveness and poor prognosis (Esposito et al., 1997; Loda et al., 1997; Steeg and Abrams, 1997; Piva et al., 1999).

4. Concluding remarks An understanding of the regulation of the cell cycle in CNS glial cells is beginning to emerge. Many of the regulatory effector mechanisms are shared with other cell types, although the control of those effectors through growth factor stimulation and contact

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inhibition of cell proliferation appear to be specific to CNS glia. Elucidation of the mechanisms of glial cell cycle control has profound implications for a wide range of pathological conditions, including neurodegenerative diseases, responses to CNS injury, and the formation of glial tumors. With increased understanding comes the identification of new potential therapeutic targets for the control of the deleterious effects of aberrant glial cell proliferation.

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Role of neuron – glia interactions in nervous system development: highlights on radial glia and astrocytes Fla´via Carvalho Alcantara Gomesp and Stevens Kastrup Rehen Departamento de Anatomia, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Centro de Cieˆncias da Sau´de, Bloco F, Ilha do Funda˜o-21941-590, Rio de Janeiro, RJ, Brazil. p Correspondence address: Tel.: þ55-21-2562-6460; fax: þ55-21-2561-7973 E-mail: [email protected](F.C.A.G.)

Contents 1. 2.

3.

4.

Introduction Neuron – radial glia interactions 2.1. Radial migration 2.2. Role of neuron– radial glia interactions in cell morphogenesis Neuron – astrocyte interactions 3.1. Role of astrocytes in neuronal morphogenesis: implications for neurogenesis, neuronal death and differentiation 3.2. Neuron –astrocyte interactions as mediators of thyroid hormone actions in NS development 3.3. Role of neurons in astrocyte morphogenesis: implications for astrogliogenesis and astrocyte differentiation Concluding remarks

Abbreviations b-Gal: b-galactosidase; BMPs: bone morphogenetic proteins; BrdU: bromodeoxyuridine; CNS: central nervous system; CNTF: ciliary neurotrophic factor; CP: cortical plate; CR: Cajal – Retzius neurons; EGF: epidermal growth factor; EGL: external granular layer; FGF: fibroblast growth factor; GDNF: glial derived neurotrophic factor; GFAP: glial fibrillary acidic protein; GGF: glial growth factor; GFP: green fluorescent protein; IF: intermediate filaments; IGL: internal granular layer; IZ: intermediate zone; ML: molecular layer; NS: nervous system; PDGF: platelet derived growth factor; RGC: retinal ganglion cells; RT –PCR: reverse transcriptase – polymerase chain reaction; SVZ: subventricular Advances in Molecular and Cell Biology, Vol. 31, pages 97–125 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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zone; T3: thyroid hormone; T3CM: conditioned medium derived from T3-treated astrocytes; TGF-b1: transforming growth factor-beta 1; TNF-b: tumor necrosis factor beta; VZ: ventricular zone. Neuron – glia interactions present great complexity and heterogeneity throughout the CNS. They play a crucial role in nervous system morphogenesis from the early stages of neurogenesis and gliogenesis to later stages of establishment of neural connections. A range of epigenetic signals are involved in determination of neural fate potential, lineage specification and cellular differentiation in the CNS and peripheral nervous system. Some of these signals, represented by diffusible factors and cell contact, are derived from neuron-glia interactions. Over the past decade, a considerable effort has been made to elucidate the mechanisms that underlie such interactions. In this chapter, we will focus on neuron-asytrocyte, neuron-radial glia interactions and their implications for cellular morphogenesis.

1. Introduction In 1846, the German pathologist Rudolf Virchow, described for the first time a connective tissue in brain and spinal cord, known as Nervenkitt (nerve glue, Virchow, 1846). To this cellular component, later called neuroglia, was attributed a merely passive, supportive function in the nervous system (NS). At that time, the neuronal doctrine designed neurons as the main functional element of the central nervous system (CNS). As a consequence, research has focused on elucidating cellular and molecular details of neuron biology, whereas glial cells have been regarded as somewhat less important companions to neurons. Today, however, more than a century after their description by Virchow, increasing evidence has accumulated indicating that neurons and glial cells have an intimate and morpho-functional relationship. The vertebrate CNS is composed of two major classes of glial cells: (1) macroglial cells that include astrocytes, oligodendrocytes and embryonic astrocytic precursors known as radial glial cells, and (2) microglial cells. Interactions between neurons and glial components play an important role in several processes of brain development such as neurogenesis (Lim and Alvarez-Buylla, 1999; Song et al., 2002), neuronal proliferation (Gomes et al., 1999b; Oppenheim et al., 2000) and migration (Hatten, 2002; Nadarajah and Parnavelas, 2002), axonal guidance (Garcia-Abreu et al., 1995; Goodman and Tessier-Lavigne, 1997; Shu and Richards, 2001); myelination (Barres, 1997), synapse formation (Na¨gler et al., 2001; Pfrieger and Barres, 1997; Ullian et al., 2001), glial maturation (Gomes et al., 1999a, 2001a; Noda et al., 2000; de Sampaio e Spohr et al., 2002; Yamada and Watanabe, 2002); and neural signaling (Alvarez-Maubecin et al., 2000; Fro´es et al., 1999; Rouach et al., 2000, 2002). One of the most compelling lines of evidence for the role of neuron– glia interactions in the NS development was the identification of the ‘glial cells missing’ (gcm) gene in Drosophila, which functions as a binary switch that turns on glial fate while inhibiting default neuronal fate (Hosoya et al., 1995; Jones et al., 1995). Its mutation causes presumptive glial cells to differentiate into neurons, whereas its ectopic expression forces virtually all CNS cells to become glial cells. Analysis of

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gcm mutants revealed, in addition to a decreased number of glial cells, a series of defects in several axonal tracts. Such defects were attributed mainly to the loss of glial signals important for axonal growth and neuronal proliferation and differentiation. Although a full elimination of glial cells has not been performed in mammals in vivo yet, the recent observation that inducible ablation of astrocytes is associated with a dramatic degeneration of granular neurons in rodent cerebellum is strongly in favor of the obligatory role of astrocyte –neuron interaction in vertebrate NS development (Cui et al., 2001). In addition, cell ablation models reported that in the absence of Cajal –Retzius (CR) neurons, the development of radial glia from cerebral cortex is severely impaired (Super et al., 2000; Xie et al., 2002). Over the past decade, a considerable effort has been made to elucidate the mechanisms that underlie neuron –glia interactions. Although interactions between neurons and oligodendrocytes and/or microglia contribute to NS morphogenesis, it will not be the scope of the present chapter to discuss their implications. In this chapter, we will focus on advances in the understanding of neuron – astrocytic cell interactions during NS development. Firstly, we discuss neuron – radial glia interactions and their implications in neuronal migration and cell differentiation. Briefly, we present some advances concerning the emerging scenario of radial glia and astrocytes as putative neural stem cells. Secondly, we summarize progress in characterizing neuron – astrocyte interactions and their role in several steps of brain development including neurogenesis, neuronal death, differentiation and astrocytic differentiation. 2. Neuron – radial glia interactions 2.1. Radial migration Introduction: radial glia and neuronal migration After exiting the cell cycle, neural cells travel from the germinal zones of the developing CNS to their final positions in the brain. This journey can occur in two different ways: by tangential and radial migration. Tangential migration is termed neuronophilic and is not confined to regional cerebral cortical boundaries. Radial migration, in contrast, is mainly gliophilic and depends on the interactions between migrating neurons and radial glial cells. This latter form of neuronal migration is the most prominent, being characterized by a radial-glia-oriented movement of early-born neurons from the ventricular zone (VZ) to the upper layers of the developing cerebral cortex (see Nadarajah and Parnavelas, 2002, for review). The cells that direct radial migration, radial glia, were first described by Magini, Ramon y Cajal, and others. Interestingly, Magini not only characterized radial glia but also described the occurrence of developing nerve cells along the radial filaments (Bentivoglio and Mazzarello, 1999). However, it was Rakic who first introduced the term ‘radial glia’ in a report examining the fetal primate neocortex using Golgi impregnation and electron microscopy (Rakic, 1972). This study strongly suggested that radial fibers provide a transient scaffold in the developing cerebral wall that promotes neuronal migration.

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Radial glia, as neuroepithelial cells, are located near the ventricular surface and have a characteristic bipolar shape and a long process that guides the migration of young neurons during development (Nadarajah and Parnavelas, 2002). Like astrocytes, these cells express the cytoskeleton proteins, vimentin and RC2 (Misson et al., 1988) and, in the primate cortex, they also express the glial fibrillary acidic protein (GFAP) (Levitt and Rakic, 1980). Nonetheless, the glial nature of radial fibers was controversial (Morest, 1970) until the advent of immunocytochemistry, which confirmed that radial cells are in fact glia (Levitt and Rakic, 1980; Levitt et al., 1981). Actually, the prevailing view of the identity of radial glia is once again under revision as recent studies from several laboratories seem to demonstrate that radial glia are neural stem cells.

Evidence of radial glia as neuronal precursors The development of neuronal and glial cells in the mammalian cerebral cortex has been the subject of investigation for over 100 years. His was the first to recognize the significance of cell migration in development of the NS and, in 1887, to describe the divergence of progenitor cells into the neuronal and glial lineages. Germinal cells visible as mitotic figures lining the lumen of the neural tube would give rise to neurons, while the syncytium of spongioblasts would produce glial cells (His, 1887 in Jacobson, 1991). The traditional view of radial glial cells was as specialized non-neuronal cells, which served solely as migratory scaffolding and disappeared early in development by transforming into astrocytes (Alvarez-Buylla et al., 2002; Parnavelas and Nadarajah, 2001). Recent experimental evidence from several laboratories suggests a surprising new role for radial glial cells as stem cells (Fig. 1; see Gotz et al., 2002 for review). Radial glia express nestin, an intermediate filament protein specifically expressed in neuroepithelial stem cells (Lendahl et al., 1990). They also divide rapidly and undergo interkinetic nuclear migration (Misson et al., 1988). Alvarez-Buylla et al. (1990) first suggested that mitotically active radial glia may give rise to neurons, but the idea failed to gain credence over the prevailing assumption that radial glia were the exclusive precursors of glial cells (Levitt et al., 1981; Alvarez-Buylla et al., 2002 for review). This assumption, however, is now coming under revision, as recent reports have demonstrated that radial glial cells can generate neurons (Hartfuss et al., 2001; Malatesta et al., 2000; Miyata et al., 2001; Noctor et al., 2002). Noctor and colleagues, for example, have investigated whether radial glia are neuronal precursors in vivo. After injecting a green fluorescent protein (GFP)-transducing retrovirus into the lateral ventricules of embryonic rats, the authors discovered clones that included mitotic radial glia and postmitotic neurons arrayed along the glial fiber (Noctor et al., 2001). In addition, radial glia have been shown to generate both neurons and glia in vitro (Malatesta et al., 2000). Radial glia are not the only cells with glial phenotypes that have recently been shown to possess neurogenic potential. Studies of the adult subventricular zone (SVZ) and hippocampal dentate gyrus indicate that astrocytic cells in these regions are neuronal precursors (Campbell and Gotz, 2002; Doetsch et al., 1999; Seri et al., 2001). Further, astrocyte monolayers derived from several regions in the perinatal mouse brain have been

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Fig. 1. Neuronal precursor cells express a mitotic radial glial marker in the VZ of the developing cerebral cortex. Cross-section of a 14-day embryonic mouse cerebral cortex demonstrates that the majority of the mitotic cells identified by the pattern of phosphorylated histone H3 labeling (A) express phospho-vimentin immunostaining (B). Overlay of the two channels (C) indicates that dividing cells in the brain are radial glia. VZ: ventricular zone, IZ: intermediate zone, CP: cortical plate. From M. Kingsbury, S.K. Rehen and J. Chun, unpublished results.

found to generate neurospheres in vitro, which can subsequently differentiate into neurons and astrocytes (Laywell et al., 2000). Thus, the prevailing view of radial glia and astrocytes is in flux: they are no longer seen as merely supporting cells, but rather as cells that give birth to new neurons at the same time as they direct their migration and support their function, respectively (Fig. 2).

Mechanisms and regulation of radial migration Neuronal translocation along radial glial fibers is an interactive, three-step process: first, leading edge extension, then nuclear translocation or nucleokinesis, and finally retraction of the trailing process. Changes in radial glial cell surface properties are thought to signal neurons to cease migration and begin their differentiation at the appropriate location in the developing cortical plate (Anton et al., 1996, 1997). A number of molecules are implicated in the control of young neuron migration along radial glial processes: astrotactin is an adhesion molecule that provides a ligand for neuronal binding to the apposed radial glial fiber. Targeted deletion of astrotactin in mice leads to a slowing of migration in the cerebellum and other defects, including dendritic abnormalities in Purkinje cells (Adams et al., 2002; Zheng et al., 1996). Neuregulins comprise a diverse group of membrane-attached and secreted peptide growth factors that are of central importance to NS development and function (Lemke, 1996). The neuregulins bind to tyrosine kinase receptors ErbB2, ErbB3 and ErbB4 and play a role in neuron – glial interactions during migration. Glial growth factor (GGF), the first isoform of neuregulin shown to participate in neuron – glia interactions, is expressed

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Fig. 2. Radial glial (1) cells are responsible for the production of both newborn neurons (2) and astrocytes (3) that can ultimately become differentiated neurons (4). Radial glia is also responsible for the guidance of its daughter neurons (curved arrow) to their destinations in the developing cortex and as astrocytes, to control neural cell survival (T) and proliferation (dark arrow) in the adult brain.

by migrating cortical neurons and promotes their migration along radial glial fibers (Anton et al., 1997). The current model of neuronal translocation along glial fibers provides the theoretical framework for a series of studies on human cortical neuronal migration malformations, likely those caused by disruptions of the cytoskeleton (for review see Hatten, 2002). Two microtubule-associated protein genes, lis1 and doublecortin (Gleeson et al., 1998) are mutated in lissencephaly, a condition characterized by abnormally shallow cortical sulci and thickening of the gray matter (Cardoso et al., 2002; Crome, 1956). Targeted deletions of lis1 in mice result in embryonic lethality but heterozygotes survive and show slowed cell migration (Hirotsune et al., 1998). The laminar patterning of the cerebral cortex is established when neurons finish their migration by signals from the Reelin pathway. Reelin is an extracellular matrix (ECM) protein, which plays a key role in the organization of architectonic patterns, particularly in the cerebral cortex (Tissir et al., 2002). The recent discovery that the cadherin-related neuronal (CNR) gene family encodes a diverse array of long-sought Reelin receptors (Senzaki et al., 1999) suggests a mechanism for the encoding of a broad range of migratory instructions by Reelin. Other known components of the Reelin pathway include two members of the lipoprotein receptor family: apolipoprotein E receptor 2 (ApoER2) and the VLDL receptor (VLDLR) (Trommsdorff et al., 1999). The intracellular adaptor disabled (mDab1), and integrin a3b1 (Dulabon et al., 2000) also appear to participate in Reelin signaling. The reeler mutation in mice leads to cortical inversion (Caviness, 1976), while in humans reelin deficiency causes a unique phenotype of lissencephaly. Though its

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cellular actions are unknown, reelin may regulate cell adhesion between radial glia and migratory neurons (Hoffarth et al., 1995) and act as a detachment signal, but not a stop or guidance cue (Hack et al., 2002). The formation of the final laminar pattern also requires the integrity of the external limiting membrane, defects of which lead to over-migration of neurons and to human type 2 lissencephaly (Gupta et al., 2002; Lambert de Rouvroit and Goffinet, 2001). Recently, a glycolipid, more specifically the 9-O-acetyl GD3 ganglioside was also shown to have a role in the migration of granule cells in the cerebellum (Santiago et al., 2001).

2.2. Role of neuron – radial glia interactions in cell morphogenesis Besides its well-described function on neuronal migration and corticogenesis, neuron– radial glia interaction plays a pivotal role on radial glia and neuronal development. It is a likely speculation that during the migration route, the intimate contact between migrating neurons and radial cells creates a special environment, which influences cell phenotype. The role of radial glia on neuronal morphogenesis has been investigated in the developing retina where radial cells were demonstrated to be instructive for neuritogenesis (Bauch et al., 1998). Ganglion cells of the chicken retina extend axons exclusively into the inner retina, whereas their dendrites grow into the outer retina. Bauch and collaborators demonstrated that ganglion cell polarity is greatly influenced by different radial glia compartments. However, whereas glial somata induced dendrite formation, neurons cultivated onto glial end feet developed mainly axons (Bauch et al., 1998). Although neuron– radial glia interactions are mainly mediated by contact as previously discussed, a few examples of soluble factors have been reported as mediators of these interactions. Hunter and Hatten have shown that the expression of radial glial cell identity in mammalian forebrain is determined by the availability of diffusible inducing signals (Hunter and Hatten, 1995). Although the factor has not been completely characterized, biochemical studies have indicated that it is different from the neural growth regulators already known. These signals act to transform mature astrocytes into a radial glia phenotype, suggesting that transformation from radial glial cell to astrocyte is reversible. These data provide support for the role of neuronal extrinsic signals in determining and maintaining a radial glial identity and suggest that the transformation of radial glia into astrocytes is regulated by the availability of neuronal signals rather than by changes in cell potential (Hunter and Hatten, 1995). Similar data were obtained from Sotelo’s group, who demonstrated that Bergmann glia differentiation was greatly influenced by Purkinje cells (Sotelo et al., 1994). More recently, by using grafting and coculture systems, they demonstrated that transplantation of embryonic CR neurons into adult cerebella induces a transient rejuvenation of host Bergmann glia into a radial-glia phenotype (Soriano et al., 1997). This process was also shown to be mediated by diffusible factors. Elucidative data on this subject have come recently from ablation assays. Degeneration of CR cells in newborn mice dramatically decreased the number of nestin-positive radial glial apical processes and increased the number of GFAP-astrocytes (Super et al., 2000). These

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findings support an essential role for CR cells in regulating the phenotype of radial glia and the radial glia – astrocyte transformation, a key step for neuronal migration. GGF/neuregulin signaling has also been implicated in neuron –radial glia fiber interactions. Rakic’s group has shown that GGF is expressed by migrating cortical neurons and promotes their migration along radial glial fibers (Anton et al., 1997). Concurrently, GGF also promotes the maintenance and elongation of radial glial cells. In the absence of GGF signaling, radial glial development is abnormal. In erbB2deficient embryos, radial glial fibers formed abnormal end feet, which were minimally arborized. The ability of GGF to influence both neuronal migration and radial glial development in a mutually dependent manner suggests that it functions as a soluble mediator between migrating neurons and radial glial cells in the developing cerebral cortex. 3. Neuron – astrocyte interactions 3.1. Role of astrocytes in neuronal morphogenesis: implications for neurogenesis, neuronal death and differentiation Radial glia have been studied predominantly in the developing brain (Alvarez-Buylla et al., 1990), and although they can persist throughout life in most vertebrates, evidence indicates that most radial glia disappear—transforming into astrocytes (Rakic, 1995)—by the end of neuronal migration. The cellular and molecular events that contribute to this transformation are not known, but the morphological change appears to coincide with the loss of vimentin, RC2 and nestin expression and the acquisition of GFAP immunoreactivity (Alvarez-Buylla et al., 2002; Gotz et al., 2002; Voigt, 1989). In the mature mammalian brain, astrocytes constitute almost one half of the total cell number, providing structural, metabolic and trophic support for neurons. Neuron – astrocyte interactions play a pivotal role in several steps of brain development such as neuronal survival, proliferation and differentiation. Astrocytes represent a potent source for most neurotrophic factors involved in these processes such as FGF, TGF and EGF families (Banker, 1980; Connor and Dragunow, 1998; Gomes et al., 2001a). Members of the EGF and FGF families are potent mitogens for multipotential neural progenitors and are profoundly implicated in several aspects of neurogenesis (Cameron et al., 1998; Kane et al., 1996; Kuhn et al., 1997). Members of the TGF-b family such as TGF-b1 itself and glial cell line-derived neurotrophic factor (GDNF) have been reported to have a broad spectrum of action during NS development (for review see Unsicker and Strelau, 2000). Both are known to favor neuronal survival in vitro as well as in vivo (Bruno et al., 1998; Choi-Lundberg et al., 1997; Tomoda et al., 1996; Oppenheim et al., 2000). Remarkably, astrocytes may also play a negative role in the maintenance of neuronal populations within the brain. Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has been previously associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS) (see Cleveland and Rothstein, 2001 for review). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase

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(iNOS), suggesting that astroglia may contribute to motor neuron degeneration in ALS (Almer et al., 1999). In fact, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitritedependent mechanisms (Cassina et al., 2002). Moreover, in Alzheimer’s disease (AD) the neurodegenerative changes that are elicited by the accumulation of b-amyloid peptides seems not only to damage neurons directly but also to activate astrocytes (and microglia) to produce inflammatory mediators (Perini et al., 2002—see also the chapter by Barger). The mechanisms that regulate the fate specification of neural stem cells are poorly understood (Gage, 2000). However, the role of astrocytes in promoting adult neurogenesis has been studied in both the SVZ (Lim and Alvarez-Buylla, 1999) and the dentate gyrus (Seri et al., 2001). In the SVZ, migratory neuroblasts and putative precursors are in intimate contact with astrocytes (Doetsch et al., 1997). Culturing dissociated postnatal or adult SVZ cells on astrocyte monolayers supports extensive neurogenesis similar to that observed in vivo. In this case, a direct cell –cell contact between astrocytes and SVZ neuronal precursors seems to be necessary for the production of neuroblasts (Lim and Alvarez-Buylla, 1999). In the hippocampus, astrocytes also actively regulate adult neurogenesis either by instructing neuronal fate commitment or by promoting proliferation of adult neural stem cells (Song et al., 2002). Indeed, the effects of astrocytes seem to be regionally specified: hippocampal or cerebral cortical astrocytes retain the potential to promote neurogenesis, but astrocytes from adult spinal cord do not. These results present an intriguing possibility that the capability for adult neurogenesis might, in part, be due to certain signals provided by regionally specified astrocytes in the adult CNS (Song et al., 2002). During CNS development, neurons must extend projections in order to establish their connections. Growing axons navigate toward their targets in response to a variety of guidance signals in their surrounding environment. These cues include diffusible attractive or repellent molecules secreted by the intermediate or final cellular targets of the axons. Glial cells have been exhaustively reported as a source of asymmetric cues during axonal navigation (Goodman and Tessier-Lavigne, 1997). Commissural and decussation formation in the NS, such as the optic chiasm and the floor plate of the NS, is dependent on the interaction of growing axons and resident glia of these regions. Several adhesion and soluble molecules involved in such interactions have already been reported, such as the laminin-related molecule netrin-1, proteoglycans, ephrins and several ECM proteins. Recently, a new class of proteins, the Slit proteins, have emerged as a pivotal component controlling the guidance of axonal growth cones and the directional migration of neuronal precursors (Piper and Little, 2003). Shu and Richards recently demonstrated that the Slit proteins are implicated in glia-mediated cortical axon guidance during the development of the corpus callosum (Shu and Richards, 2001), when cortical axons from one cerebral hemisphere cross the midline to reach their targets in the opposite cortical hemisphere. A population of midline glial cells act as intermediate guideposts for callosal axons, which avoid this glial region. The authors demonstrated that the chemo-repellent activity of glial cells is due to expression of slit-2 by these cells, whereas cortical axons express their receptors (Shu and Richards, 2001).

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In agreement with in vivo studies, several lines of evidence obtained in vitro have implicated astrocytic soluble factors in neuronal morphogenesis. Neuronal polarity, which is crucial for neural circuits, is modulated to a great extent by glial cells. Sympathetic neurons maintained in vitro in the presence of astrocytes extend axons and dendrites, while in the absence of astrocytes they extend solely axons. Such astrocyte-induced dendritic growth has been reported to be mediated by bone morphogenetic proteins, a subclass of the TGF-b superfamily involved in many aspects of neuronal maturation (Lein et al., 2002; Prochiantz, 1995). Additional evidence for the influence of astrocytes on neuronal morphogenesis has been provided by the studies of Garcia-Abreu and colleagues (Garcia-Abreu et al., 1995, 2000), who demonstrated that astrocytes derived from distinct regions of the midbrain can differently modulate neurite extension. Astrocytes derived from the lateral region of the mesencephalon are permissive to neurite outgrowth, whereas those derived from the midline proved to be restrictive to neuritogenesis (Fig. 3; Garcia-Abreu et al., 1995). These astrocyte populations exhibited great heterogeneity in the content of soluble proteoglycans secreted in the medium, which may account for the differences observed in their ability to support neurite outgrowth (Garcia-Abreu et al., 2000; Mendes et al., 2003). Taken together, the data presented above provide evidence that soluble factors released from astrocytes play an important role in several steps of neuronal morphogenesis from the early events of neuronal precursor proliferation until later periods of neuronal differentiation and establishment of neural circuits. A greater understanding of the interaction between neurons and astrocytes may help advance the therapeutic use of glial cells for both the regulation of neural stem cell proliferation and the modulation of neuronal cell death.

Fig. 3. Heterogeneity of neuron–glia interaction patterns in the mesencephalon in vitro. Mesencephalic neurons cultivated onto astrocytes derived from distinct regions of the mesecephalon: (A) lateral glia, and (B) medial glia. Neurons are marked by b-Tubulin III immunostaining. Note that lateral glia are more permissive for neuritogenesis. Arrows (A) and arrowheads (B) shows neurite length (kindly provided by J. Garcia-Abreu).

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3.2. Neuron –astrocyte interactions as mediators of thyroid hormone actions in NS development Thyroid hormone (3,5,30 -triiodothyronine, T3) is essential for development of the vertebrate NS, influencing diverse processes of brain development such as neuronal migration, neurite outgrowth, synapse formation, myelination and glial cell differentiation (Bernal and Nunez, 1995; Forrest et al., 2002; Gomes et al., 2001b; Lima et al., 2001). The finding that thyroid hormone receptors are present in the developing brain suggests that it exerts its effects by regulating the expression of specific genes (THresponsive genes). (For reviews see Gomes et al., 2001b; Koibuchi and Chin, 2000; Ko¨nig and Moura Neto, 2002.) However, the molecular mechanism of hormone action is still controversial (Anderson et al., 1997; Koibuchi et al., 1999; Potter et al., 2001). Evidence has accumulated over the last 10 years indicating that such endocrine regulation of brain development might be the result of T3-dependent modulation of secretion of several growth factors such as NT3 (neurotrophin 3), NGF (nerve growth factor), IGF (insulin-like growth factor), BDNF (brain derived neurotrophic factor) (Beck et al., 1993; Figueiredo et al., 1993) and FGF (fibroblast growth factor) (Trentin et al., 2001). This proposition is highlighted by the fact that injection of NT-3 or BDNF results in some rescue of cerebellar development in hypothyroid animals (Neveu and Arenas, 1996). The fact that astrocytes have been considered for a long time as a potential source of growth factors makes them a candidate for mediators of T3 action on NS. Several studies have shown that thyroid hormone has direct effects on astroglia in vivo such as regulation of astrocyte number (Clos and Legrand, 1973) and the maturation of Bergmann cells in the rat cerebellum (Clos et al., 1980). A great step towards the understanding of T3-glia-mediated action on neural cells has been provided by the work of Moura Neto and collaborators (for review see Ko¨nig and Moura Neto, 2002). They have demonstrated that treatment of cultured astrocytes by thyroid hormone elicits distinct responses, depending on the origin of the cells. Protoplasmic astrocytes derived from cerebral cortex are morphologically transformed into process-bearing cells upon hormone treatment (Trentin et al., 1995, 1998; Lima et al., 1997). On the other hand, thyroid hormone induces cerebellar astrocyte proliferation in vitro, instead of its morphological differentiation (Trentin et al., 1995; Trentin and Moura Neto, 1995; Lima et al., 1997, 1998). Such effects seems to be mediated by the growth factors secreted by the hormone-treated cells, suggesting an indirect autocrine mechanism underlying the T3 mode of action (for review see Gomes et al., 2001b; Ko¨nig and Moura Neto, 2002). In order to gain insights into T3 effects on the CNS we have focused on the ontogenesis of cerebellum, which is one of the most dramatically affected brain structures in hypothyroidism (Nicholson and Altman, 1972). Most of the granular cells of the cerebellum arise from the external granular cell layer (EGL). Postnatally, these cells migrate from the premigratory zone of the EGL to the internal granular layer (IGL) leaving their axons behind to produce the molecular layer (ML). These events are accompanied by a progressive morphological differentiation of Purkinje cells characterized by perisomatic extensions and dendritic trees (Anderson, 2001; Komuro et al., 2001;

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Miale and Sidman, 1961). Although cerebellar histogenesis is well studied, the molecular mechanisms that control proliferation and differentiation of granular cells are still unknown. These processes have been shown to undergo dramatic modulation by thyroid hormone (Anderson, 2001; Cayrou et al., 2002; Nicholson and Altman, 1972). Besides a series of abnormalities found in the cerebellar cortex, hypothyroidism causes a decrease in EGL proliferation rate, increased neuronal death in the IGL, impaired migration of granular cells, and a deficiency in the elaboration of Purkinje cell dendritic trees, spines and synapses (Nicholson and Altman, 1972). We have recently reported that TNF-b and EGF secreted by cultured cerebellar astrocytes in response to T3 treatment can modulate EGL-neuronal proliferation (Gomes et al., 1999b). Culture of 19-day embryonic cerebellar rat neurons on conditioned medium derived from T3-treated astrocytes (T3CM) increased the neuronal population by 60 – 80% (Fig. 4). Bromodeoxyuridine (BrdU) proliferation assays revealed that this increase was mainly due to neuronal precursor proliferation. The proliferation index was three times higher for neuronal cells maintained in T3CM than in control medium and neuronal survival was not affected by EGF suggesting a prior function for TNF-b and EGF in glia-mediated neuronal proliferation (Gomes et al., 1999b; Martinez and Gomes, 2002). The early germinative zone of the mouse EGL (E15-19) lacks T3 receptors, which will be expressed later in development in the premigratory zone of post-mitotic EGL and IGL (Bradley et al., 1992). These observations highlight the importance of a T3action mediator (possibly glial cells) at least in the early events of cerebellum ontogenesis. Besides modulating neuronal precursor proliferation, thyroid hormone (via astrocytes or not) is also involved in ECM deposition and neuronal migration. The ECM helps to regulate cell migration, survival, differentiation and axonal pathfinding in the NS (Reichardt and Tomaselli, 1991). It has been suggested that astrocytes produce most of the ECM components in the CNS including fibronectin and laminin (Liesi et al., 1983, 1986). In addition, it has been demonstrated that thyroxine (T4) regulates the pattern of integrin distribution in astrocytes by modulating the organization of microfilaments (Farwell et al., 1995) and controls the extracellular deposition and organization of laminin on the surface of astrocytes (Farwell and Dubord-Tomasetti, 1999). Moura Neto’s group has shown that T3-induced cerebellar astrocyte proliferation is accompanied by alterations in the GFAP filaments and fibronectin (Trentin and Moura Neto, 1995). More recently, we provided new insights into T3 modulation of laminin and fibronectin expression (Martinez and Gomes, 2002). Using a neuron –astrocyte coculture model, we have investigated the effects of T3-treated astrocytes on cerebellar neuronal differentiation in vitro. Neurons plated onto T3-treated astrocytes presented a 40– 60% increase of total neurite length and an increment in the number of neurites (Fig. 5). Treatment of astrocytes with EGF yielded similar results, suggesting that this growth factor might mediate T3-induced neuritogenesis. EGF and T3 treatment increased fibronectin and laminin expression by astrocytes, suggesting that astrocyte-induced neurite permissiveness elicited by these treatments is mostly due to the modulation of ECM components (Fig. 5).

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Fig. 4. Soluble factors secreted by astrocytes treated by thyroid hormone induce neuronal proliferation. Conditioned medium derived from thyroid hormone-treated astrocytes (T3CM) increases neuronal population (A) and incorporation of the proliferation marker BrdU (B). Cerebellar neurons were cultivated onto T3CM and CCM (control conditioned medium derived from nontreated astrocytes) and immunostained for the neuronal marker cytoskeleton protein b-tubulin III and BrdU (C,D). Note the presence of dividing neurons double-labeled for both markers (arrow) and nondividing neurons (*) (modified from Gomes et al., 1999b).

Our data on neuronal proliferation and axonal growth together provide evidence that EGF secreted by astrocytes in response to T3 performs a dual role in cerebellar ontogenesis (Fig. 5): acting directly on neurons, EGF promotes proliferation of granular cell precursors; and indirectly, EGF increases neuronal morphological differentiation, by modulating the content of two astrocytic ECM proteins, laminin and fibronectin. Thus, our work gives glial cells a novel role as mediators of the endocrine-regulated cerebellar development and describes an additional role for EGF on brain morphogenesis.

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Fig. 5. (A) Astrocytes treated by thyroid hormone or EGF increase cerebellar neuritogenesis by modulating production of ECM proteins. Thyroid hormone induces astrocyte secretion of EGF. Astrocytic EGF modulates laminin and fibronectin secretion thus strongly enhancing astrocyte permissiveness to neurite outgrowth. (B) Cerebellar neurons from embryonic rats cultivated onto control and EGF-treated astrocyte monolayers. Cells were immunostained using a monoclonal antibody to the neuronal marker cytoskeleton protein, b-tubulin III (modified from Martinez and Gomes, 2002). C, D, and E show fibronectin immunostaining. Note that T3 and EGF greatly potentiated ECM production by astrocytes.

3.3. Role of neurons in astrocyte morphogenesis: implications for astrogliogenesis and astrocyte differentiation A central aim in developmental biology is to elucidate the mechanisms that specify the form of particular cell types during animal development. Although the developmental genetic program is decisive, it does not account for the extraordinary cell-type specific patterns present in the CNS. Within this context, neuron – glia interactions are of fundamental importance in determining cell identity. Only very recently, however, outstanding progress has been made to understand how glial cell fate becomes specified, and what the role is of cell –cell interactions in this process. Further, little is known about

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the molecular mechanisms controlling the differentiation of astrocytes, which is hindered by the considerable diversity and heterogeneity of this cell population. While there is compelling evidence of the effects of astrocytes on neurons, the effects of the neuronal environment on glial cells is still far from being well understood. Most of our knowledge concerning neuron– glial interactions is based on the effects of glial cells on neuronal morphogenesis. However, evidence has accumulated in recent years pointing to a mutual influence between these two cell types. A great part of our understanding concerning effects of neurons on gliogenesis was provided by studies of axon –oligodendrocyte and Schwann cell interactions (for a review, see Barres, 1997). At present, it is widely recognized that the survival and proliferation of oligodendrocytes and Schwann cells are highly dependent on neuronal contact and neuronal soluble factors (Barres, 1997). A variety of newly identified soluble signals, including ciliary neurotrophic factor (CNTF), BMPs and neuregulin-1, were shown to direct multipotential stem cells to become glial cells (McKay, 1997; Morrison et al., 1999). In addition, contact-mediated signaling such as the Notch pathway has been found to induce differentiation of glial cells, including Schwann cells, retinal Mu¨ller cells, and radial glial cells in the cerebral cortex (for a review see Wang and Barres, 2000). Similar neuronal soluble and contact factors, however, are only now beginning to be identified as important cues for astrogliogenesis. Notch is an ancient protein used by vertebrate and invertebrate organisms in controlling multiple aspects of development (Artavanis-Takonas et al., 1995, 1999). During mammalian cerebral cortical development, Notch signaling is implicated early in the prevention of precocious neurogenesis and preservation of precursor pools (Artavanis-Takonas et al., 1995, 1999). Recently, by introducing a retroviral vector containing an activated form of Notch1 (NIC) into the mouse forebrain, Gaiano et al. (2000) demonstrated that the NIC-infected cells became radial glial cells. Gaiano data, together with the observation of Notch1 expression in endogenous radial glia, raise the questions as to how Notch1 is normally activated in this cell type. A tentative explanation given by the authors is that newly generated neurons, expressing high levels of a Notch ligand, activate Notch1 in radial glia during migration along the radial processes. This activation would allow glia to respond to environmental cues, such as GGF or others, which might then maintain glial morphology and gene expression. Studies of Tsai and McKay (2000) found that cell contact helps to regulate the fate choice of cortical stem cells strongly favoring astrocyte generation. Although these investigators did not identify the contact-mediated signal, Notch signaling is an obvious candidate for mediating such a process. Although Notch signaling clearly enhances astrocyte differentiation, its mechanism of action is not clear. One possibility is that by inhibiting neurogenesis, Notch preserves progenitor pools so that the remaining cells can respond to other astrogliogenic cues and subsequently differentiate into astrocytes. Alternatively, Notch might instructively trigger an astrogliogenic pathway by directly activating the transcription of astroglia-specific genes. This idea is in concert with the recent finding that the Notch pathway directly activates the GFAP gene (Ge et al., 2002). Since neurogenesis precedes gliogenesis during NS development, this suggests

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that a molecule expressed by neurons or neuronal precursors might modulate astrogliogenesis (for reviews see Luskin, 1998; Temple, 2001). Whereas knowledge of neuronal effects on astrogliogenesis still suffers from lack of conclusive data, effects of neurons on astrocyte differentiation have proved to be an expanding field over the past decade. The first evidence of neuronal influence on astrocyte morphogenesis was provided by the work of Hatten and collaborators in the 1980s. By establishing a model system to study neuron –glia interactions in vitro, this group clearly demonstrated that granular neurons regulate proliferation and differentiation of cerebellar astroglia. In the absence of granule neurons, cerebellar astroglia assume a flat morphology and proliferate rapidly (Hatten, 1985). Addition of neurons to the cultures rapidly arrested glial cell proliferation and induced its morphological differentiation into profiles resembling the cerebellar astroglia seen in vivo. Although the decrease in proliferation and induction of differentiation occurred simultaneously, those processes were mediated by distinct mechanisms. While neuronal membranes mediate inhibition of glial cell division, glial morphological differentiation requires living neurons (Hatten, 1985, 1987). Several proteins found on neuronal membranes were associated with cell contact regulation of astrocyte functions; however, at that time, the question remained unanswered whether neurons were able to secrete a growth factor that could modulate astrocyte development. Today, however, the list of soluble factors secreted by neurons is being extended, as we will discuss soon. The finding that astrocytes exhibit receptors for a series of soluble factors, previously solely attributed to neurons, enhances the possibility that their development could be modulated by neuronal signals. These signals might include soluble growth factors and neurotransmitter substances. In the following section we will discuss some of the emerging literature on the effect of neuronal soluble factors on astrocyte differentiation. Effect of neuronal growth factors on astrocyte differentiation Some of our knowledge about effects of neuron-derived growth factors on astrocyte biology came from studies of the visual system. The vertebrate eye provides an interesting system to study cell – cell communication. During development, cells from several different sources come together in a coordinated fashion to form the final structure of the eye (Karshing et al., 1986). The retina itself is composed of cells of different origins, where cell numbers must presumably be matched to one another by cell –cell interactions. Most of the cells of the neural retina are generated from multipotential neuroepithelial precursors that reside near the outer surface of the retina. In contrast, retinal astrocytes originate from the optic stalk and migrate across the inner surface of the retina. The migrating astrocytes form a glial network that spreads radially in close association with the retinal ganglion cell (RGC) axons (see also the chapter by Stone and Valter). This invasion by astrocytes has been reported to be mediated by secretion of platelet-derived growth factor (PDGF) by RGCs. This factor is expressed and secreted by RGCs, while PDGF receptor alpha (PDGFRa), an isoform of the tyrosine kinase PDGFR, is expressed in retinal astrocytes. By inhibiting PDGF signaling with a neutralizing anti-PDGFRa or a soluble extracellular fragment of PDGFRa, Fruttiger and collaborators impaired the development of the astrocyte

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network (Fruttiger et al., 1996). Apparently, PDGF mediates a paracrine interaction between RGCs and astrocytes during retinal development. RGC axons exert a strong influence not only on migration of retinal astrocytes, but also on their morphology. At the periphery of the cat retina, where RGC axons are sparse, astrocytes adopt a stellate shape in contrast to the strongly elongated form present in RGCrich regions. Gargini et al. (1998) provided evidence that the axon-dependent morphology of the astrocytes is induced by a signal derived from neuronal spikes. Although the nature of the trophic signal from RGC axons has not been identified, a possibility considered by the authors is that it might be a released polypeptide acting through astrocyte receptors. As far as brain development is concerned, following the morphological data obtained by the Hatten group, an increased amount of evidence has been accumulated pointing towards neurons as modulators of astrocyte gene expression and differentiation (Gomes et al., 1999a; Kvamme et al., 1982; Mittaud et al., 2002; Rouach et al., 2000, 2002; Swanson et al., 1997). A way to study astrocyte differentiation is by evaluating levels of proteins which expression patterns vary during development such as the intermediate filament GFAP (Eng et al., 1971), the enzyme glutamine synthetase (Kvamme et al., 1982) and glutamate transporters (Wu¨rdig and Kugler, 1990), among others. In order to gain insight into astrocyte differentiation induced by neuronal cells, we have focused on GFAP expression. GFAP is the major component of the astrocytic intermediate filaments (IF) (Bignami et al., 1972; Eng et al., 1971; Fig. 6). In general, it is widely

Fig. 6. General pattern of GFAP immunostaining of cultured astrocytes. Cultured cortical astrocytes derived from newborn rats presenting distinct morphology: (A) a protoplasmatic, and (B) a process-bearing astrocyte. Note that GFAP filaments extend from the perinuclear region to all over the cytoplasm (A). In (B), the GFAP filaments are reorganized through cytoplasmic processes (F. Gomes, unpublished results).

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accepted that astrocyte maturation is followed by a switch in IF protein expression. Astrocyte precursors of the rodent embryonic CNS usually express vimentin, which is replaced by GFAP during astrocyte maturation (Dahl, 1981; Pixley and De Vellis, 1984). By using transgenic mice bearing 2 kb of the 50 flanking region of the GFAP gene linked to the b-galactosidase (b-Gal) reporter gene, we have demonstrated that cortical neurons can induce the GFAP gene promoter followed by transgenic astrocyte differentiation in vitro. Addition of embryonic neurons from cerebral hemisphere to transgenic astrocyte monolayers increased by 60% b-Gal positive astrocytes (Fig. 7). This event was dependent on the brain origin of the neurons and was followed by an arrest of astrocyte proliferation and induction of glial differentiation. Addition of conditioned medium derived from cortical neurons had a similar effect, suggesting that a soluble factor derived from neurons might be responsible for the induction of the GFAP gene promoter (Gomes et al., 1999a). Recently, we identified TGF-b1 (transforming growth factor b1) as the major mediator of this event (de Sampaio e Spohr et al., 2002). The TGF-b superfamily comprises multifunctional polypeptide members, which perform critical functions in regulating CNS developmental processes such as cell adhesion, migration and proliferation (Abreu et al., 2002; Bo¨ttner et al., 2000; Massague´, 2000; Unsicker and Strelau, 2000). TGF-b1 inhibits astrocyte proliferation, increases GFAP expression in vivo and in vitro, and modulates several ECM proteins and ionic channels (Laping et al., 1994; Perillan et al., 2002; Rich et al., 1999). Despite the widespread

Fig. 7. Neurons induce GFAP gene promoter and astrocyte differentiation by secreting TGF-b1. (A) An astrocyte culture derived from transgenic mice bearing part of the GFAP gene promoter linked to the bgalactosidase (b-Gal) reporter gene. Cells were immunostaining for GFAP (brown cytoplasm) (arrow) and reacted with X-Gal (blue nuclei, arrowhead) (modified from Gomes et al., 1999a). (B) Addition of neurons, CM or TGF-b1 dramatically increased b-Gal astrocyte number whereas addition of anti-TGF-b1 prevented this effect. G: astrocytes cultured alone; N: astrocytes cultured with embryonic neurons; CM: astrocytes cultured with conditioned medium; TGF-b1: astrocytes cultured with TGF-b1; C: in the absence of neutralizing antibodies to TGF-b1; þ aTGF-b1: in the presence of antibody against TGF-b1.

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effects reported for TGF-b1 in CNS injury, where it has been implicated in the organization of the glial scar (Moon and Fawcett, 2001; Zhu et al., 2002; see also the chapter by Kalman), relatively little has been reported on its role in physiological situations. Recently, TGF-b1 secreted by hypothalamic neurons was reported to modulate the oxytocin receptor in cultured rat astrocytes (Mittaud et al., 2002). Our work was pioneering in revealing a physiological function of TGF-b1 on astrocyte development and GFAP expression. We demonstrated that both cell types, neurons and astrocytes, synthesize and secrete this factor. However, addition of neurons to astrocyte monolayers greatly increased TGF-b1 synthesis and secretion by astrocytes. Further, by taking advantage of the cell culture system we investigated the influence of the developmental stage of neurons and astrocytes on this interaction. We demonstrated that younger neurons derived from 14-day-old embryos of wild type mice were more efficient in promoting astrocyte differentiation than those derived from 18-day-old mouse embryos. Similarly, astrocytes also exhibited a timed responsiveness to neuronal influence with embryonic astrocytes being more responsive to neurons than newborn and late postnatal astrocytes. RT –PCR assays identified TGF-b1 transcripts in young but not in old neurons, suggesting that the ability to induce astrocyte differentiation is related to TGF-b1 synthesis and secretion. Our data support the concept that within the context of brain development, neuronal signals might provide a source responsible for astrocyte development and strongly implicates TGF-b1 in this process. The role of neurotransmitters on astrocyte differentiation In addition to the traditional growth factors, recognition of the role of nonconventional trophic factors such as neurotransmitters on astrocyte differentiation has been growing for the last 10 years. This idea is greatly supported by findings that astrocytes exhibit a large variety of neurotransmitter receptor systems previously thought to be unique to neurons (for a review see Nedergaard et al., 2002; see also the chapter by Hansson and Ro¨nnba¨ck). Several studies demonstrated that the inhibitory neurotransmitter GABA secreted by neurons might act as a diffusible factor inducing the differentiation of neighboring astrocytes (Matsutani and Yamamoto, 1997; Mong et al., 2002). As previously observed by Hatten, addition of isolated neurons to monolayers of cultured astrocytes induced a morphological change (Hatten, 1985). Treatment of cocultures with a GABA-receptor antagonist inhibited the neuron-induced astrocyte differentiation whereas treatment with GABA agonists mimicked the neuronal effect. These results might suggest that GABA released by neurons serves as a signal, which triggers morphological changes in astrocytes (Matsutani and Yamamoto, 1997). Glutamate, the major excitatory neurotransmitter of the mammalian CNS, and its receptors and transporters are likely to be part of the complex network of signals that regulates astrocyte development in vivo. Increasing evidence has demonstrated that synaptically released glutamate could have two different actions on neurons: one direct and well known, through activation of postsynaptic receptors and another indirect, heterocellular action, mediated by the activation of glutamate transporters and receptors on glia, as we will discuss in the following paragraphs.

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Although glutamate has a crucial role in several processes of brain development, excessive accumulation of extracellular glutamate leads to neuronal death and has been implicated in various neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer disease and Huntington disease. The maintenance of normal glutamatergic neurotransmission and the prevention of glutamate-induced neurodegenerative disorders depends primarily on the presence of active glutamate transport in glial cells (McLennan, 1976; Rothstein, 1996; see also the chapter by Schousboe and Waagepetesen). Two main subtypes of glutamate transporters have been described in glia, i.e., GLAST, with expression predominating at early stages, and GLT-1 the expression of which progressively increases with maturity. Swanson et al. (1997) have reported that neurons can modulate astrocyte glutamate transporter expression in vitro. In the absence of neurons, cortical astrocytes maintain polygonal shapes and express only the GLAST transporter. When cocultured with a neuronal layer, many of the astrocytes assume a stellate shape and express GLT-1. These findings support the general principle that normal expression of GLT-1 protein by astrocytes requires a neuronal signal, suggesting that neurons can modulate astrocyte differentiation. Although the nature of this neuronal signal remains to be identified, recent reports have clearly demonstrated that GLT-1 and GLAST expression is modulated by neuronal soluble factors rather than by cell contact (Gegelashvili et al., 2000; Perego et al., 2000). These data suggest a regulatory loop tuning between glutamate physiology in the brain and astrocyte differentiation. Another target in the mediation of neuron –astrocyte interaction are the glutamate receptors, especially the metabotropic receptors, mGlu. They form a family of eight subtypes (mGlu1-8) that have been subdivided into three groups: I, II and III. Members representative of all groups are expressed by astrocytes and are apparently involved in mediation of neuronal signaling (Bruno et al., 1997; Besong et al., 2002; Kommers et al., 2002). Elucidative data on mGluII function has come from works by the Nicoletti group. They demonstrated that activation of group II mGlu receptors in astrocytes in vitro is associated with the presence of neuroprotective factors in astrocyte conditioned medium (see also the chapter by Peng). Medium collected from cultured astrocytes after a brief exposure to mGlu3 receptor agonist is highly neuroprotective against NMDA toxicity (Bruno et al., 1997). Neuroprotection was attenuated after treating the astrocytes with the protein synthesis inhibitor cycloheximide suggesting that astrocytes produce and release a proteic neuroprotective factor in response to mGlu3 receptor activation. More recently, these protective factors were identified as TGF-b1 and TGF-b2 (Bruno et al., 1998; D’Onofrio et al., 2001). Studies in Nicoletti’s group on glutamate physiology together with our own investigations provide new insight on TGF-b1 mediated neuron –astrocyte interactions and create a new scenario for neurotransmitter-growth factor actions on NS development. As illustrated in Fig. 8, a transient activation of group II mGlu receptor in astrocytes leads to an increased production and release of TGF-b1, which in turn protects neighboring neurons against excitotoxic death. Additionally, neurons might also potentiate astrocytic TGF-b1 secretion by release of small amounts of TGF-b1, which activate TGF-b

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Fig. 8. TGF-b1-glutamate putative pathway. Neurons release small amounts of TGF-b1 and glutamate, which act on the astrocytic membrane through TGF-b receptors (TGFbR), glutamate receptors (mGlu) and glutamate transporters (GLT-1 and GLAST). A transient activation of group II mGlu receptor in astrocytes leads to an increased production and release of TGF-b1, which in turn protects neighboring neurons against excitotoxic death. Additionally, neurons might also potentiate astrocytic TGF-b1 secretion by release of small amounts of TGF-b1, which activate TGF-b receptor on the astrocytic membrane. In addition to promoting neuronal survival, TGF-b1 induces GFAP gene expression and the astrocytic differentiation program. Such interaction between TGF-b1 and glutamate signaling suggests that astrocyte differentiation and neuronal development are strictly intricate processes.

receptors on astrocyte membrane. In addition to promoting neuronal survival, TGF-b1 induces GFAP gene expression and the astrocytic differentiation program. Such an interaction between TGF-b1 and glutamate signaling may provide new insights into the mechanism of neuronal degeneration. The interplay between these two pathways may suggest that astrocyte differentiation and neuronal development are processes much more intricate than we previously thought.

4. Concluding remarks Taken together, available literature data support the concept that within the context of brain development, neuron –glia interactions are not of a single type, but rather present great complexity and heterogeneity throughout the CNS. Currently, it is widely accepted that neuron– glia interactions play an important role in several steps of NS morphogenesis from the early stages of neurogenesis and gliogenesis to later stages of establishment of neural connections. A dynamic interplay between neurons and glial cells undoubtedly helps to shape developing neural circuits by controlling the survival and morphology of neurons, the growth of their axons, and the number and efficacy of their synapses. Although we have learned much about the physiology of glial cells over the last 10 years, our knowledge of the function and development of glia is still rudimentary. Several growth factors involved in gliogenesis have been identified and this will certainly be crucial for a better understanding of glial cell functions and interactions with neurons. The recent

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finding that glial cells can act as neural stem cells in the adult mammalian brain clearly highlights the new view of glial cells held by neurobiologists (for a review see Taupin and Gage, 2002). Whereas glial cells have been regarded so far as elements of structural and trophic support, today they might represent a key element in neural cell origin. One key issue in developmental neurobiology is to understand how the brain orchestrates the differentiation of various cell types. A range of epigenetic signals are involved in determination of neural fate potential, lineage specification and cellular differentiation in the CNS and peripheral nervous system. Some of these signals are initiated very early in development due to the diffusible factors and cell contact found in the developmental environment. Within this context, it will be very useful in the future to elucidate the mechanisms involved in Neuronal –glia interactions, since these are among the most relevant interactions neural cells will experience during development. Although several molecules involved in such interactions have already been identified, we are still clueless regarding neuronal effects on glial cells particularly those involved in astrocyte development. It would be worth exploring gene expression in neural cells in order to understand how Neuronal –glia interactions might modulate developmental genes during construction of the NS. Until recently there was no way to fully eliminate mammalian glial cells in vivo in order to explore how the brain develops and functions without them, as done in Drosophila after gcm identification. However, as previously discussed, the recent ablation of astrocytes in the cerebellum and CR neurons in cerebral cortex, which severely impaired granular neuron and radial glial cell development, respectively, clearly demonstrated the mutual dependence of neurons and astrocytes in the mammalian brain. We have taken a great step from the passive glia described by Virchow more than a century ago to the ‘astrocytic stem cells’ of today. The close association between neurons and glial cells during NS development suggests that deep inside these interactions might be hidden the secret of NS organization. Acknowledgements We thank Dhruv Kaushal and Cecı´lia Hedin-Pereira for comments on the manuscript and Marcy Kingsbury for generous help. References Abreu, J.G., Ketpura, N.I., Reversade, B., De Robertis, E.M., 2002. Connective tissue growth factor (CTGF) modulates cells signaling by BMP and TGF-b. Nat. Cell Biol. 4, 599–604. Adams, N.C., Tomoda, T., Cooper, M., Dietz, G., Hatten, M.E., 2002. Mice that lack astrotactin have slowed neuronal migration. Development 129, 965–972. Almer, G., Vukosavic, S., Romero, N., Przedborski, S., 1999. Inducible nitric oxide synthase up-regulation in a transgenic mouse model of familial amyotrophic lateral sclerosis. J. Neurochem. 72, 2415–2425. Alvarez-Buylla, A., Theelen, M., Nottebohm, F., 1990. Proliferation “hot spots” in adult avian ventricular zone reveal radial cell division. Neuron 5, 101– 109. Alvarez-Buylla, A., Seri, B., Doetsch, F., 2002. Identification of neural stem cells in the adult vertebrate brain. Brain Res. Bull. 57, 751–758.

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Cells lining the ventricular system: evolving concepts underlying developmental events in the embryo and adult Francis G. Szelea,* and Sara Szuchetb a

Department of Pediatrics, 2430 N. Halsted, No. 209, CMIER Neurobiology Program, Children’s Memorial Hospital, Feinberg School of Medicine, Northwestern University, Chicago, IL 60614-3394, USA p Correspondence address: Tel.: þ 1-773-880-3791; fax: þ1-773-868-8066 E-mail: [email protected] b Department of Neurology and the Brain Research Institute, Pritzker School of Medicine, The University of Chicago, Chicago, IL 60637, USA Tel.: +1-773-702-6396 E-mail: [email protected]

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction Mitotic cells that line the ventricles during development generate the nervous system: the role of RG in neurogenesis Morphological and gene expression pattern complexity of the ventricular system increases during development The birth of EC and their lineal relationships to VZ cells Adult EC are functionally and morphologically heterogeneous NOVOcan: a novel molecular link among RG, EC, tanycytes and immature OLG Embryonic and adult subventricular zone cells Concluding remarks

The proliferative and migratory cells that line the ventricular system in the embryo give rise to the nervous system. We discuss some new concepts concerning the role of radial glia (RG) in neurogenesis. The transition between ventricular zone (VZ) cells in the embryo and ependymal cells (EC) in the adult is not clear. We review current ideas concerning the lineage relationships between these cells and present data on NOVOcan, a novel protein that may shed light on the differentiation of EC. Adult EC, which are morphologically and possibly functionally heterogeneous, play a role in forming the brain –cerebrospinal fluid barrier. We also address the contribution EC might have to events taking place in the subependymal layer of the lateral ventricles, where developmental features of proliferation and migration continue throughout life. Advances in Molecular and Cell Biology, Vol. 31, pages 127–146 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction The cerebrospinal fluid (CSF)-containing ventricular system of the brain interior and spinal cord is lined by a thin layer of epithelial cells of neuroectodermal origin. During development, these ‘ventricular zone’ (VZ) cells are proliferative and give rise to the majority of the nervous system (Kintner, 2002). The functions of adult ‘ependymal cells’ (EC) lining the ventricular system are more enigmatic, yet it is generally accepted that they constitute the brain – CSF barrier (Bruni et al., 1985; Del Bigio, 1995a,b; Bruni, 1998). Although it is thought that EC cells are derived from VZ cells, there is relatively little known about the lineal relationships between developmental and adult cells lining the ventricles. VZ cells form a morphologically simple neural tube during embryogenesis, which becomes gradually more complex in shape as the brain develops; this is especially true in the forebrain (Figs. 1 and 2). VZ cells express genes in exclusive or overlapping patterns, which often correspond to morphologically distinct regions that give rise to specific cell types with distinct migratory pathways (Fig. 3). As the embryo develops, the molecular and morphological heterogeneity of VZ cells and their progeny increases. Successive, yet overlapping, spatial and temporal waves of neurodevelopmental events occur in the VZ: gene expression patterning, differential proliferation and migration of cells away from the ventricles. These processes are responsible for determining cell identity along the neuraxis, growth and morphogenesis of the central nervous system (CNS), and final positioning of neural cells, respectively. In the adult, a single layer of ECs comprise the ventricular system. The morphologically identifiable EC cells can be found along the entire neuraxis, from the lateral ventricles to the caudal spinal cord. They are thought to serve as selectively permeable physical and biochemical barriers between the neural parenchyma and CSF. The function and morphology of cells that line the ventricles varies considerably between mammalian and nonmammalian species. For example, whereas most ECs of mammals are regular, cuboidal, and postmitotic, the ECs of many ‘lower’ vertebrates are proliferative and are morphologically and functionally similar to radial glia (RG). Evolutionary complexity and other topics relating to ventricular cells are interesting; they have been extensively covered in other reviews concentrating on phylogeny (Reichenbach and Robinson, 1995; Garcia-Verdugo et al., 2002), development (Bruni, 1998), injury (Bruni et al., 1985; Del Bigio, 1995a,b; Sarnat, 1995), histology (Sarnat, 1998), cell junctions (Mugnaini, 1986), and heterogeneity (Mitro and Palkovits, 1981). There is also a wealth of literature on the in vitro culture of embryonic VZ cells, which provides fundamental insights into mechanisms of neural development. There are fewer, but nevertheless informative, culture studies of postnatal EC (Gabrion et al., 1998). Herein the emphasis is on: (1) new concepts and findings concerning the development and lineage relationships of RG and other brain cells, and (2) the heterogeneity and function of embryonic VZ, adult ependymal and adult subependymal cells. Additionally, we present data on a novel gene— novocan—whose expressed protein—NOVOcan—establishes an intriguing link between RG, EC, tanycytes and young oligodendrocytes (OLG).

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2. Mitotic cells that line the ventricles during development generate the nervous system: the role of RG in neurogenesis There is a major developmental shift in the cells that form the lumen of the ventricles from proliferative embryonic cells to nonproliferative adult cells. Early in embryogenesis, the nervous system is essentially a tube made of one layer of pseudostratified neuroepithelial cells (Fig. 1A). Both the CNS and the peripheral nervous system are derived from the ‘neural tube’. The precursors of the peripheral nervous system, neural crest cells, are specified dorsally around the period of neural tube closure (Fig. 1A). Neural crest cells, some of which express stem cell properties, then migrate away from the dorsal neural tube to distant and diverse organ systems. Thus, immediately after a neural tube, i.e., a ventricle, has formed, stem cells appear that give rise to neurons with robust migratory behavior. At the early stages of development, when the neural tube is one cell layer thick, VZ cells extend processes from the ventricular lumen to the pial surface (Fig. 1A). The nuclei of VZ cells ascend and descend as they go through the cell cycle, a process known as interkinetic nuclear migration (Sauer, 1935; Sauer and Chittenden, 1959; Sauer and Walker, 1959). Mitosis occurs when the nucleus is near the ventricular surface. The VZ cells first undergo symmetric and then later asymmetric division (Temple, 2001; Sommer and Rao, 2002). The former pattern of division gives rise to two daughter cells, both of which are mitotic, which results in exponential increase in cell numbers. In asymmetric division, one mitotic daughter cell remains in the VZ and one postmitotic daughter cell migrates away from the ventricles along radial glial fibers (radial migration, Fig. 1B). RG are amongst the earliest born neural cells and have traditionally been classified as glial cells. They are found throughout the CNS and appear as a dense pallisade of fibers

Fig. 1. (A) Scanning electron micrograph of a 2-day old chick neural tube. The tube consists of a single layer of proliferating cells. Note that the tube is about to close dorsally. The neural tube gradually thickens and the mitotic VZ cells remain proximal to the ventricle. The cell bodies of RG are also found in the ventricular zone (vz) (arrow in B). A dense system of radial glial fibers extends to the pial surface (ps). Neuroblasts (blue cell) use the RG processes as physical substrates for migration away from the VZ. A: adapted from Alberts et al. (2002); B: adapted from Sidman and Rakic (1973).

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(Fig. 1B). RG cell bodies contact the lumen of the ventricles and have processes that extend to the pial surface (Rakic, 1971a,b; 1972). With progressive thickening of the brain, their processes get longer but their cell bodies stay in the VZ. Whereas VZ cells also contact the lumen of the ventricles, in contrast to RG, VZ cell processes merely span the thickness of the VZ, but do not extend beyond it. In summary, cells that line the ventricles during development include mitotic VZ cells, RG and postmitotic daughter cells, which migrate away from the ventricles along RG. RG have been known since the classic anatomical studies of Ramon y Cajal and others in the 19th century. However, with the advent of immunohistochemistry and other modern techniques, their molecular phenotype and function have been characterized in greater detail. The RC2 antibody (Chanas-Sacre et al., 2000) specifically immunolabels RG cells and has allowed more detailed comparison of this cell type with precursor cells (nestinpositive) and astrocytes (positive for the intermediate filament proteins vimentin and glial fibrillary acidic protein (GFAP)) (Misson et al., 1988a,b; Hartfuss et al., 2001). Towards the end of development, RG retract their processes and either die or differentiate into astrocytes (Levitt and Rakic, 1980), leading to the accepted notion that they are gliogenic. Interestingly, RG, similar to VZ neuroepithelial cells, are mitotic and exhibit interkinetic nuclear migration (Basco et al., 1977; Misson et al., 1988a,b; Kamei et al., 1998). This begs the question of which cells arise from them. Recently, evidence has accumulated from a number of laboratories that RG in the cerebral cortex are neurogenic (Hartfuss et al., 2001; Miyata et al., 2001; Noctor et al., 2001; Tamamaki et al., 2001; Noctor et al., 2002). RG processes were labeled from the pial surface with DiI (a lipophilic dye that diffuses through the cell membrane) and their cell bodies were sorted by FACS (fluorescent-activated cell sorting) from the VZ (Malatesta et al., 2000). When these cells were isolated from young embryos, they could give rise to neurons in vitro suggesting that RG may also be neurogenic in vivo (Malatesta et al., 2000). However, when RG cells were isolated from older embryos they gave rise to astrocytes (Malatesta et al., 2000), which fits well with their accepted role as gliogenic cells. The in vivo neurogenic potential of RG was put on firmer footing by Noctor and colleagues with retroviral labeling showing that RG and neurons could be clonally related (Noctor et al., 2001). Then, using time-lapse microscopy of embryonic brain slices, they showed that RG could divide and give rise to young neurons that migrate along them (Noctor et al., 2001). It has been proposed that similarly to RG, astrocyte-like cells adjacent to the adult lateral ventricles in the subependymal layer (see below) give rise to neurons which then migrate in juxtapostion to them (Alvarez-Buylla and Garcia-Verdugo, 2002). Since most cells lose their complex morphology and round up before they divide, it seemed that RG would behave similarly and retract their processes before division. The question of RG morphology before division was addressed by Miyata et al., who found in slice preparations of E14 mouse cortex that RG processes do not retract from the pial surface before division, but become thinner (Miyata et al., 2001). If the division was neurogenic then, surprisingly, the neuronal daughter cell inherited the RG process (Miyata et al., 2001). In a subsequent study, Kriegstein’s laboratory has drawn into question whether the distinction between VZ cells and RG is valid. They showed that both cell types share molecular, mitotic and electrophysiological characteristics (Noctor et al., 2002). Taken together, these studies have forced a major revision in our understanding of

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the nature of cells lining the ventricles during development. However, the biology of RG at levels of the developing neuraxis other than the anlagen of the cerebral cortex is less well understood, and it remains to be seen whether all RG share the features of those in the dorsal telencephalon. Radial migration and the recently discovered neurogenic potential of RG favor the radial unit hypothesis, which states that positional information for cortical subregions is embedded in the VZ (the VZ contains ‘protomaps’) (Rakic, 1988). Retroviral studies and videomicroscopy have elucidated additional migratory patterns which are independent of RG (tangential migration) (Walsh and Cepko, 1988; Austin and Cepko, 1990; O’Rourke et al., 1992; O’Rourke et al., 1995). Tangential migration can occur within the VZ (Reid et al., 1995; O’Rourke et al., 1997) as well as in the cerebral cortex and other developing nuclei (Austin and Cepko, 1990; Walsh and Cepko, 1992; Szele and Cepko, 1998). As we will see, migration in close proximity to the ventricular lumen continues throughout life in the subependymal layer.

3. Morphological and gene expression pattern complexity of the ventricular system increases during development Whereas the simple architecture of the neural tube remains in the spinal cord as the central canal, more anterior portions of the ventricular system undergo complex morphogenesis (Fig. 2). In the forebrain, the ventricles become subdivided to form the bilateral and C-shaped lateral ventricles (Fig. 2B –D). The third ventricle is a midline, doughnut-shaped, structure (Fig. 2C,D). The intricate shapes of the telencephalic and diencephalic (lateral and third) ventricles are complemented by large uneven surface areas. Ventricular system morphogenesis is characterized by bulges in the VZ, which in part are created by differential proliferation. Two such regions, the lateral and the medial ganglionic eminences (LGE and MGE) (Fig. 3A) appear in the lateral ventricles early in embryogenesis. The LGE and the MGE give rise to the basal ganglia, whereas the dorsal telencephalic VZ gives rise to excitatory projection neurons of the cerebral cortex (Fig. 3A) (Corbin et al., 2001). Recently, another level of complexity was discovered: the ganglionic eminences give rise to neurons that migrate dorsally into the cerebral cortex and differentiate into interneurons (Anderson et al., 1997a,b). Thus, some cells generated in distinct subregions of the germinal neuroepithelium end up in adjacent nuclei whereas others migrate far away. The subependymal layer is an example of the latter case, in the adult. The morphological complexity of the walls of the embryonic ventricular system is often very well correlated with specific gene expression patterns (Fig. 3B). The earliest and best studied example of this is in the hindbrain rhombomeres, a transient series of bulges that selectively express homeobox genes in sequential anterior – posterior patterns (Nieto et al., 1992; Krumlauf et al., 1993). These patterning transcription factors take part in complex genetic regulatory pathways to influence a variety of developmental events in the hindbrain. For example, if misexpressed, they can alter rhombomeric identity or, if deleted, alter routes of migration out of the rhombomeres (Zhang et al., 1994; Studer et al., 1996).

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Fig. 2. The morphological complexity of the ventricular system increases with development. The human ventricular system is shown during early embryogenesis (A and B) and in the adult (C and D). In A and B, the regions of the brain derived from the ventricular wall are indicated on the left. (A– C) dorsal views; (D) sagittal view. A and B: adapted from Carpenter and Sutin (1983); C: adapted from Pinel (2000).

Other parts of the developing brain, such as the telencephalon, have also been found to have complementary ventricular bulges and gene expression patterns (Fig. 3B), suggesting that similar genetic regulatory mechanisms of regionalization occur throughout the neuraxis (Rubenstein et al., 1994; Schuurmans and Guillemot, 2002). The dorsal germinal neuroepithelium selectively expresses genes such as Pax6, Gli3, and Emx1, and 2 (Fernandez et al., 1998; Schuurmans and Guillemot, 2002). Conversely, the MGE and LGE express other genes, such as Nkx2.1, Dlx1 and 2, and Shh, which are not expressed by the dorsal germinal neuroepithelium (Shimamura et al., 1997; Fernandez et al., 1998; Rubenstein and Beachy, 1998; Schuurmans and Guillemot, 2002). Deletion of Dlx genes causes disruptions in the migration and differentiation of cells derived from the ganglionic eminences (Anderson et al., 1997a,b). In the adult subventricular zone (SVZ), Dlx2 is expressed by transit-amplifying progenitor cells and migratory neurons, suggesting that it may have similar roles as in the ganglionic eminences (Doetsch et al., 2002).

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Fig. 3. Proliferative subregions surround the ventricles and have distinct molecular expression patterns during embryonic development. (A) Coronal section of the rat forebrain. Note that a cell dense (dark Nissl stain) ventricular zone surrounds the lateral ventricles (lv). The red line shows the approximate demarcation of germinal neuroepthelia fated to primarily give rise to the cerebral cortex or the basal ganglia. Ventrally, two distinct bulges, the LGE and MGE, generate cells which end up in specific nuclei of the basal ganglia. The subregions of the VZ express many distinct molecules. (B) Pax-6 expression in ventricular zone cells (arrowhead) of the dorsal chick telencephalon (which is homologous to the mammalian cerebral cortex). B: adapted from Szele et al. (2002).

4. The birth of EC and their lineal relationships to VZ cells During development, different classes of neural cells become postmitotic in a stereotyped order. It is generally accepted that neurons are born first, followed by astrocytes, with OLGs being born last. A number of studies have sought to determine the birthdates of ependymal and related cells at various levels of the neuraxis by using 3 H-thymidine incorporation during S-phase (Rakic and Sidman, 1968; Altman and Bayer, 1978a,b; Das, 1979; Korr, 1980; Rutzel and Schiebler, 1980). Similar to the rest of the brain, EC are generally born in a caudal to rostral gradient (Das, 1979; Altman and Bayer, 1990). Birthdating studies in the third ventricle of rats showed that the majority of EC are born late in development at E16-19, after neurogenesis has ceased (Altman and Bayer, 1978a,b; Das, 1979). The majority of EC in the lateral ventricles are born a few days later, between E20 and E21 (Das, 1979). However, some studies have shown that a few EC are born as early as E10 –12 in the third ventricle (Rakic and Sidman, 1968; Korr, 1980) and E14 in the cerebral aqueduct and fourth ventricle (Das, 1979). In contrast to EC, tanycytes are primarily born postnatally (Altman and Bayer, 1978a,b). The lineage relationship of adult EC (and other cells lining the ventricles) to embryonic VZ cells remains a relatively understudied topic. However, it is generally assumed that EC are derived from VZ cells (Kintner, 2002). If it is true that around 90% of VZ cells are RG (Noctor et al., 2002), then most EC may derive from RG, either via differentiation or division. This notion is not altogether preposterous as some specialized EC (tanycytes, see below) resemble RG. Also, in lower vertebrates, most ependymoglial cells extend processes to the pial surface (Reichenbach and Robinson, 1995). If RG differentiate into EC, then the most obvious phenotypic changes would be loss of pial processes and

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establishment of cilia. Recently, it was found that the neonatal mouse lateral ventricle has cells which express markers of RG, undergo interkinetic nuclear migration and divide and thus resemble both RG and VZ cells (Tramontin et al., 2002). It seems likely, but as of yet remains unproven, that these cells then differentiate to form EC. A few retroviral studies have shown that adult EC and neurons can be derived from the same progenitor cells. In the spinal cord, ventral motorneurons were found that were clonally related to EC (Leber et al., 1990). Retroviruses injected during the period of neurogenesis in the mouse marked postnatal day 21 EC in the third ventricles (McCarthy et al., 2001). Thus it is likely that EC are derived from the same VZ or RG cells that give rise to neurons. The development of the human ependymal layer has been examined with immunohistochemistry, and although lineage relationships between VZ cells, RG, and adult EC were not clear, a cell with intermediate morphology suggested a gradual transformation from VZ-RG to adult EC (Gould and Howard, 1987; Gould et al., 1990). 5. Adult EC are functionally and morphologically heterogeneous Ependymal cells, also known as ependymoglial cells, are similar to epithelial cells; they are a single layer of regularly spaced cuboidal cells (Fig. 4) (Bruni et al., 1985). However, whereas most epithelial cells self-renew in the adult, EC generally do not divide (Bruni et al., 1985). EC cells are connected by tight junctions and gap junctions, and they bear cilia. Ependymal cells and other cells lining the ventricles are quite heterogeneous with

Fig. 4. (A) Electron photomicrograph of two EC (one highlighted in pink, one in light green) in the adult mouse lateral ventricle showing cilia (large arrow) and microvilli (small arrow). Two basal bodies, the insertion of cilia in the cell body, are indicated by asterisks. Electron dense zonula adherens is shown by red arrow. (B) Dog EC in the cerebral aqueduct with one exhibiting a process (P) emanating away from the ventricle. Note the zonula occludens (arrows). B adapted from Carpenter and Sutin (1983).

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some cells including tanycytes, the choroid plexus epithelial cells and cells of the circumventricular organs being very specialized (Mitro and Palkovits, 1981). Finally, the ECs of the lateral ventricles are flanked by the subependymal layer, which contains the largest concentration of adult brain stem cells. Although much is known about the morphology, ultrastructure and molecular expression patterns of EC, their functions in the adult still remain largely enigmatic and speculative (Bruni et al., 1985; Del Bigio, 1995a,b; Bruni, 1998). CSF is primarily generated by choroid plexus cells in the lateral, third and fourth ventricles, and probably to a small extent by diffusion of extracellular fluid from the brain through EC. However, adult EC, in contrast to embryonic cells lining the ventricles, do not express secretory proteins (Sarnat, 1998). CSF is replenished at a rate of 0.5% of the total volume per minute, which in humans corresponds to a daily production of 450– 700 ml (Oldendorf, 1972). CSF is slowly moved through the ventricular system by the coordinated beating of cilia on the apical surface of EC (Oldendorf, 1972). The cilia have a characteristic 9 þ 2 microtubule arrangement and are powered by the molecular motor dynein. In addition to cilia, EC have numerous microvilli (Fig. 4) (Mitro and Palkovits, 1981). The density of cilia and microvilli on EC varies with EC juxtaposed to white matter having fewer apical specializations (Page et al., 1979). Ependymal cells have tight junctions that serve to establish the CSF –brain barrier (Fig. 4) (Mitro and Palkovits, 1981; Carpenter and Sutin, 1983; Mugnaini, 1986). The zonula occludens at the apical junction of juxtaposed EC completely occlude any extracellular space whereas the adjacent zonula adherens is characterized by more intracellular space (Mitro and Palkovits, 1981). ECs are thought to regulate the flow of water, ions and small molecules between the brain parenchyma and the CSF (Del Bigio, 1995a,b); however there are microregions in the ventricular system which are more porous. ECs also express specific transport proteins, which serve to regulate the brain – CSF transit of molecules. In addition, they may detoxify or otherwise serve as a metabolic barrier between brain and CSF (Del Bigio, 1995a,b). Adult EC and embryonic VZ cells are connected to one another via gap junctions (Privat, 1977; Mugnaini, 1986; Yamamoto et al., 1990; Gabrion et al., 1998; Bittman and LoTurco, 1999). During early development, almost all RG and neural precursors that are in G1 or S-phase in the VZ are coupled (Bittman et al., 1997). As development progresses, VZ cells become uncoupled during G1 and S, but become coupled during G2. Disruption of gap junction communication with octanol caused a decrease in the fraction of VZ cells entering S phase, prompting the proposal that gap junctions in the VZ may contribute to neurogenesis. Connexin-26, a gap junction protein, has also been found in subependymal layer astrocytes (Mercier and Hatton, 2001). Whereas it is not known what functions gap junctions may have in EC, it is tempting to speculate that they are coordinated with the syncytium of gap junctionally connected astrocytic and meningeal cells with which they form a continuum (Mercier and Hatton, 2001; also see chapter by Mercier and Hatton). Cells lining the ventricles express a number of glial markers, including vimentin, GFAP, and S-100b (Roessmann et al., 1980; Sarnat, 1998). However, these markers are expressed in a heterogeneous fashion throughout the developing and in the adult neuraxis, further suggesting that there is molecular heterogeneity in VZ/EC (Sarnat, 1998). Young RG express nestin (a marker of neural precursors) and vimentin. Then, as they mature and

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transform into astrocytes they start to express GFAP. The fact that most adult murine EC are positive for vimentin suggests that at least some EC are derived from RG. Tanycytes are a special class of EC with a morphology reminiscent of RG (Fig. 5) (Card and Rafols, 1978; Seress, 1980; Bruni et al., 1983). In the adult mouse, they express both vimentin and GFAP (Fig. 5), which distinguishes them from EC that primarily express vimentin. Tanycytes are primarily found around the third ventricle and extend processes that can wrap around blood vessels, terminate on neurons or glia, or extend to the glia limitans (Bruni, 1998). Because of their specialized morphology, quite a few studies have been conducted to determine their function (reviewed in Bruni, 1998). For example, it has been hypothesized that tanycytes transport CSF from the third ventricle to hypothalamic neurons (Lofgren, 1959; Bruni et al., 1985). They may also be involved with hormonal release from the hypothalamic portal system (Wittkowski, 1998). Tanycytes are juxtaposed with luteinizing hormone releasing hormone (LHRH or GnRH) axon terminals and it has thus been suggested that they influence the physiology of those neurons (Kozlowski and Coates, 1985). Finally, tanycytes in the mediobasal, but not in the dorsal hypothalamus, may participate in repair of lesioned monoaminergic neurons by promoting sprouting along their processes (Chauvet et al., 1998).

Fig. 5. Tanycytes express vimentin, GFAP, and NOVOcan. (A) Vimentin immunoreactivity; (B) GFAP immunoreactivity; (C) merge of A and B. The arrows in A –C point to a tanycyte process that expresses both intermediate filament proteins, arrowhead to one that only expresses vimentin (A– C: adult mouse). D and E: NOVOcan immunoreactivity in postnatal day 16 rat is found in cells lining the third (D) and fourth (E) ventricles. Note that some cells are weakly labeled (arrowhead) and others are strongly labeled (arrows) (D and E: P16 rat).

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6. NOVOcan: a novel molecular link among RG, EC, tanycytes and immature OLG As part of an effort to identify genes that are important in the process of OLG regeneration, we screened an OLG cDNA library with oligonucleotide probes designed to identify proteoglycans important for the biology of other cell types (Szuchet et al., 2000). One of the isolated cDNA (3500 nts) hybridized specifically to a probe modeled after perlecan, a heparan sulfate proteoglycan important for peripheral myelination. However, the sequence of the isolated cDNA proved to be novel, unrelated to perlecan except for the short segment corresponding to the probe. We named the gene novocan to stress the fact that it is new (novo) and can—a suffix for proteoglycans—because the sequence contains glycosaminoglycan binding sites. We refer to the expressed protein as NOVOcan. Novocan is a member of a novel family of developmentally regulated, species conserved, genes. Southern blot analysis of rat genomic DNA indicated that it is the product of a single copy gene; northern blot analysis of rat brain mRNA revealed at least three bands most likely representing alternatively spliced forms. Brain shows the highest level of novocan expression, which occurs at early postnatal times (Szuchet et al., 2001). The predicted amino acid sequence of NOVOcan was screened for antigenicity, surface exposure and absence of cross-reactivity with database entries. This search yielded a segment of 28 amino acids close to the N-terminus that was selected as an immunogen for the generation of a panel of monoclonal antibodies (mAbs). When these mAbs were used in a Western blot analysis on rat-brain extracts, they detected three bands with molecular masses of 400, 240 and 200 kD. Preliminary biochemical studies indicate NOVOcan to be a keratan sulfate proteoglycan. In order to determine the in situ cell and tissue localization of NOVOcan, we employed immunohistochemistry on brain sections at different stages of development. The results (Figs. 5 –7) are intriguing for they attest to current concepts that link, developmentally, RG, EC and tanycytes. As shown in Fig. 6A, NOVOcan antibodies detected degenerating RG in the cerebral cortex at P6. In neonatal rats, NOVOcan was also expressed in cell bodies lining the lateral ventricles and a few cell diameters from the ventricle, suggesting that it is expressed by a combination of VZ cells, RG and SVZ cells (Fig. 6B –E). Many cell nuclei looked like they were wound with fibers of NOVOcan. Interestingly, NOVOcan-positive fibers resembling RG emanated from the periventricular region into the adjacent brain parenchyma. At later ages (P16), NOVOcan was expressed by tanycytes of the third and fourth ventricles (Fig. 5D,E). These cells were very similar in morphology to those detected in the lateral ventricles at P6. In the adult, NOVOcan was primarily expressed by EC (Fig. 7D). Very few NOVOcanpositive RG- or tanycyte-like processes were immunodetected at P60 (Fig. 7D). Whereas NOVOcan and GFAP seemed to be coexpressed at P6 (Fig. 7A –C), they were not at P60. At P60 NOVOcan was expressed almost exclusively by EC, and GFAP was expressed primarily by SVZ astrocytes (Fig. 7D – G). The transition from an RG-like NOVOcanpositive cell in early postnatal lateral ventricle, and its expression by EC in the late postnatal period, lends credence to the notion that EC derive from RG. Furthermore, though tanycytes and RG have been distinguished based on their selective expression of markers, our studies of NOVOcan expression suggest that they may belong to the same

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Fig. 6. NOVOcan immunohistochemistry (red fluorescence) in early postnatal brain. (A) Retracting/degenerating NOVOcan þ radial glial fibers in the cerebral cortex of a 6-day old rat. (B) NOVOcan þ cells in the wall of the lateral ventricle (LV) of a 3-day old rat. Note that NOVOcan appears to be distributed in fibrous strands (arrows) around the nucleus (blue DAPI counterstain). Also note that NOVOcan-positive fibers (arrowheads) emanate from the ventricular wall into the adjacent parenchyma. (C) NOVOcan immunofluorescence; (D) DAPI counterstain; (E) merge of C and D, 6-day old rat. NOVOcan is expressed in EC (arrowheads in C–E) as well as in SVZ cells (arrows in C–E). NOVOcan þ processes extending into the striatum are seen in C and E. cp ¼ Choroid plexus, str ¼ striatum.

lineage. Continuing studies should further delineate the anatomy and function of NOVOcan during development and in the adult. 7. Embryonic and adult subventricular zone cells The majority of the adult ventricular system is lined by a single cell layer of EC, which is flanked by neuropil. However, the EC of the lateral ventricles are flanked by a layer of specialized periventricular cells (Fig. 6) known as the subependymal layer (1970) or SVZ (Gates et al., 1995; Alvarez-Buylla and Garcia-Verdugo, 2002). The adult SVZ contains a nestin-positive progenitor cell, two GFAP-positive subtypes of astrocyte-like cells, and a class III b-tubulin-positive neuroblast (Jankovski and Sotelo, 1996; Doetsch et al., 1997). Unfortunately very few cell-type specific markers exist that permit light microscopic identification of SVZ cell types; however, ultrastructural studies allow clearer distinctions to be made (Doetsch et al., 1997). In vivo, SVZ cells exhibit the highest rate of proliferation in the intact adult brain (Fig. 8A,B). The neuroblasts move long distances in the SVZ in a specialized ‘chain migration’ to the olfactory bulbs where they become

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Fig. 7. The coexpression of NOVOcan and GFAP changes during development A– C: (A) NOVOcan, (B) GFAP, (C) merge in the lateral ventricle at P6. Note that some NOVOcan-positive cells in the wall of the lateral ventricle also express GFAP (example shown by arrowheads in D –F). Weak NOVOcan immunoreactivity is also found in the choroid plexus (cp). D –G: Novocan immunoreactivity is largely confined to EC in the P60 rat lateral ventricle; (D) novocan immunofluorescence; (E) GFAP immunofluorescence; (F) DAPI counterstain; (G) merge of D –F. Arrows show three novocan-positive/GFAP-negative EC lining the lateral ventricle (lv). Only a few of the processes emanating from the subventricular zone (svz) into the striatum (str) are positive for both novocan and GFAP (arrowheads).

functional interneurons (Fig. 8C,D) (Jankovski and Sotelo, 1996; Alvarez-Buylla and Garcia-Verdugo, 2002). Thus, the SVZ can be considered to be a periventricular subregion of the adult brain where ‘development’ never ceases; the processes mentioned above continue throughout life.

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Fig. 8. The adult SVZ surrounds the lateral ventricles and contains mitotic and migratory neuroblasts. (A) Coronal section of an adult mouse brain showing the location of SVZ cells (red dots) labeled with, 3Hthymidine. Note the relatively high density of cells in S-phase in the dorsolateral angle of the SVZ (dl svz), in between the corpus callosum (cc), the striatum (str), and surrounding the ventral lateral ventricles (lv). (B) BrdU labeling of cells in S-phase results in similarly large numbers of dorsolateral SVZ cells (arrows). Ependymal cells are not labeled with BrdU (arrowheads). (C) Sagittal view of the brain showing the location of the SVZ (shaded region) in relation to the lateral ventricle (light blue). The SVZ extends into the olfactory bulb along a pathway called the rostral migratory stream (RMS). PSA-NCAM is expressed by neuroblasts migrating from the SVZ to the olfactory bulbs via the RMS. (D) PSA-NCAM immunofluorescence in the rostral migratory stream. Note that these cells are arranged in chains of cells (Lois et al., 1996). A and C: adapted from Smart (1961).

The adult SVZ may derive from the embryonic SVZ, a secondary germinal neuroepithelium, which is not in direct contact with the ventricular lumen but overlies the VZ. In contrast to the VZ, cells in the embryonic SVZ do not exhibit interkinetic nuclear migration and have slower rates of mitosis. Another difference is that whereas the VZ is thought to be primarily neurogenic, the embryonic SVZ is mainly gliogenic. Elegant retroviral studies showed that OLGs and astrocytes that migrate to the striatum, corpus callosum, and cortex continue to be generated from the SVZ during early postnatal development of the rat (Levison and Goldman, 1993). At approximately the same time, several laboratories showed in vitro stem cell activity (self-renewal and wide fate potential) in cell preparations from the adult mammalian brain (Reynolds et al., 1992; Richards et al., 1992). Lois and Alvarez-Buylla microdissected the SVZ from surrounding tissues and showed that in contrast to cortical and striatal explants, SVZ explants were able to give rise to neurons and glia (Lois and Alvarez-Buylla, 1993). Since then, many laboratories have grown SVZ cells as floating neurospheres in the presence of epidermal growth factor and/or basic fibroblast growth factor (Gritti et al., 1996; Suslov et al., 2002).

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If neurospheres are allowed to adhere to a substrate and the mitogenic growth factors are removed, cells migrate away from the neurospheres and differentiate into the three major lineages of the adult CNS; astrocytes, OLGs and neurons. The search for the identity of the stem cell in the wall of the adult lateral ventricle has pointed to several candidates, including the EC themselves (Johansson et al., 1999). This possibility was especially interesting, since EC are probably derived from VZ cells and thus should have the molecular machinery necessary for neurogenesis (Kintner, 2002). Ependymal cells were labeled with DiI or adenovirus and, subsequently, labeled cells were found in the SVZ and olfactory bulb, suggesting that the EC had generated them (Johansson et al., 1999). However, DiI can spread from cell membrane to cell membrane and both labels may have labeled the SVZ astrocytes, which occasionally extend CSFcontacting processes between EC (Doetsch et al., 1997). Then EC were separated with immuno-magnetic beads and were able to give rise to neurospheres in vitro (Johansson et al., 1999). However, others have shown that EC grown in vitro cannot self-renew and only give rise to glia (Chiasson et al., 1999; Capela and Temple, 2002). Whereas the present data suggest that EC are not the stem cells that reside in the walls of the lateral ventricle, they may interact with the SVZ to provide an environment conducive to stem cell functioning. For example, EC make noggin, an inhibitor of gliogenic BMP signaling, thus allowing neurogensis to occur within the SVZ (Lim et al., 2000). An alternative proposal has been that the SVZ astrocyte-like cells are the stem cells and that SVZ progenitor cells are an intermediate transit amplifying population (Doetsch et al., 1999a,b, 2002). Mitotic poisons were used to deplete the SVZ of all cells except one of the astrocyte-like subtypes, which were then able to gradually reconstitute the SVZ, with the progenitor cells reappearing first and then the neuroblasts (Doetsch et al., 1999a,b). However, it is unclear whether the lineage of cells is similar in the intact SVZ to the ‘lesioned’ SVZ. An avian retrovirus was next used to specifically infect astrocyte-like cells in the SVZ of transgenic mice that had an avian retrovirus receptor tagged to the GFAP promoter (Doetsch et al., 1999a,b). Surprisingly, the astrocyte-like cells were again able to give rise to all the other cell types in the intact SVZ (Doetsch et al., 1999a,b). This is very strong evidence that astrocytes are stem cells in the SVZ. Whether the astrocytes are the only type of SVZ cells with stem cell properties in vivo remains unclear. Regardless of their identity, it is clear that the largest population of stem cells in the adult brain is adjacent to the adult lateral ventricles.

8. Concluding remarks Cells that line the embryonic ventricles are proliferative, migratory, and the source of the peripheral and CNS. Many of the features present in the VZ of the entire neuraxis during development, such as self-renewal/proliferation, wide fate potential, and migration, are maintained in an anteriorly restricted region: the adult subependymal layer. Some of the data reviewed here are forcing a reconsideration of the lineage relationships and developmental roles of ventricular cells (neuroepithelial cells, RG, astrocytes, SVZ cells and ependyma). A hypothesis has been put forward suggesting a periventricular lineage consisting of transitions from neuroepithelial cells to RG to astrocytes. In this model, all

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three cell types in the lineage are neurogenic (Alvarez-Buylla et al., 2001, 2002). The expression of a novel gene, NOVOcan, may shed some light on questions of periventricular cell identity. Identification of the precursor of EC may lead to a better understanding of their role in the adult. Retroviral lineage studies, carefully timebracketed from late embryogenesis through adulthood, may provide some answers. This, coupled with the new technology of multiphoton microscopy, which allows long-term imaging without tissue damage, may clarify patterns of division and differentiation of fluorescent protein-marked cell subtypes to reveal which cells give rise to which.

Acknowledgements The authors would like to thank members of the Szele laboratory (G. Goings, N. Peters, H. Chin) and of the Szuchet laboratory (D. Plachetzki) for their help in generating the figures. Supported by RO1 NS/AG42253-01 (F.G.S.) and RO3 HD40832 (S.S.).

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The perisynaptic astrocyte process as a glial compartment-immunolabeling for glutamine synthetase and other glial markers A. Derouiche Institute of Anatomy, University of Dresden, Fetscherstr. 74, D-01307, Dresden, Germany

Contents 1. 2.

3.

4. 5.

Introduction The peripheral astrocyte process (PAP) compartment 2.1. Structural features of PAPs 2.2. Glutamine synthetase and PAP functions PAPs: other markers and functions 3.1. Preferential versus additional PAP labeling 3.2. Preferential PAP labeling 3.3. Additiional PAP labeling Pathological alterations of PAP integrity and GS staining Concluding remarks

This review focuses on the compartment of the ‘peripheral astrocyte process (PAP)’, and on its properties. This compartment includes astrocytic filopodia, and it accounts for a large fraction of the total astrocytic surface area. PAPs have been shown to be highly dynamic in response to neuronal activity. The localization of some glial antigens will be described, which are important either for the function of the PAP or for its characterization. Ezrin and radixin, two proteins present in astrocytes, are virtually restricted to PAPs indicating that the filopodia constitute a separate glial compartment, where these proteins are localized. Other proteins, e.g., glutamine synthetase are present in PAPs, but are not restricted to this compartment. The usefulness of a compartmentoriented approach is pointed out by the functional and pathological relevance of the PAP. 1. Introduction Since early on, a primary concern in the study of neuronal functions has been their allocation to the well-known neuronal compartments. This is not so in the case of Advances in Molecular and Cell Biology, Vol. 31, pages 147–163 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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astrocytes, since astrocytic compartmentalization is only beginning to emerge. Protein localization and biochemical and physiological functions are mostly considered as ‘glial’, but it would be very important to know the exact localization of, e.g., enzymes, transmitters, receptors and transporters. Since the terms ‘compartment’, ‘domain’ and ‘polarization’ are partly overlapping, the definition of a compartment may be exemplified by the neuronal dendrite. As a compartment, this structure is endowed with a set of proteins that are particularly targeted at this specific location, and define its morphology and physiological properties. Based on its branching pattern, the dendrite may be subdivided into several topical domains, which together constitute the dendritic compartment. Axon terminals, for example, constitute a single neuronal compartment, which, however, consists of several discontinuous topical domains. In an astrocyte, several processes are traditionally distinguished on grounds of their morphology and structural relationship (i.e., subpial, subependymal, ependymal, perivascular, perisynaptic, perineuronal); however, it is not clear how far they represent separate compartments or only topical domains. The main part of this review focuses on the compartment of the ‘peripheral astrocyte process (PAP)’, and on its properties. The localization of some glial antigens will be described, which are important either for the function of the PAP or for its characterization. The usefulness of a compartment-oriented approach is pointed out by the functional and pathological relevance of the PAP. Although region specific differences between astrocytes are known, and subtypes of astrocytes are being described within a given region, based on electrophysiology and antigen expression (Zhou and Kimelberg, 2001), astrocytes will be considered as a single entity, without presently established consensus regarding astrocytic subtypes. 2. The peripheral astrocyte process (PAP) compartment 2.1. Structural features of PAPs The well-established ultrastructural features (Peters et al., 1991) of PAPs are the paucity in organelles, in particular mitochondria, which gives them a clear appearance. They display extremely fine dimensions and a mostly concave shape, complementary to the round neuronal structures. PAPs often consist of merely 50– 100 nm wide membrane appositions apparently not enclosing any cytoplasm volume, which results in an extremely high surface/volume ratio (Chao et al., 2002) that is of great functional importance. These morphological characteristics would per se not specify the PAP as a compartment, especially since there are gradual transitions at the ultrastructural level between the PAPs and the main processes, containing glial filament and organelles. Based on their comparable dimensions, filopodia of cultured astrocytes have been regarded as the in vitro equivalent of the extremely fine PAPs in vivo. Video microscopy has demonstrated that filopodia can form and elongate within 30 s (Cornell-Bell et al., 1990), whereas changes in gross shape, such as the classical transition from flat to stellate astrocytes (after appropriate provocation) requires several hours. Similarly, PAPs have been shown to be highly dynamic in response to neuronal activity. They can rapidly elongate and retract in synchrony with the activity of hypothalamic neurons during the

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processes of lactation, parturition or circadian changes (Serviere and Lavialle, 1996; Theodosis and Poulain, 2001—see also chapters by Mercier and Hatton, Salm et al. and Prevot et al.), or within 60 min in response to lesion-induced changes of neuronal activity in cerebral cortex (Landgrebe et al., 2000; Laskawi et al., 1997). The search for the molecular basis of the extremely fine filopodial structure and its motility has focused on the ERM protein family (acronym for ezrin, radixin and moesin), which were discovered during studies of similar phenomena occurring in epithelial microvilli (Berryman et al., 1993). Interestingly, immunoreactivity for ERM proteins in astrocytes is predominantly found in their filopodia (Derouiche and Frotscher, 2001), which in cultured astrocytes appear detached from the main processes and perinuclear region, when the cell is not double-stained for example, by anti-GFAP (Fig. 1). Thus, the restricted localization of ezrin and radixin, the two ERM proteins present in astrocytes, indicates that the filopodia constitute a separate glial compartment, where these proteins are localized. Apart from being filopodial marker molecules, ERM proteins are part of the molecular mechanism required for filopodia formation (Takeuchi et al., 1994) and maybe motility. This is because the actin-binding ERM proteins (reviewed by Tsukita et al., 1997), of which ezrin is the main representative, link the actin cytoskeleton to the plasma membrane, by binding intracellularly to adhesion molecules. Ezrin and radixin are selectively localized to the PAPs in the brain. Together with their particular morphology and structural plasticity, this has lead to the recognition of the PAPs as a separate astroglial compartment (Derouiche and Frotscher, 2001), further underlining their correspondence to filopodia in vitro. The predominant, if not selective labeling, of PAPs by anti-ezrin or other markers (see below) has caused some problems, since these processes are submicroscopic. Thus, these processes collectively form a diffuse ‘background’ staining at the light microscopic level (allowing a more generalized view), which is occasionally misinterpreted as nonspecific immunoreactivity. This is particularly so, since the ‘classical glial’ pattern, i.e., stem processes and perikarya is only vaguely apparent, if at all (Fig. 2a). Thus, the interpretation of selective PAP labeling clearly needs ultrastructural confirmation. At higher light microscopic magnification, a clear indication of the presence of PAP labeling is the typically granular or punctate pattern (Fig. 2b), which can be observed with suitable technique (see below). The

Fig. 1. Anti-ezrin labels the filopodia (A) of cortical astrocytes in primary cultures. This is often selective, so that they appear isolated. Their relation to one or several cells has to be verified in the other channel (B). Double immunofluorescence for anti-ezrin (A) and the potassium channel Kv3.1b (B), which stains the perinuclear region and the stem processes.

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Fig. 2. Selective demonstration of PAPs by staining with anti-ezrin. At medium magnification of CA3 from rat hippocampus (A), the neuropil layers appear diffuse, only unlabeled neuronal profiles stand out negative. At higher magnification (B, boxed area from A), the typical punctate pattern is visible. Unlabeled dendrites in cross and oblique section can be seen within and above the pyramical cell layer in (C).

distinction from nonspecific background is evident only at high magnification, when neuronal profiles such as cross-sectioned dendrites and even axon terminals on dendrites and perikarya are outlined by the immunoreactive glial processes (Fig. 2c). For the reliable visualization of PAPs, a combination of details in histological processing has proven essential, since these structures are at the limit of light microscopic resolution. The following recommendations are unpublished observations by the author, but they have been confirmed both positively and negatively by many reports. Tissue integrity is best conserved by perfusion fixation and preparation of vibratome sections. For detection of immunoreactivity using peroxidase or fluorescence methods, the absence of tissue permeabilization (by triton £ 100 etc.) is recommended. The resulting staining is limited to the two surfaces of the section and thus faint, but free of out of focus information, yielding structural detail comparable to semithin sections. In cryostat sections, blurring of the fine PAPs by the abundant signal from extrafocal planes is seen, which can only be overcome by high performance deconvolution. However, preservation of fine detail integrity in cryostat sections is inconsistent, depending on cryoprotection and snap freezing. Structural preservation for PAPs is clearly insufficient in paraffin sections. Surprisingly, the visualization of PAPs is enhanced in vibratome sections from immersion-fixed material,

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Fig. 3. Glutamine synthetase immunoreactivity reveals astrocytes and some degree of diffuse ‘background’ staining (fig. A). At higher magnification (fig. b), astrocytic stem processes display spiny or bud-like ecrescences, which are frequently cut-off appearing as isolated puncta (circles). The specimen has been deliberately fixed by immersion for obvious demonstration of these peripheral processes (cf. ‘Technical Comments’, below). Rat hippocampus, CA1.

e.g., in human brain. The PAPs are strongly immunoreactive, appearing as round puncta or buds (Fig. 3b). Whereas this is clearly a swelling-induced phenomenon, the appearance of PAPs in vibratome sections from perfusion-fixed brain is fine and granular (for comparison see the puncta in Fig. 2c and 3b). Immersion fixation may under extreme circumstances even lead to varicose swellings or even fractionation of radiating glial-stem processes.

2.2. Glutamine synthetase and PAP functions 2.2.1. Functional aspects It is an interesting phenomenon that there is a diffuse ‘background’ staining located to ultrastructurally confirmed astrocytic processes, when glutamine synthetase (GS) is determined immunocytochemically (Derouiche and Frotscher, 1991). GS is considered a cytoplasmic marker enzyme for astrocytes (see, however, below), and anti-GS antisera label the entire astrocyte, including its PAPs (Fig. 3). The immunochemical stain in the PAPs appears to be relatively independent of that of the astrocytic stem processes and somata, since the staining intensity of the ‘background’ could vary in a lamina-dependent way with clear-cut boundaries, for example, after hippocampal deafferentation (Derouiche, 2000). However, there were no obvious differences in number of cells or stem processes that might account for these differences. These findings and observations in

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the normal brain (see below) showing lamina-specific differences in the diffuse ‘background’ staining by GS has initiated studies focusing on the PAPs, and the search for selective PAP markers. In addition, the intriguing intense GS-immunoreactivity in the PAPs suggests that the function of this enzyme is particularly associated with these processes. GS activity represents a key function of astrocytes in the CNS, since it catalyses the formation of glutamine from glutamate and ammonia; however, its physiological role might not be the same in the different astrocyte processes (e.g., perisynaptic, perivascular). Since the first localization of GS in the CNS (Martinez-Hernandez et al., 1977), it has been suggested that its function is related to the inactivation of astrocytically accumulated transmitter glutamate and the return to neurons of a glutamate precursor (see chapter by Schousboe and Waagepetersen). In support of this point of view, regional GS immunoreactivity is strongest in retina, hippocampus and olfactory bulb (Norenberg, 1979), CNS regions displaying the highest amounts of glutamatergic activity. When the region-dependent diffuse ‘background’ representing the PAPs is investigated at the level of clearly delineated glutamatergic terminal fields within regions, variations of GS immunoreactivity clearly correspond to such terminal fields, for example, to the primary olfactory termination in lamina I of the perirhinal cortex, the molecular layer in the cerebellar cortex, the perforant path terminal field in the outer (but not the inner) molecular layer of the fascia dentata, or to the smooth lamination of the dense thalamocortical input fields in the somatosenory cortex (Derouiche et al., 1996). At the ultrastructural level, GS is localized in the perisynaptic glial processes around identified glutamatergic synapses (Derouiche and Frotscher, 1991) (Fig. 4). These processes participate in defining glomerular arrangements of synapses in various regions (reviewed by Chao et al., 2002). Pow and Robinson (1994) showed that in explant retina, glutamate localization is restricted to neurons, and glutamine to glia (Mu¨ller cells), and that after GS inhibition glutamine can no longer be detected and glutamate is restricted to Mu¨ller cells. This and a body of biochemical evidence (reviewed by Hertz et al., 1999) support the GS reaction as a key step in the glutamate– glutamine shuttle to replenish the neuronal transmitter pool. Physiologically, however, the GS reaction is not the sole source of transmitter refuel in many other CNS regions, where intracellular glutamate synthesis from glucose in a collaboration between astrocytes and neurons can compensate for the lack of glutamine formation from glutamate accumulated in astrocytes (Hertz et al., 1999). In the hippocampal slice culture, irreversible GS inhibition only leads to approximately 50% depletion of glutamate from axon terminals (Laake et al., 1995), in contrast to the complete glutamate depletion in the retina (Pow and Robinson, 1994). The role of GS in pathophysiology is often considered to be ‘beneficial’ in terms of glutamate-induced excitotoxicity, since the enzyme is involved in the degradation of extracellular glutamate (after transporter-mediated uptake). This would be its primary role, the synthesis of glutamine as a transmitter precursor for glutamatergic and GABAergic neurons being an energy-saving side effect. In the view of recent data, this assumption needs to be revised. Even though several experimental studies have shown that GS inhibition can induce seizures (for example, Tiffany-Castiglioni et al., 1990), recent experiments (Bacci et al., 2002) demonstrate that the decisive glial mechanism in regulating neuronal activity is not the clearance of extracellular glutamate but the supply

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Fig. 4. Perisynaptic glial process (bold arrows) containing glutamine synthetase immunoreactivity (silverintensified DAB reaction product). The asymmetrical synapse on a dendritic spine (s) is situated in the outer molecular layer of the rat fascia dentata, most probably a glutamatergic perforant path terminal. Such processes often extend a fingerlike protrusion (open arrows) towards the synaptic cleft.

of the glutamate precursor glutamine. It appears that the glia-dependent spontaneous oscillatory activity of cultured neurons is critically based on the astrocyte-to-neuron recycling of glutamine (Verdio et al., 1999). The neuronal system A neutral amino acid transporter required for neuronal glutamine uptake can be selectively inhibited (Armano et al., 2002). The epileptiform activity induced in the hippocampal slice culture by blockade of GABAergic transmission can be inhibited by blocking either GS activity or system A neutral amino acid transporter (Bacci et al., 2002). Since the blockade of epileptiform activity by GS inhibition can be restored by exogenous glutamine, and glutamate addition can counteract system A neutral amino acid transporter inhibition (Bacci et al., 2002), GS activity may even enhance neuronal glutamatergic mechanisms. This represents a novel aspect of the role of GS in pathophysiology. In addition to intense staining for GS in perisynaptic astrocyte processes, there is also very intense GS-ir in the perivascular glial processes (Fig. 5), an integral part of the blood – brain barrier, suggesting a role of GS in detoxification of blood-derived ammonia, which can be most effectively achieved here (within physiological limits). This indicates

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Fig. 5. The perivascular cuffs are labeled particularly intense by glutamine synthetase immunoreactivity, indicating its function in this location. Astrocytic end feet can be seen around a vessel in cross-section (bold arrow), or connected to glial-stem processes (small arrows) alongside a vessel. Human hippocampus.

two roles of astrocytes with regard to GS function in situ, which would be localized to different compartments. Astrocytes may display different types of processes also in culture (Derouiche and Frotscher, 2001), but metabolic flux studies on the role of GS in whole cultures cannot differentiate between them or their related ‘physiological’ functions in the brain (transmitter degradation and ammonia detoxification). Thus, overall in vitro measurements (reviewed by Schousboe et al., 1993; Hertz et al., 1999) have shown that the degree to which glutamate is metabolized by GS and by oxidative metabolism, respectively, depends critically on the concentrations of glutamate (McKenna et al., 1996) and ammonia (Schousboe et al., 1993). As a corollary, GS function might be different in the perivascular and perisynaptic processes since it is reasonable to assume that in the brain glutamate concentrations differ between the respective microenvironments. The GS reaction may also trigger pathophysiologically relevant events in addition to metabolism of glutamate in the perisynaptic processes, and detoxification of blood-borne ammonia in the perivascular cuffs. For example, inhibition of GS attenuates the ammonia-induced mitochondrial permeability transition in cultured astrocytes (Bai et al.,

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2001), a transition which is associated with mitochondrial dysfunction and may initiate apoptosis. Similarly, the formation of free radicals observed in a similar situation is suppressed by GS inhibition (Murthy et al., 2001). It remains unknown whether these adverse effects result from glutamine as such or from indirect mechanisms triggered by the GS reaction. 2.2.2. Glutamine synthetase-ir in astrocytes and/or oligodendrocytes Although not directly related to the role of GS in the PAPs, the confusing literature on GS-ir cell types will be overviewed. The specifically astrocytic localization of glutamine synthetase (GS), first demonstrated by Norenberg and Martinez-Hernandez (1979) is of fundamental importance both histologically and metabolically. The overall staining by GS clearly reveals astrocytes, including all cells of the astroglial family (Fedoroff, 1986), i.e., Bergmann glia, Mu¨ller cells (Riepe and Norenberg, 1977), tanycytes (Derouiche, 1997), retinal pigment epithelium (Derouiche and Rauen, 1995) and ependymal cells. The star shaped morphology from classical silver impregnations relates to cortical and hippocampal astrocytes, which display a comparable pattern in material stained for GFAP. However, the dense population of GS-stained astrocytes found in all diencephalic and mesencephalic regions (unpublished observations) indicated that while apparently all astrocytes contain GS, they normally have GFAP-ir filaments only in a region-dependent pattern. This discrepancy is complicated by the emerging view that ‘astrocytes’ constitute a heterogeneous population, even within a given region. In the rat hippocampus, combined immunostainings have revealed that the ‘classical’ GFAP-ir astrocyte constitutes a subpopulation of GS-ir astrocytes, which thus can also lack GFAP staining (direct double staining: Walz and Lang, 1998). However, an inverse relation between GFAP and GS labeling has been observed by Mearow et al. (1989). In view of several astroglial subtypes and/or glial precursors present in the adult rodent brain, anti-GS appears to be the most general astrocyte marker. The general consensus is that GS is a glial marker, which definitely excludes neurons and microglial cells. Neuronal cultures from chick brain display GS activity, which they do not in vivo (Tholey et al., 1987). Except for the recent reports by Robinson (2000, 2001) of GS expression in brains from Alzheimer patients (which is discussed below), the only neuronal localization of GS in situ has been reported recently in a proteomic analysis of squid optic lobe synaptosomes, a definitely glia-free preparation (Jimenez et al., 2001). Some controversy exists regarding the additional labeling of oligodendroglial subtypes, which might render use of anti-GS for the definite identification of astrocytes problematic, for example, in physiological experiments. The question of oligodendroglial expression of GS has not been reviewed systematically. The authors observing oligodendroglial GS localization rely mostly on the morphology of ‘ovoid cells’ in the gray matter, and only sometimes on the unambiguous alignment of interfascicular oligodendrocytes. Only one study each is based on GS mRNA in situ hybridization, which was observed exclusively in astrocytes (Mearow et al., 1989), or on colocalization of GS-ir with oligodendroglial markers. The reports on GS-ir in oligodendrocytes by three groups (Cammer, 1990; D’Amelio and Eng, 1990; Miyake and Kitamura, 1992) do not agree with each other, which might result from the use of different antisera and/or divergent interpretations of

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morphology. Thus, Cammer (1990), applying a proprietary anti-sheep brain GS, observed clearly intrafascicular oligodendrocytes, but only faint white matter astrocytes in rat spinal chord. A similar pattern was evident in rat forebrain white matter (proprietary GS antiserum; specimens prepared by Dr M. Lavialle). Anti-rat liver GS (Cammer, 1990) produced the most convincing intrafascicular oligodendrocytes displaying also immunoreactivity for CNPase, an established oligodendrocyte marker. In gray matter, GS-ir oligodendrocytes were present, but hardly any astrocytes were detectable. Using a different rabbit anti-sheep brain GS antiserum in cat brain, D’Amelio and Eng (1990) found GS localization only in gray matter oligodendrocytes, but not in interfascicular oligodendrocytes. These cells were identified by light microscopic morphological criteria, most of them in perineuronal position. A localization of GS in gray (but not white) matter oligodendrocytes, mostly perineuronal and perivascular was confirmed by plausible ultrastructural criteria, using another rabbit anti-sheep brain GS in the mouse brain (Miyake and Kitamura, 1992). The GFAP-negative and NG2-ir, putative oligodendrocyte precursor cells in the rat forebrain (Ong and Levine, 1999) are devoid of GS-ir (Reynolds and Hardy, 1997). Altogether, the morphological identification of the GS-ir cell types has been unclear in several reports based on the absence of a typical light microscopical pattern distinguishing astrocytes from oligodendrocytes. Although white matter oligodendrocytes are unambiguous, this applies particularly for astrocytes and oligodendrocytes in perineuronal position in cortex or hippocampus, and in most nontelencephalic regions, where astrocytes are generally nonstellate. In the cerebellum, even an ‘oligodendrocytelike astrocyte’ has been described (Palay and Chan-Palay, 1974). As evidence in favor of a GS localization restricted to astrocytes, absence of oligodendrocyte labeling in white and gray matter has been reported by the Norenberg group, in particular at the ultrastructural level (Norenberg, 1979; Norenberg and Martinez-Hernandez, 1979), and the author’s group, who investigated vibratome sections from human (Derouiche and Ohm, 1994), and rat (Derouiche, 1997), using a previously characterized anti-GS antiserum (Hallermayer and Hamprecht, 1984) or commercial GS antibodies (Chemicon mAb, Santa Cruz goat pAb; unpublished observations). In particular, GS-ir in the conspicuous interfascicular oligodendrocytes would not have been overlooked in these studies. Also, a clear distinction by GS-ir between astroglial cells and oligodendrocytes was maintained in the tumors derived from these cells, since all astrocytomas and ependymomas but no oligodendrogliomas were GS-ir (Pilkington and Lantos, 1982). Another astrocytic antigen, ezrin (see above), which labels predominantly the PAPs of all astrocytic cells but not oligodendrocytes (Derouiche and Frotscher, 2001), has a corresponding, clear-cut specificity within the range of human glial tumors (Geiger et al., 2000). Thus, most available data do converge on a clear astrocytic identification by GS, at least in the rodent hippocampus (see below). 3. PAPs: other markers and functions 3.1. Preferential versus additional PAP labeling Glial markers for antigens in PAPs can label either the entire astrocyte in a way comparable to GS, including the submicroscopic PAPs, or selectively the PAPs, as with

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anti-ezrin. There are a variety of astroglial antigens localized in the PAPs. These proteins define the specialized properties and functions of PAPs. The following two paragraphs enumerate some of them, categorizing them into the two labeling patterns (preferential or additional PAP labeling). The list does not aim at being complete, and contribution of the respective antigens to the PAP’s functional properties is only hinted at.

3.2. Preferential PAP labeling One of the selective PAP markers is the actin-binding protein alpha-actinin. Abd-El-Basset et al. (1991) have shown the preferential labeling of highly motile actincontaining structures, such as lamellipodia and filopodia, in cultured astrocytes. Here, it is strictly colocalized with phalloidin-labeled actin bundles (Abd-El-Basset et al., 1991); this is in contrast to the actin-binding protein, ezrin, which is mostly punctate and lining cell boundaries (Derouiche and Frotscher, 2001). However, in contrast to the neuronal alphaactinin 2, localization of alpha-actinin has not been investigated in vivo. The patchy, faint neuropil staining typical of PAPs is also revealed by the astrocyte specific carbohydrate epitope CD15, an observation confirmed at the ultrastructural level, in the rat (Gocht et al., 1994). The restricted PAP staining is apparent only in gray matter astrocytes. In white matter, CD15 positive astrocytes are obvious, although in a region specific way. For example, CD15 staining is absent in cerebellar white matter (Gocht et al., 1994). In the optic nerve, CD15 appears to be related to distinct to cell-to-cell contact sites of astrocytes, supporting a role of CD15 in intercellular recognition processes (Gocht et al., 1994). Interestingly, the regional specificity may be related to developmental events, since CD15 expressed on a subset of radial glia could play a role in developmental demarcation of prosencephalic regions (Mai et al., 1998). Certain extracellular matrix (ECM) components (glycoproteins and lectin-binding proteoglycans) in the adult CNS can basically assume two forms under the light microscope (Derouiche et al., 1996), as visualized by lectin-staining or immunocytochemistry for ECM components. The very conspicuous perineuronal nets (Celio et al., 1998) cover neuronal somata and proximal dendrites, with holes occupied by perisomatic and dendritic axon terminals (Bru¨ckner et al., 1993). An analogous, but less obvious form of ECM staining, is diffuse and present, e.g., in the hippocampal principal cell layers (Derouiche et al., 1996). These ECM patterns are closely related to and even clearly congruent with GS-ir PAPs (but not with GS-ir glial-stem processes; Bru¨ckner et al., 1993; Derouiche et al., 1996). However, a large body of evidence has established that the perisynaptic lectin-binding ECM is primarily deposited by neurons (reviewed by Celio et al., 1998). It has been hypothesized that the perineuronal nets, in conjunction with the PAPs, participate in regulating ionic diffusibility and availability in the perisynaptic microenvironment (Ha¨rtig et al., 1999), thereby supporting a subset of parvalbumincontaining, fast-spiking neurons, on which the perineuronal nets are predominantly found. The connexins, in particular connexins 43 and 30, constitute the glial gap junctions, which establish the functional astrocyte syncytium (reviewed by Dermietzel, 1998). Connexin-ir does not outline astrocytes, and at high magnification, connexin 43 staining is more punctate and less diffuse than the ezrin pattern (Rohlmann et al.,

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1993; Nagy et al., 1999). Structurally, this reflects that the membrane organization of the connexins is clustered, and that the glial gap junctions form preferentially at the level of the PAP compartment (see also chapter by Scemes and Spray). Connexin 43 labeling in primary cultures of astrocytes displays a corresponding preponderance for filopodia, or in polygonal astrocytes for the very cell boundary (Wolff et al., 1998; Hofer and Dermietzel, 1998), suggesting the presence of specific mechanisms targeting connexin 43 to the PAPs or to the filopodia compartment. Application of connexin 43 labeling at low magnification as a tool to study the diffuse, field-specific labeling of rat cortex has revealed short term plasticity of PAPs, and Landgrebe et al. (2000) observed a shift in cortical field labeling as rapidly as 60 min following facial nerve lesion. Of the many glial transporters, only the high-affinity glutamate transporters (Rothstein et al., 1994) will be discussed here. In particular EAAC-1 and GLT-1 display the preferential PAP pattern, which is characteristically diffuse and was even overlooked in the initial description (Rothstein et al., 1994) and only reported later (Conti et al., 1998). The selective PAP localization of glutamate transporters obviously reflects their role in glutamate clearance from the perisynaptic microenvironment (Conti et al., 1998; Rothstein et al., 1994), a function which is less important in the glial-stem processes. Co-distribution of glutamate transporters and GS in the PAPs, as observed in the retinal Mu¨ller cell processes around the glutamatergic photoreceptor terminals, indicates that GS metabolizes the transmitter glutamate taken up by the transporter (Derouiche and Rauen, 1995), a situation that can be extrapolated to the brain based on the extant data. A variety of glial receptors and ion channels also appear to specifically contribute to the functions of the PAPs. As one example, Kir6.1, a pore-forming subunit of the ATP-dependent Kþ channels, is another selective PAP marker (Thomzig et al., 2001).

3.3. Additional PAP labeling Labeling of the entire astrocyte by the well-established markers anti-GS and antiS100b appears darker than labeling of filaments by anti-GFAP or anti-vimentin, since the additional labeling of the submicroscopic PAPs induces a ‘background’. These markers are effective tools for PAP labeling, if selective labeling is not required. Thus, PAPs intervening between perisomatic axon terminals, and their relation to perineuronal nets have been studied by aid of these markers (Bru¨ckner et al., 1993; Derouiche et al., 1996). Markers yielding this staining pattern are often molecules considered cytosolic, such as GS and S100b (Rickmann and Wolff, 1995), a Ca2þ-binding protein, enriched in astrocytes. Similarly, the astrocyte specific enzyme glycogen phosphorylase is cytosolic, labeling the entire cell, including the PAPs (Richter et al., 1996). The importance of PAP localization of this enzyme is functionally interesting, since the degradation of glycogen by this enzyme is related to neuronal activity. The enzyme initiates production of lactate or pyruvate, which may be oxidized by the astrocytes and perhaps also transferred to adjacent neuronal elements as a substrate for their energy metabolism (Dringen et al., 1993; Pellerin and Magistretti, 1994). A glial pyruvate/lactate transporter (monocarboxylate

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transporter2) displays a comparable light microscopic pattern, although its presence appears to be region-dependent (Gerhart et al., 1998). The localization of CD44, an astroglial adhesion protein, has not been systematically investigated in the rodent brain. In the human brain, it labels astrocytes according to the ‘GS-pattern’ (Akiyama et al., 1993). The PAP localization of CD44 is interesting, since it is known in other cell types to interact with ezrin (Yonemura et al., 1998), although this interaction remains to be demonstrated for the astrocyte. If so, it may be speculated that the PAP might be selectively involved in binding of hyaluronic acid and T cells, by ezrinbased mechanisms and CD44 (Asher and Bignami, 1992). 4. Pathological alterations of PAP integrity and GS staining It might provide fruitful insight to apply the above-mentioned view of glial compartments to glial pathology. For example, compartmentalized (axonal or axon terminal) pathology in the neuron has greatly added to the understanding of disease mechanisms of multiple sclerosis and myasthenia gravis. Selective dysfunction of proteins enriched in PAPs or impaired structural integrity of PAPs might similarly be associated with key functions of the nervous system, such as metabolism of transmitter glutamate and rapid glial plasticity. In human autopsy material, microscopy of the GS-ir PAPs and perisynaptic glial sheaths is facilitated, since they are swollen by immersion-fixation, appearing as ‘perisynaptic buds’ (Robinson, 2001). These structures are clearly reduced in Alzheimer’s disease brain (in inferotemporal cortex) although overall astrocytic morphology appears normal (Robinson, 2001). In addition, the intensity of GS-staining of the remaining PAPs was reduced, which might be related to the likewise reduced presence of glutamate transporters in PAPs, in Alzheimer’s disease (Lauderback et al., 2001; Robinson, 2001). Thus, in addition to other glial roles in Alzheimer’s disease (see chapter by Barger) one feature of this disorder appears to be the selective dysregularation of glia-synaptic interaction, at the level of the PAP. It has recently been reported that neuronal perikarya, particularly of pyramidal cells were intensely labeled for GS in human autoptic material from individuals suffering from Alzheimer’s disease, (Robinson, 2000, Robinson, 2001). Previous evidence and technical considerations do not support this observation, which contrasts with previous human data (Pilkington and Lantos, 1982; Smith and Lantos, 1985; Derouiche and Ohm, 1994). However, neuronal GS labeling similar to that reported by Robinson (2000, 2001)) has been observed in sections from rat brain (perfusion or immersion-fixed, vibratome) or human hippocampus (vibratome, paraffin), applying various anti-GS antisera in two laboratories (author’s unpublished observations; Dr. M. Lavialle, personal communication). This neuronal labeling was regarded as spurious, since it occurred inconsistently after storage (exceeding one week), often without the expected glial staining, even in vibratome sections from the same block that had yielded the exclusive astroglial pattern in staining runs before. This might indicate the recognition of distinct epitopes, displaying independent physicochemical properties. Interestingly, the anti-GS mAb (Chemicon) used by Robinson (2000, 2001)) has been found to cross-react with a ‘GS-like protein’ (Boksha et al., 2000) different from GS; however, its cellular localization in the brain has not been established.

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Ezrin, normally a PAP specific protein, is upregulated in glial tumors of astrocytic origin, and there is a high degree of correlation between anti-ezrin staining intensity and astrocytoma malignancy, whereas ezrin is not present in oligodendrogliomas at all (Geiger et al., 2000). Astrocytoma cells display an increasingly rarified process tree, glioblastoma cells being plump and devoid of processes, and ezrin is distributed in the remaining stem processes and the perinuclear region. (Geiger et al., 2000). Since ezrin also is restricted to astrocytes in the normal brain, its role in malignant transformation might be related to its physiological functions, e.g., filopodia formation, cell motility and shape changes (Tsukita et al., 1997). It is consistent with this concept that ezrin mediates motility and invasiveness of glioma cells in vitro (Wick et al., 2001), and that malignant behavior in cultured fibroblasts can be induced by and is dependent on ezrin (Lamb et al., 1997). 5. Concluding remarks Recently developed immunohistochemical methods have allowed the determination of a separate astrocytic compartment, the PAPs, which in many respects corresponds to the filopodia and lamellipodia of astrocytes described by classical anatomical techniques. It is intriguing that these processes form a separate astrocytic compartment, as indicated by the selective expression of specific proteins. In addition it expresses surface-bound proteins, like glutamate transporters, and part of the glutamine synthetase expression in astrocytes. This compartment is likely to be of key importance for many astrocytic functions and it may be a target of astrocytic dysfunction. Acknowledgements The author wishes to thank Andrea Hufschmidt for assistance in bibliography. Supported by Deutsche Forschungsgemeinschaft (DFG De 676/2-3)

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The astrocytic syncytium Eliana Scemesa,* and David C. Sprayb a

Department of Neuroscience, Albert Einstein College of Medicine, Room 203 Kennedy Center, 1300 Morris Park Avenue, Bronx, New York 10461 p Correspondence address: Tel.: þ1-718 430-3303; fax: þ1-718 430-8594 E-mail: [email protected] b Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Contents 1. 2. 3. 4.

5.

Introduction: Do astrocytes form a syncytium? Gap junctions represent a compromise between syncytium and cellularization Structural features of the astrocyte syncytium What are the substances that are distributed through the astrocytic syncytium? 4.1. Kþ siphoning 4.2. Distribution of energy sources and metabolites in the CNS 4.3. Modulation of neuronal activity: waves of intracellular calcium in the astrocytic network 4.4. Transmission of death signal versus neuroprotection Concluding remarks

In this chapter, we have summarized evidence that gap junctions between astrocytes provide a pathway for direct intercellular exchange of ions, nutrients and signaling molecules. We have also provided arguments that this direct intercellular pathway may be part of the machinery responsible for delivery of nutrients to neurons, ridding the extracellular space of excess of Kþ and glutamate, spreading intercellular waves of Ca2þ, and exchanging cell death signals. Although this intercellular network is not truly syncytial, as it consists entirely of discrete cellular elements, the interconnections extend cellular functions, such as enlarging the buffer volume for dilution of Kþ and toxins, as well as integrating cells with different classes of nonjunctional proteins into functional units.

1. Introduction: do astrocytes form a syncytium? A syncytium is defined as a multinucleated mass of cytoplasm that is surrounded by a continuous plasma membrane with no individualized units (cells). In the truest sense of the Advances in Molecular and Cell Biology, Vol. 31, pages 165–179 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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word, astrocytes accordingly do not form a syncytium. However, as it will be discussed below, they can be regarded as forming a functional syncytium, because intercellular channels (gap junctions) span the membranes of adjacent cells, allowing direct exchange of substances from the cytosol of one cell to that of its coupled neighbors.

2. Gap junctions represent a compromise between syncytium and cellularization There are numerous authentic types of syncytial tissues in vertebrates, invertebrates and plants. In invertebrates, many species start off their lives as multinucleated embryos, where considerable regional differentiation can occur even in the absence of cellularization, including patterning of subcellular regions (see Mazumdar and Mazumdar, 2002; Holloway et al., 2002). In both lower- and higher plants, tissues are syncytial, with plasmodesmata (fine strands of cytoplasm surrounded by plasma membrane) providing portals penetrating the cellulose walls that encase the plasmalemma (see Haywood et al., 2002). In mammals, examples of a true syncytium include the multinucleated spermatogonia from which mature spermatocytes differentiate in the seminiferous tubule; skeletal muscle fibers, where fusion of myoblasts precedes differentiation; and the socalled villous trophoblast that forms the maternal side of the placenta (see Moens and Hugenholtz, 1975; Jaggi et al., 1997; Huppertz et al., 2001). Under pathological conditions, multinucleated giant cell formation can occur due to viral infection not only in the hematopoetic system, but also in the CNS, where fusion of microglia and resident macrophages results from HIV infection (see Orenstein, 2001). Such syncytial formations are the neurological hallmark of HIV infection and a prognostic indicator of dementia. The transformation from a syncytium to individualized cells, a process called cellularization, is a feature common to several morphogenetic pathways with each nuclei of the multinucleated blastoderm becoming enclosed in its surrounding cytoplasm by a growing membrane (see Mazumdar and Mazumdar, 2002; Holloway et al., 2002). During this process, a fully functional epithelium containing membrane compartments is gradually formed with specialized and distinct junctions that will ultimately separate each cell into a functional unit. In a syncytium, the ratio of volume to surface area is maximized, so that metabolic supplies and energy sources can be efficiently utilized by a massive embryo or by the contractile machines that are skeletal muscle fibers. However, the steepness of gradients of proteins and other molecules that can be generated within a syncytium by asynchronous activity in individual nuclei is greatly compromised by the absence of cell membranes (Fig. 1; nevertheless, see Kerszberg and Changeux, 1994, and Holloway et al., 2002 for models by which steep gradients can be generated without cellular boundaries). True cellularization seem to have arisen as a mechanism to increase molecular gradients from the nucleus of one cell to the nucleus of another (see Fig. 1A), providing the opportunity for greater cellular diversity and permitting the accumulation of higher concentrations of cytoplasmic compounds. Gap junction channels allow diffusional exchange of ions and small molecules from one cell to the next and appear to represent an adaptive compromise between a true

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Fig. 1. Hypothetical intra- and intercellular gradients (black lines) of a gap junction permeant, intracellularly metabolized molecule generated in the center of a group of three cells or cellular regions (gray boxes, labeled Cell 1, 2 and 3 and Region 1, 2 and 3). (A) In the case of cells with no leakage of the molecule from the cell in which it is generated, there is a shallow gradient within the cell, due to its metabolism, and no intercellular diffusion. (B) In the absence of cellular boundaries, the molecule diffuses from its source at a constant rate. (C) In the presence of gap junctions connecting the cells, the molecule diffuses intracellularly with a shallow gradient that is steeper at the junctional membrane due to impeded diffusion.

syncytium and cell individuality (see Figs 1B and C). Gap junctions are a common feature of most vertebrate and invertebrate tissues, where they connect cells into large networks capable of exchanging ionic and metabolic signals. For example, gap junctions in the heart serve to relay depolarizations necessary for synchronization of ventricular contraction, in the liver serve to coordinate the glycolysis and gluconeogenesis that are performed by separate cells in the lobules, and in the lens to provide nutritional support in the absence of vascular supply. In astrocytes, such interconnections play multiple roles, as discussed in Section 3.

3. Structural features of the astrocyte syncytium Although astrocytes, as well as other tissues whose cells are connected by gap junction channels such as cardiac myocytes in the heart, hepatocytes in the liver and outer lens cells in the eye, are not considered to be syncytial, they may be viewed as a functional syncytium with regard to certain tissue demands; this is so, because these intercellular channels span the membranes of joining cells, allowing direct exchange of ions, signaling molecules and metabolites from the cytosol of one cell to that of its coupled neighbors.

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In such a conceptual view of a functional syncytium, it would be expected that astrocytes would coordinately and cooperatively react to long-range neuronal activity and environmental stimuli. The understanding of the structural organization of the glial network has been radically changed by the very recent study of Bushong et al. (2002), in which individual protoplasmic astrocytes in brain slices were injected with fluorescent dyes following 4% paraformaldehyde fixation. This preparation leads to blockade of gap junction channels and superior preservation of cell structure, both of which were critical to the findings reported. Somewhat surprisingly, the dye injections (with Lucifer Yellow or the newer Alexa fluorescent compounds from Molecular Probes) revealed an intricate web of ‘spongiform’ astrocyte processes extending much farther and more densely than had been apparent from previous studies using the astrocyte marker GFAP, the estimated volume occupied by a single astrocyte being about 66,000 mm3. Although previous studies had obtained abundant Connexin(Cx)43 immunostaining within the spherical or oblate domains of individual astrocytes (Rohlmann and Wolff, 1996; Wolff et al., 1998), the Bushong et al. (2002) study clearly demonstrated that inter-astrocytic contact is limited to the margins between the fine spongiform processes, so that only the outermost processes of astrocytes are in true intercellular contact (see Fig. 2). This finding indicates that individual astrocytes occupy virtually nonoverlapping volumes, with little or no interdigitation of the major astrocyte processes. As pointed out by the authors, one implication of these findings is that a single astrocyte might be in contact with more than 100,000 neuronal synapses and not share this direct contact with other astrocytes. In apparent contradiction to these nonoverlapping astrocyte – astrocyte domains is that extensive immunostaining for Cx43, the major astrocytic gap junction protein, that has been reported within the domains of a single astrocyte, both in brain sections and in

Fig. 2. Discrete regions of interaction between the fine processes of protoplasmic astrocytes, which have been filled with red or green dye. Pixels containing both green and red (and therefore corresponding to overlapping astrocytic processes) were pseudocolored in bright yellow to mark their presence. Scale bar, 20 mm. Modified very slightly from Bushong et al. (2002).

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cultured cells (Rohlmann and Wolff, 1996; Wolff et al., 1998). These domains were termed ‘autocellular zones’ by Rohlmann and Wolff (1996), consistent with their observation that the abundant localization of Cx43 within the autocellular space is, in part, due to small astrocytic processes forming autaptic gap junctions onto other fine processes or onto major branches of the same astrocyte. Such an arrangement is similar in principle to the autaptic or reflexive gap junctions formed by Cx32 between cytoplasm-containing regions squeezed off by compact myelin in Schmidt –Lantermann incisures and paranodal loops of myelinating Schwann cells and in paranodal regions of oligodendrocytes (see Spray and Dermietzel, 1995; Scherer et al., 1999). The function of such junctions is presumed to lie in nutrient delivery from nucleus to innermost regions of the Schwann cell (or, alternatively, for exchange of signaling molecules generated at the contacts between axons and the innermost Schwann cell membrane with the Schwann cell body), thereby shunting the tortuous route created by as many as 100 wraps of the Schwann cell around the axon (Balice-Gordon et al., 1998). A somewhat similar role might be played by reflexive gap junctions in astrocytes, where changes in coupling strength might isolate or integrate microdomains, as could occur where fine astrocytic processes surround synapses. For example, functional microdomains have been demonstrated in Bergmann glia, to which the spread of Caþ2 elevations is regionally limited in response to neuronal activity (Grosche et al., 1999). Two other findings reported by Bushong et al. (2002) are noteworthy. First, the topologies of the volumes proscribed by the individual astrocytes were found to be variable, ranging from roughly spherical to quite oblate. Just as the extracellular space has been shown to exhibit anisotropic permeation by tracer molecules (Sykova, 1997; Nicholson and Sykova, 1998), such differences in form of astrocytes would be expected to produce anisotropic intercellular diffusion of ions and small molecules. Secondly, although processes of neighboring astrocytes were not found to penetrate the outer processes of adjacent astrocytes, oligodendrocyte processes freely intermingled within the ‘auto-cellular zone’. Direct oligodendrocyte –astrocyte gap junctions have long been known from thin section and freeze –fracture studies (Massa and Mugnaini, 1985), and recent studies using freeze –fracture immunolabeling (FRIL) have confirmed their abundance (Nagy and Rash, 2000), giving rise to the concept of global functional intercellular communication throughout the brain, the so-called ‘panglial’ syncytium (Rash et al., 1997). A number of different types of gap junction proteins (connexins) connect astrocytes and oligodendrocytes among themselves and between one another. The gap junction gene family currently has about 20 identified members in mammals, more than half of which are found in the brain (Willecke et al., 2002; Spray et al., 2003). The primary gap junction protein in astrocytes is Cx43. Although Cx26, Cx30, Cx40, Cx45 and Cx46 have also been reported to be expressed in astrocytes in vivo or in culture, total junctional conductance between cortical astrocytes from Cx43 null mice is 95% less than between astrocytes from wildtype siblings (Scemes et al., 1998), indicating that this gap junction protein ordinarily supplies the vast majority of the intercellular communication. In oligodendrocytes, Cx32 and Cx45 were reported initially (Dermietzel et al., 1997; Kunzelmann et al., 1997), but it now appears that the Cx45 probes used in those studies may have crossreacted with the newly identified Cx47. Although Cx47 was initially described as neuronal in its

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distribution (Teubner et al., 2001), recent evidence suggests that it is mainly (or exclusively) expressed in myelinating cells of the CNS and PNS (D.L. Paul, personal communication). Because the most abundant astrocytic connexin, Cx43, does not form functional channels when paired with cells expressing Cx32 (White and Bruzzone, 1996), it seems likely that astrocytic –oligodendrocytic coupling is established by heterotypic gap junctions, with Cx43 on the astrocyte side and Cx47 contributed by the oligodendrocytes. Such connections between oligodendrocytes and astrocytes may explain in part the abundance of Cx43 staining within the autocellular astrocytic zones, although it is certainly likely that the demonstrated autaptic contacts between fine processes of a single astrocyte also contribute to the Cx43 staining in these zones. 4. What are the substances that are distributed through the astrocytic syncytium? Although individual types of gap junction channels may be somewhat selective with regard to whether anions or cations permeate the channels, all types of connexin channels allow direct communication of virtually all molecules with molecular weights less than 1000 Da. Despite this broad range of permeability, gap junctions impose a resistance to permeation, establishing gradients throughout a tissue (Fig. 1C). This resistance enables a high cytosolic concentration of gap junction permeant molecule in one cell to dissipate only gradually in time and space through the coupled network. Spatial gradients may be established either through generation of these molecules at specific locations or through their uptake from extracellular space in a subpopulation of cells. Dissipation of gradients driven by uptake of potassium ions (Kþ) from areas around active neurons may be the most paramount function of astrocytes, with gap junctions mediating siphoning of that Kþ, a process that is well established in the retina (Fig. 3). Because there is a great variety of molecules that can cross cell boundaries through gap junction channels, (Kþ ions, cAMP, IP3, Ca2þ, glucose, glutamate, etc.) the functions performed by the interconnected astrocytic syncytium may be quite diverse. Some of the roles performed by astrocytes which involve the participation of gap junctions are summarized below. 4.1. Kþ siphoning The concept that spatial buffering of extracellular Kþ is one of the key roles played by astrocytes in CNS function was first proposed by Dick Orkand, John Nichols and Steve Kuffler (Orkand et al., 1966; Kuffler et al., 1966). Through this mechanism, glial cells were proposed to remove the excess Kþ released at synaptic sites (source) to regions of low extracellular Kþ (sink). Evidence that glia cells mediate such Kþ flux has especially come from experiments performed in the Mueller cells (Fig. 3A), showing that these modified retinal glial cells transported Kþ from the plexiform layers to the vitreous body, blood vessels and subretinal space (Newman, 1985; Reichenbach et al., 1992; Newman and Reichenbach, 1996). Although the Naþ/Kþ ATPase, the Naþ, Kþ, 2Cl2 transporter, and passive uptake of KCl may also participate in Kþ re-distribution (Walz, 2000; Chen and Nicholson, 2000—see also chapter by Walz), siphoning of Kþ into glia is likely

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Fig. 3. Spatial buffering of potassium (Kþ) by glia. (A) Neuronal activity in the outer and inner plexiform layers (OPL, IPL) leads to increased Kþ concentration in the extracellular space, which is siphoned by the Mueller cells. The influx of extracellular Kþ (white arrows) into the Mueller cells is facilitated by the high density of Kir2.1 potassium channels expressed in domains of the Mueller cell facing the OPL and IPL. The efflux of Kþ (black arrows) occurs at the end-feet of the Mueller cells, facing the vitreous humor and the capillaries and at the apical end of the cell facing the subretinal space, regions with a high density of Kir4.1 potassium channels. Modified from Newman and Reichenbach (1996) and Kofuji et al. (2002). (B) Hypothetical model for astrocytic Kþ siphoning in which gap junction channels would assemble subpopulations of astrocytes expressing Kir2.1 and Kir4.1 potassium channels. In this model, similarly to a Muller cell, the influx of Kþ into the astrocytic network (white arrows) would occur at sites where the strongly rectifying Kir2.1 potassium channels are expressed. The efflux of Kþ (black arrows) would occur through the weakly rectifying Kir4.1 potassium channels located at the end-feet of astrocytes facing the vasculature. Thus, Kþ ions would be redistributed from sites of high extracellular Kþ concentration (indicated as 12 mM, corresponding to intense neuronal activity) to regions of low extracellulat Kþ levels (the two ends of the chain of astrocytes, representing the end-feet). [Although the spatial buffering provided by the Mueller cells is quantitatively important for removal of extracellular Kþ in the retina, astrocytic spatial buffering in the brain is probably performed over shorter distances; see text for details].

to be mainly mediated by inward rectifying Kþ channels (Newman, 1985; Reichenbach et al., 1992; Kofuji et al., 2002). Immunohistochemical studies performed on mouse retina have identified two different subtypes of inward rectifying Kþ channels differentially expressed in the Mueller cells. These are (i) the strongly rectifying Kþ channel (Kir2.1) highly expressed in membrane domains of the Mueller cells extending into the (source) plexiform layers that would mediate the influx of Kþ into the Mueller

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cell, and (ii) the weakly rectifying Kþ channel (Kir4.1) expressed predominantly at (sink) the end feet located at the vitreous body and blood vessels, which will favor the efflux of Kþ from the Mueller cells (Kofuji et al., 2002). In brain, the contribution of the spatial buffer mechanism to Kþ homeostasis is more uncertain (see also chapter by Walz). However, in various brain regions, Kir4.1 immunoreactivity was found in about half of the astrocytes, being mainly expressed at regions where the astrocytic end-feet meet the blood vessels (Takumi et al., 1995; Poopalasundaram et al., 2000; Higashi et al., 2001; Schroder et al., 2002; Kofuji et al., 2002), whereas Kir2.1 transcripts were observed in subpopulations of astrocytes in different brain regions (Kofuji et al., 2002; Schroder et al., 2002). It is conceivable that the distribution of these two inward Kþ channels are confined to different subpopulations of astrocytes in the brain, and that gap junction channels by linking the members into a network would assemble the different specialized membrane areas. This arrangement would form a multicellular astrocytic network functionally equivalent to a Mueller cell (see Fig. 3B). Although the space constant for spatial buffering is unknown, it has been estimated that the astrocytic syncytium is far more likely to support Kþ transport than the extracellular diffusion (Gardner-Medwin, 1983; Gardner-Medwin and Nicholson, 1983) and propagates Kþ waves faster than the extracellular space (Amzica et al., 2002). Gap junction channels are permeable to Kþ, and thus are likely to provide the astrocytic network with the necessary volume to accommodate the focal influx of high concentrations of Kþ. In this regard, it has been shown that exposure of astrocytes to high Kþ solutions increase the coupling strength (Enkvist and McCarthy, 1994; De Pina-Benabou et al., 2001), thus expanding the effective volume of the interconnected astrocytes while maintaining a high surface area for Kþ uptake.

4.2. Distribution of energy sources and metabolites in the CNS Glucose together with lactate, ketone bodies and glutamate/glutamine are the main sources of energy utilized by the brain (Williamson, 1982; Lopes-Cardozo and Klein, 1985; Vicario et al., 1993). Although glucose is the only blood-borne substrate used by the brain as an energy source, lactate and other nonoxidized products of glucose metabolism are consumed as fuel during elevated brain activity (see Dienel and Hertz, 2001). A selective transport system localized in the Blood Brain Barrier (BBB) provides glucose to the cells within the brain. Given that astrocytes are interposed between the capillaries and neuronal elements and thus are the first cellular elements that glucose entering the brain encounters after crossing the endothelium of the BBB, astrocytes were assigned a nutritive role for neurons. Astrocytes express the glucose transporter Glut1 (Maher et al., 1994) and store glucose in the form of glycogen. Given that astrocytes lack the enzyme that transforms glucose-6-phospate into glucose during glycogen breakdown, the glycolytic product lactate can be delivered from astrocytes to neurons as a source of energy (see Giaume et al., 1997), or it can be transported within the astrocytic syncytium, including autaptically, and subsequently be oxidized by astrocytes (see chapter by Peng et al.). Glutamate, the main excitatory neurotransmitter released by CNS neurons, is taken up by astrocytes and together with ammonia is converted in glutamine, which is then

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delivered to neurons (see Broer and Brookes, 2001 and chapter by Schousboe and Waagepepetersen). Although glucose, lactate, glutamate and glutamine can diffuse through the extracellular space and then be taken up by neuronal cells, it is also likely that these fuels travel more efficiently through the astrocytic network, by diffusing through gap junction channels (for review see Dienel and Hertz, 2001). Permeability of gap junction channels to 2-deoxyglucose has been demonstrated in cell lines (Pitts and Finbow, 1977), in the lens (Goodenough et al., 1980), and between smooth muscle cells (Cole et al., 1985; Cole and Garfield, 1986). The passage of glutamine and glutamate, lactate and glucose through astrocytic gap junction were shown using the scrape loading technique applied to cultured astrocytes (Tabernero et al., 1996; Giaume et al., 1997), and in vivo by the use of gap junction channel blockers, which caused a 50% reduction of (14C) glucose spread through the brain of a conscious rat (Cruz et al., 1999; Dienel et al., 2001).

4.3. Modulation of neuronal activity: waves of intracellular calcium in the astrocytic network Recently, several lines of evidence have indicated that astrocytes, besides their role in regulating the levels of extracellular Kþ and glutamate (see above), produce calcium waves (Cornell-Bell et al., 1990; Charles et al., 1991) and modulate neuronal activity (Nedergaard, 1994). Intercellular calcium wave spread is a phenomenon characterized by an increase in cytosolic calcium levels within one cell, that is followed by increased intracellular Ca2þ levels in adjacent cells in a wave-like, propagating fashion (see chapter by Cornell-Bell et al. and Shuai et al.). Coordinated endogenous neural activity in the form of propagating calcium waves has been observed in developing retina, cortex and in hippocampal-slices (Galli et al., 1988; Meister et al., 1991; Yuste et al., 1992; Feller et al., 1996; Harris-Whyte et al., 1998; Kandler and Katz, 1998; Graschuck et al., 2000). Using brain slices from transgenic mice, in which astrocytes were labeled by a green fluorescent protein expressed under the GFAP promoter, it was shown that local electrical stimulation induced intercellular wave propagation that spread not only through the GFAP fluorescent cells, but also to other nonastrocytic glial cells over a distance of 100 mm (Schipke et al., 2002). Propagating intercellular calcium waves have not only been observed in response to mechanical, electrical and chemical stimulation, but they also occur spontaneously, independent of neuronal activity. In the presence of tetrodotoxin to block synaptic activity, astrocytes from the ventrobasal thalamic slices of rat were shown to display spontaneous Ca2þ oscillations (Parri et al., 2001; Thashiro et al., 2002), which could be propagated to other astrocytes, and which could induce neuronal activity along the wave path (Parri et al., 2001). Modulatory action on neuronal activity corresponding to calcium wave spread between astrocytes have been shown to be related to the release of glutamate and ATP from these glial cells, acting on glutamatergic and purinergic receptors expressed in neuronal plasma membranes (Parpura et al., 1994; Kang et al., 1998; Fields and Stevens-Graham, 2002). There are two ways by which calcium signals can be transmitted from one astrocyte to another: (i) through gap junction channels due to the diffusion of calcium releasing molecules, such as Ca2þ and/or IP3, generated in one cell, and directly spreading to

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adjacent cells to which it is connected, and (ii) through the extracellular space by the diffusion of neuroactive substances released from one cell, such as ATP and glutamate, and the stimulation by the released neuroactive substance of receptors in the membrane of neighboring nonconnecting cells (see Scemes, 2000). Gap junction channels have been shown to be permeable to IP3, Ca2þ and cAMP. Micro-injection of IP3 and Ca2þ into one hepatocyte was shown to induce cytosolic calcium rise in the adjoining coupled cells (Saez et al., 1989); in C6 glioma cells, permeability of Cx43 and Cx32 gap junction channels to IP3 have been shown by photoreleasing caged IP3 and imaging of intracellular calcium rises in coupled cells (Fry et al., 2001). Although several lines of evidence indicate that gap junction channels participate in the propagation of calcium signals between cultured astrocytes, their participation in calcium wave propagation in slice preparation have been somewhat questionable. Different from their effects under culture conditions, application of gap junction channel blockers (octanol, etc) did not prevent electrically induced calcium waves between astrocytes in slice preparations, which instead were blunted by the application of purinergic receptor blocker Reactive Blue (Schipke et al., 2002). Such conflicting data may be related to the way by which astrocytes establish their connection in situ and in culture; as mentioned above, the majority of gap junction channels in astrocytes in brain slices are found as autaptic junctions, rather than between pairs of astrocytes, which would be expected to reduce the range to which IP3 will diffuse through the syncytium. Interestingly, under certain pathological conditions, a switch from a gap junctiondependent to a gap junction-independent, purinoceptor-dependent mechanism for calcium wave propagation was observed in cultured human astrocytes. Such an alteration occurred after interleukin1-b treatment of cells, and it was paralleled by a decreased expression of Cx43 levels and upregulation of P2 receptors (John et al., 1999). A similar situation was reported in cultured spinal cord astrocytes from Cx43 knockout astrocytes and in wild-type cells treated with Cx43 anti-sense oligonucleotides (Scemes et al., 2000; Suadicani et al., 2003). Thus, it is likely that such differential contribution of the two pathways for calcium wave propagation observed in slices and in culture preparations may be related to the degree of gap junctional communication between astrocytes from distinct brain regions. For example, junctional conductance between astrocytes has been shown to vary from about 17 nS in cortical astrocytes to about 9 nS in spinal cord astrocytes (Scemes et al., 1998, 2000).

4.4. Transmission of death signal versus neuroprotection Although gap junctions have been implicated in the transmission of damage signals from injured cells to normal cells, as observed in alpha-irradiated cells (Azzam et al., 2001), the issue of whether gap junctional communication confers a neuroprotective role is still controversial. In a stroke model, glial cell death occurring during secondary expansion of infarction was shown to be reduced by gap junction channel blockers (Rawanduzy et al., 1997; Saito et al., 1997). Similar results were obtained by comparing the extent of cell death in an in vitro trauma model (organotypic slice culture), in which the contribution of gap junctional communication to cell death was evaluated using gap junction channel

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blockers (heptanol and carbenoxolone) and by down regulation of Cx43 expression either by the use of antisense-oligonucleotides or from molecularly engineered Cx43 KO mice (Frantseva et al., 2002). Furthermore, although junctional conductance is decreased by intracellular acidification, as a rapid gating response (Spray et al., 1981; Ek-Vitorin et al., 1996) and also possibly due to the activation of a protein kinase that phosphorylates Cx43 on serine residues (Yahuaca et al., 2000), astrocytic gap junction channels were shown not to be totally closed at the penumbra of an ischemic region and to participate in the amplification of the damaged area (Cotrina et al., 1998). Contrary to expectations, however, studies performed in Cx43 heterozygotes (HT), which express less than half the levels of Cx43 protein compared to wildtype (WT), have indicated that gap junctions may be neuroprotective. These studies performed on Cx43 HT mice 4 days after obstruction of the right middle cerebral artery (Siushansian et al., 2001) or after traumatic injury (Frantseva et al., 2002) indicated that the infarct area was significantly increased, when compared to WT brains, and the authors suggested that the reduced gap junctional communication in the Cx43 HT compromised the astrocytic syncytium, favoring neurotoxicity (Siushansian et al., 2001). Furthermore, in cocultures of neurons and astrocytes, the blockade of gap junctional communication with either carbenoxolone or alpha-glycyrrhetinic acid resulted in increased glutamate-induced neurotoxicity, indicating that gap junctions may have a neuroprotective role against glutamate toxicity (Ozog et al., 2002). Gene therapy methods have been applied to glioblastoma treatment. One of these, treatment of Herpes thymidine kinase (TK) transduced tumor cells with ganciclovir (GCV), is very efficient, especially because of the bystander effect that it generates, leading to tumor regression after GCV metabolites generated by TK diffuse through gap junctions to neighboring cells (Estin et al., 1999; Andrade-Rozental et al., 2000; Mesnil and Yamasaki, 2000). Although loss of gap junction-mediated intercellular communication has been long believed to be a common, even causative, occurrence in tumor cells (Lowenstein and Rose, 1992; Rose et al., 1993), it is now clear that tumor cells retain functional coupling and that this coupling pathway can be used therapeutically, essentially as a cellular drug delivery device.

5. Concluding remarks The studies summarized in this review indicate that astrocytes form a cellularized functional syncytium, with gap junctions serving to interconnect these glial cells. Although intercellular gradients of proteins and smaller molecules generated within single cells are steeper than would be achieved by a true syncytium, the gap junction channels provide the sharing of signaling molecules throughout the coupled astrocytic network. New studies carefully defining the surfaces of interactions between individual astrocytes indicate that each has its own private volume, where intercellular communication is limited to the outermost fringes surrounding the autocellular space. Within this space, hundreds of oligodendrocytes may intermingle with fine astrocyte processes and tens of thousands of synapses are surrounded by individual processes of a single astrocyte.

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Because there are various molecules that can cross cell boundaries through gap junction channels, (Kþ, cAMP, IP3, Ca2þ, glucose, glutamate, lactate, etc.), the functions performed by the interconnected astrocytic syncytium may be quite diverse. By providing the interconnections between the cytosol of individual astrocytes gap junction channels allow faster dissipation of potassium gradients around active neurons than the diffusion through the extracellular space (although the spatial spread of potassium may be less in brain than that achieved by single Mueller cell in the retina); moreover, gap junctions serve as a route for transport of energy sources and metabolites, for the spread of second messengers involved in the propagation of intercellular calcium waves and for the transfer of both death and neuroprotective signals within the interconnected network. Therefore, gap junctions provide ways for astrocytes to extend their individualized cellular functions into an integrated and larger functional unit.

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Structural plasticity of nonneuronal cells in the hypothalamo-neurohypophyseal system: in the right place at the right time A.K. Salm,* A.E. Ayoub and B.E. Lally Department of Neurobiology and Anatomy, West Virginia University School of Medicine, P.O. Box 9128, Morgantown, WV 26506-9128, USA p Correspondence address: Tel.: þ 1-304-293-2435; fax: þ 1-304-293-8159. E-mail: [email protected](A.K.S.)

Contents 1. 2.

3.

Introduction: structural plasticity in the central nervous system (CNS) Structural plasticity of astrocytes in the HNS 2.1. HNS 2.2. Glial retraction 2.3. Correlates of glial retraction in the SON and posterior pituitary 2.4. Reorientation of astrocytes in the SON-VGL 2.5. Immunocytochemical studies 2.6. Role of GFAP messenger RNA in astrocyte shape changes 2.7. Proliferation of astrocytes in the activated SON and posterior pituitary 2.8. Breakdown of basal lamina and tenascin 2.9. Activity dependent plasticity of microglia in the activated HNS? Concluding remarks

Structural plasticity of astrocytes in the normally functioning brain is widespread. Two of the best-studied regions where structural plasticity occurs are the supraoptic nucleus (SON) and posterior pituitary. In this chapter, we review recent developments in our understanding of how this structural remodeling comes about, including new findings that show microglia also participate in structural plasticity of the SON. Other brain areas where structural plasticity of astrocytes has been demonstrated are also briefly introduced. 1. Introduction: structural plasticity in the central nervous system (CNS) As this volume attests, glial cells maintain the intracellular milieu of the CNS in diverse ways. Central to their ability to perform their regulatory functions is the capacity to be in Advances in Molecular and Cell Biology, Vol. 31, pages 181–198 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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‘the right place at the right time’. Whether removing dopamine from synaptic clefts, buffering extracellular potassium or providing glutamine to neighboring neurons, glial cells must first have their processes placed in strategic locations. Structural plasticity of glia, predominantly that of astrocytes, occurs throughout the nervous system (see Salm et al., 1998 for review). Sites where structural plasticity of astrocytes has been documented in normal brains include the hippocampus with long term potentiation induction (Wenzel et al., 1991), in the suprachiasmatic nucleus across the diurnal cycle (Lavialle and Serviere, 1993), in the cerebellar cortex with motor learning (Black et al., 1990; Anderson et al., 1994), in the arcuate nucleus of the rat (Garcia-Segura et al., 1994; Olmos et al., 1989) and monkey (Witkin et al., 1991), across changing estrogen levels, and extensively, in the hypothalamo-neurohypophyseal system (HNS) in response to diverse conditions leading to the synthesis and release of the peptides oxytocin (OX) and vasopressin (VP). A feature common to all of these examples is that the neurons in the vicinity are vigorously challenged to perform their specific functions, e.g., transmission of photic information (in the suprachiasmatic nucleus) or maintenance of fluid homeostasis (in the HNS). Given this, one might predict that similar changes would occur in any brain region where neurons can be selectively challenged. Structural plasticity of astrocytes is not stereotypic and varies with region. This is especially true with respect to glial coverage versus synaptic contacts. For example, in the cerebellar cortex, motor learning is accompanied by formation of synapses, an increase in astrocytic surface volume, and increased coverage of synaptic elements (Anderson et al., 1994; Jones and Greenough, 1996). Conversely, in the arcuate nucleus, a decrease in astrocytic coverage of neuronal somata is inversely related to synaptic coverage (Olmos et al., 1989). Many of the functional systems where structural plasticity of astrocytes has been found are in the hypothalamus (Salm et al., 1998). In fact, the hypothalamus might be prone to such changes due to the perpetual challenge of maintaining homeostasis. An enhanced degree of ‘neural nimbleness’ may be required to respond to sometimes rapid changes in the internal milieu. The hypothalamus is also experimentally convenient, with specific functions being performed by discrete, easily identifiable, nuclei. These features, in particular, have made the HNS especially attractive for the study of structural plasticity of glia. 2. Structural plasticity of astrocytes in the HNS 2.1. HNS The HNS consists primarily of magnocellular neuroendocrine cells (MNCs) in the hypothalamic paraventricular nucleus (PVN) and the supraoptic nucleus (SON). These cells send their long axons through the hypothalamus to terminate in the posterior pituitary. It also includes the nucleus circularis (Hatton, 1976; Tweedle and Hatton, 1977) as well as accessory MNCs nearby in the hypothalamus. Many studies have been done examining structural remodeling of astrocytes and neurons in the PVN, SON and posterior pituitary. Especially interested readers are encouraged to refer to a number of exhaustive reviews (Theodosis and Poulain, 1993; Hatton, 1997; Theodosis et al., 1998; Hatton, 1999). Structural plasticity of glia has been characterized most thoroughly in

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the SON and posterior pituitary. The SON is relatively homogeneous compared to the multiple subnuclei described for the PVN (Armstrong et al., 1980). MNCs produce OX and VP, as well as other peptides colocalized with OX and VP (Levin and Sawchenko, 1993). The function of OX is to produce the smooth muscle contractions of parturition, lactation and orgasm, whereas VP acts at the kidneys to promote water reabsorption at the renal tubules in response to increased plasma or extracellular osmolality or hemorrhage. Therefore ‘activated’ MNCs can be studied in the HNS of animals who are recently postparturient or lactating, or animals who have been dehydrated via osmotic challenges. Dehydration regimens used have included prolonged water deprivation, chronic substitution of drinking water with 1.5– 2.0% saline solutions, or acutely, injection of hypertonic sodium chloride (Beagley and Hatton, 1992). However, other activating stimuli include factors surrounding expression of maternal behaviors (Salm et al., 1988) and restraint stress (Miyata et al., 1994). Since normally cycling female rats experience fluctuations in estrogen and other hormones that may affect astrocytes (Stone et al., 1997—see also chapter by Melcangi et al.), all of our work described below has used male rats. Dehydration is induced by 2% saline administration for varying time periods. Rehydration uses the same manipulation, followed by access to normal drinking water for varying times. The SON is populated by two distinct populations of astrocytes (Figs 1 and 2). One of these consists of the stellate cells that occupy the magnocellular region of the SON (the SON proper), where they envelope nearby MNCs. These astrocytes are immunopositive for the astrocytic marker glial fibrillary acidic protein. A second population consists of those astrocytes with cell bodies arrayed along the glial limitans ventral to the SON (SONVGL). Under basal conditions, the processes of SON-VGL astrocytes form a dense local meshwork. These cells also send long processes dorsally, sometimes as far as 500 mm, through the SON proper where they envelope the MNCs (Figs 1 and 2). These cells are also GFAP þ , but they are additionally reactive with antibodies to vimentin

Fig. 1. Light micrograph showing the SON adjacent to the optic chiasm (OC). The section has been stained with antibodies to glial fibrillary acidic protein (GFAP) and astrocyte fibers can be seen throughout the nucleus. The densely stained ventral glial limitans subjacent to the SON (SON-VGL) is seen most ventrally.

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Fig. 2. High-power micrograph of the SON from a control animal that has been stained for GFAP. A dense meshwork of fibers is seen throughout the nucleus, many of which are emanating dorsally from the SON-VGL. Asterisks denote regions where MNCs can be seen in relief.

(Bonfanti et al., 1993) and taurine (Decavel and Hatton, 1995). In the SON, MNC somata and dendrites are segregated, with MNCs most dorsal in the nucleus (arrow in Fig. 3). SON dendrites course as a group ventrolateral to the somata (DZ in Fig. 3), and therefore can be easily studied (Armstrong et al., 1982).

2.2. Glial retraction Magnocellular neurons of the SON are tightly packed together, and under basal conditions this space is occupied by astrocyte processes. Modney and Hatton (1989) estimated that 84% of MNC membrane in the SON is covered by glial processes under basal conditions. In 1976, Charles Tweedle, Glenn Hatton and colleagues at Michigan State University began publishing a series of electron microscopic studies describing remarkable changes in the dehydration-activated HNS (Tweedle and Hatton, 1976). Shortly thereafter, Dionysia Theodosis, Dominique Poulain and colleagues at INSERM in Bordeaux reported similar observations in lactating rats (Theodosis et al., 1981; Theodosis and Poulain, 1984). Both groups quantified a reduction in glial coverage of neuronal elements with activation that led to increased direct apposition of the membranes of somata and dendrites in the SON (although ‘direct appositions’ were first noticed in a report from Lafarga et al., 1975). Tweedle and Hatton further reported a reduction in glial envelopment of axon terminals in the posterior pituitary which accompanied activation of the HNS (Tweedle and Hatton, 1980a,b). They hypothesized that the changes in the activated SON and posterior pituitary were due to ‘glial retraction’ whereby the astrocytes actively retracted their processes from around the neurons and axon terminals, and then

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Fig. 3. Light level micrographs of 1 mm thick, plastic embedded, toluidine blue stained sections depicting the organization of the SON and a dehydration associated decrease in SON-VGL thickness. (A) Tissue from a normally hydrated male rat. Magnocellular neurons (open arrow) can be seen at the top of the picture, overlying the dendritic zone (DZ), where many dendrites can be seen in cross-section. Astrocyte cell bodies comprising the VGL are arrayed along the dotted line (p). SAS: subarachnoid space. (B) Tissue from a 9-day dehydrated rat. Note the reduction in thickness of the VGL, delineated by carets. This enables MNCs to be in close proximity to the SAS. Magnification bars ¼ 10 mm (after Bobak and Salm, 1996).

reinserted them when stimulation ceased (Tweedle and Hatton, 1976). We now know that glial retraction occurs rapidly and can be detected by electron microscopy within as little as five hours following an injection of hypertonic saline (Beagley and Hatton, 1994). Remarkably, these changes are reversible following cessation of activation, i.e., adequate periods of rehydration or post weaning. In the region of the cell bodies, this enables the neurons to form direct appositions. Likewise, in the DZ of the SON, ventral to the somata, dendrites also become directly apposed and form bundles of up to 13 dendrites (Perlmutter et al., 1984, 1985; Salm et al., 1988). In the SON, the functional consequence of the reduction in astrocyte coverage is to synchronize neuronal activity. Physiological studies have shown that activation of OX and VP neurons leads to the emergence of distinct firing patterns for each type of neuron. In lactating rats, OX neurons change from a slow, continuous firing rate to synchronized, high frequency discharges that precede the milk ejection reflex. This in turn leads to a bolus of OX release and subsequent contraction of the mammary myoepithelial cells (Wakerley and Lincoln, 1973; Lincoln and Wakerley, 1975). Vasopressin neurons, when activated, change from a slow continuous firing pattern to synchronized phasic-bursting (Poulain and Wakerley, 1982). Glial retraction may promote these changes in several ways. One is through the formation of novel synaptic contacts or ‘double synapses’ where one presynaptic profile forms synapses with two or more post-synaptic dendrites, somata, or both (Tweedle and Hatton, 1984). Under basal conditions, the majority of synapses onto

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MNC somata are single synapses. However, with 10 days of 2% saline administration there is a significant increase in the amount of somatic membrane contacted by double synapses. A concomitant, approximately equal, decrease in amount of membrane contacted by single synapses suggests that the retraction of a glial process allows double synapses to be formed from single synapses (Modney and Hatton, 1989). In the DZ, the appearance of bundles is accompanied by formation of gap junctions, and this too would serve to synchronize the activity of coupled neurons (Andrew et al., 1981). In addition to these changes that directly coordinate the MNCs, the absence of astrocyte processes where they normally exist leads to changes in the extracellular environment that heighten MNC excitability. The lack of astrocytes to buffer potassium (Tang et al., 1980) and glutamate (Oliet et al., 2001) is also excitatory to MNCs. In both the SON (Deleuze et al., 1998) and posterior pituitary (Miyata et al., 1997), the retraction of those astrocyte processes that contain taurine, an inhibitory amino acid, would also serve to enhance MNC excitability. In the posterior pituitary, axons and axon terminals are normally engulfed by the cytoplasm of pituicytes, which separates the terminals from fenestrations in the capillary bed of the pituitary. With dehydration, far more terminals are directly apposed to these portals to the general circulation (Tweedle and Hatton, 1980a,b). This too is reversible with rehydration or postweaning. Hence, retraction of pituicytes in the neural lobe mirrors astrocyte plasticity in the SON and allows OX and VP access to the circulation.

2.3. Correlates of glial retraction in the SON and posterior pituitary Our laboratory and others have been engaged in exploring factors surrounding structural remodeling in the SON (see Salm, 2000 for review). Factors that we have investigated so far include reorientation of astrocytes, reentry of glial cells into the cell cycle, downregulation of morphoregulatory molecules, dissolution of the basal lamina, changes in the GFAP, concomitant changes in expression of GFAP message and changes in the population of resident microglia.

2.4. Reorientation of astrocytes in the SON-VGL We have carried out extensive electron microsopic investigation of changes in SONVGL astrocytes in the activated SON (Bobak and Salm, 1996; Salm and Bobak, 1999). From montages of the SON-VGL constructed at 1100 £ magnification we learned that SON activation is accompanied by a remarkable reorientation of these cells (Fig. 4). Under basal conditions they are oriented predominantly vertically, with processes extending up through the MNCs. With two days of 2% saline administration we see this population assume a 458 orientation relative to the pial surface. By 7 days of activation, the SON-VGL astrocytes are seen in a predominantly horizontal position. With 14 days of rehydration, astrocytes of the SON-VGL once again are seen extending vertically into the MNCs. Functionally, we believe that this reorientation is one means by which glial processes retract from around MNCs.

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Fig. 4. Left: electron micrograph montage of two (A and B) vertically oriented astrocytes in the SON-VGL of a normally hydrated rat. Right: Electron micrograph montage of a horizontally oriented astrocyte in the SON-VGL of a 9-day dehydrated rat. Astrocytes resume their vertical orientations upon rehydration. Magnification bars equal 5 mm (after Bobak and Salm, 1996).

A second significant finding of our electron microscopic studies was that the SON-VGL undergoes significant thinning when the SON is activated (Bobak and Salm, 1996; Fig. 3). The result of this is to permit the MNCs to abut very closely to the underlying subarachnoid space and cerebrospinal fluid (CSF). It is already known that molecules may pass from the CSF deeply into the SON-VGL (Brightman, 1965). We speculate that this thinning might facilitate even more communication between MNCs and the CSF. However, rigorous investigation of this remains to be done. Another consequence of reorientation of SONVGL astrocyte processes may be to permit a continued glial interface between an enlarged SON proper and the underlying CSF. It has been known for many years that MNCs hypertrophy when activated (Hatton and Walters, 1973; Modney and Hatton, 1989) and the overall result is a significantly enlarged nucleus. Recent measurements in our laboratory of the mediolateral extent of the SON have found it significantly wider with activation (Hawrylak and Salm, 1999). With rehydration, again, the SON-VGL returns to normal width and thickness (Bobak and Salm, 1996) and the SON regains its normal mediolateral dimensions (Hawrylak and Salm, 1999).

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2.5. Immunocytochemical studies We have applied immunocytochemical methods and antibodies to the astrocytespecific GFAP (Uyeda et al., 1972; Dahl and Bignami, 1973) to the SON and posterior pituitary (Salm et al., 1982, 1985). Because GFAP is a major constituent of the astrocyte cytoskeleton, and is specific to astrocytes, it seemed reasonable that changes in astrocyte morphology in the activated SON would be reflected by changes in this protein. Studies performed on SON tissue from lactating rats revealed a significant reduction in immunoreactivity for GFAP relative to female estrous controls (Salm et al., 1985), consistent with the idea that astrocytes retract their processes in the activated SON. In the posterior pituitary, we and others (Suess and Pliska, 1981; Salm et al., 1982) also demonstrated for the first time that pituicytes of the posterior pituitary were, as long suspected, specialized astrocytes, as they also contain GFAP. This observation was somewhat surprising since, under normal conditions, few intermediate filaments are seen in rat pituicytes when examined by electron microscopy (Tweedle and Hatton, 1980a,b). As seen with immunostaining for GFAP, pituicytes possess fewer, and more stubby, processes than do normal stellate astrocytes throughout the CNS. This hampered efforts to document changes in cell shape. However, Miyata and colleagues have recently found that these cells stain robustly with antibodies to microtubule-associated protein 2 (MAP2), and they have been able to verify process retraction in response to dehydration (Miyata et al., 1999; Matsunaga et al., 1999). Interestingly, astrocytes in the SON do not stain for this protein. Thus, the presence of MAP2 in pituicytes may play a unique role in enabling shape changes in that region. In recent years, we have revisited the impact of MNC activation on GFAP immunoreactivity in the SON using dehydration as the stimulus (Hawrylak et al., 1998). As with lactation, a significant reduction in GFAP immunoreactivity was found with seven days of 2% saline substitution for drinking water. We also assessed recovery of staining and observed robust immunoreactivity with seven days of reintroduction to drinking water. As was the case with the earlier lactation study (Salm et al., 1985), our impression was that there were fewer, and thinner astrocyte processes remaining in the activated state. We then applied stereologic methods to the tissue from the dehydration study and confirmed a reversible reduction in astrocyte surface volume, i.e., a reduction of mostly astrocyte processes, in the dehydrated state (Hawrylak et al., 1999; Fig. 5). 2.6. Role of GFAP messenger RNA in astrocyte shape changes In two separate studies, we have found that reductions in GFAP immunoreactivity accompany activation of the SON (Fig. 5). One question that persists is whether there are commensurate changes in the GFAP mRNA that also occur, reflecting regulation of GFAP at the molecular level by events surrounding MNC activation. To answer this question, we have constructed a probe to GFAP mRNA from a plasmid containing linearized GFAP cDNA probe (Chen and Liem, 1994) to perform in situ hybridization on tissue from control, 2 and 7 day 2% saline administered, and 21 day rehydrated rats (Lally et al., 2001; Fig. 6). It appears that the GFAP mRNA in SON astrocytes is as labile as the protein derived from it. In normally hydrated control animals, there is a relatively low-level of

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Fig. 5. Micrographs of GFAP immunostaining in the SON of: (A) Control, (B) 6-day dehydrated, and (C) 6-day dehydrated/6-day rehydrated male rats. There is a significant reduction of immunoreactivity in the activated SON (B), which is reversible with the cessation of stimulation (C) (after Hawrylak et al., 1999).

message being expressed. The level of message then increases gradually at 2 days of activation to a peak at 7 days of saline treatment. GFAP mRNA expression in the SON of rats given saline for 7 days and then allowed to rehydrate for 21 days is essentially back to control levels in the majority, but not all, of the animals in this group. Hence, there appears to be an inverse relationship between message expression and protein expression—at least as seen with immunocytochemistry. These data suggest a feedback mechanism exists that upregulates message in response to decreased protein levels. The increase in message seen at 7 days of dehydration may represent a ‘stockpiling’, whereby the cell becomes prepared to produce protein at a rapid rate upon cessation of SON activation. Indeed, we have seen in our rehydration studies that at 7 days of rehydration, there is an immense, albeit not fully organized, network of immunoreactive astrocyte processes in the SON. The precise mechanisms of the relationship between GFAP and its mRNA in this population of astrocytes remains to be elucidated.

2.7. Proliferation of astrocytes in the activated SON and posterior pituitary One of the earliest observations in the literature of glial plasticity was that of increased glial numbers in the cerebral cortices of rats housed in enriched environments (Altman and Das, 1964; Diamond et al., 1966). Murray (1968) and Paterson and LeBlond (1977) investigated the possibility that glial cells also proliferate in the SON of male rats given 1.5% saline solutions in lieu of drinking water. Both studies found that a significant proliferation of nonneuronal cells accompanied activation of the SON. Using triple fluorescence labeling, we revisited this issue in the posterior pituitary with antibodies to bromodeoxyuridine (BrdU), GFAP, and the DNA marker DAPI (Murugaiyan and Salm, 1995). In the posterior pituitary, we found a significant increase in proliferated pituicytes by 9 days of saline treatment, but a trend was already evident already after 3 days.

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Fig. 6. Low-magnification micrographs depicting in situ hybridization for GFAP mRNA in the SONs of hydrated, 2- and 7-day dehydrated, and 7-day dehydrated/21-day rehydrated rats. Compare with Fig. 5 and note that during dehydration, when GFAP immunostaining is low, GFAP mRNA expression is high. Arrows delineate the boundaries of the nucleus.

With rehydration, numbers of BrdU þ pituicytes declined to control levels in 7 days. An unexpected observation in this study was that cells captured in the process of dividing exhibited only thin perisomatic cytoplasm and usually only two processes. Pituicytes that were not labeled with BrdU exhibited the usual array of short stubby processes. From this

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we concluded that dividing pituicytes temporarily lose their processes and that this may underlie some glial process retraction in the neural lobe. Since it has been established that SON MNCs are capable of dendritic release of OX and VP (Pow and Morris, 1989; Neumann et al., 1993), we have also investigated whether either peptide might have mitogenic effects on astroglia in culture (Lucas and Salm, 1995). A significant effect of OX was established for both hypothalamic and cortical astroglia, whereas VP only had an effect on cortical astroglia. Thus, it seems that OX could be a signal for that portion of structural remodeling attributable to reentry into the cell cycle of SON astrocytes. In this regard, Theodosis et al. (1986) have also presented evidence that OX is an activating signal in the structural remodeling of the SON.

2.8. Breakdown of basal lamina and tenascin In the course of our electron microscopic studies, we observed an intricate and extensive basal lamina in the dense array of glial processes in the SON-VGL (Salm and Bobak, 1999). It is well established that basal lamina and its associated molecules play a role in maintenance of cell polarity (Ojakian and Schwimmer, 1994; Klein et al., 1998). We therefore carried out a stereological study of the basal lamina across activation states in the SON-VGL. A significant reduction in basal lamina occurred with only two days of 2% saline substitution. With longer treatment, the extent of the basal lamina was progressively reduced, until it was virtually absent in the D7 group. With 9 days of access to normal drinking water, the basal lamina returned. It is interesting that the significant reduction in basal lamina at D2 precedes the findings of significant reorientation (Bobak and Salm, 1996), although trends in this direction are evident at D2. This is consistent with the idea that basal lamina reduction is permissive for structural plasticity (see chapter by Mercier and Hatton). Another contributing signal for structural remodeling in the SON-VGL may be the breakdown of the morphoregulatory molecule tenascin. Tenascin, a 210 – 220 kDa glycoprotein, has been implicated in neurite outgrowth regulation and boundary formation during development (Erickson and Lightner, 1988; Steindler et al., 1988). This molecule has received attention both from our group (Singleton and Salm, 1996) and Theodosis et al. (1997) as a possible regulator of plasticity in the SON. Painstaking cell cultures of tissue dissected solely from the SON revealed that tenascin is manufactured and secreted by SON astrocytes. Double labeling of tissue sections with antibodies to tenascin and GFAP revealed that in the SON-VGL, tenascin is only detectable in the residing astrocytes, and that immunoreactivity was reduced in tissue sections from six-day dehydrated animals. Protein biochemistry and Western blots further revealed that the reduction in the highmolecular weight forms was accompanied by the appearance of low 50– 60 kDa proteins, which were also immunoreactive to tenascin antibodies. With 6 days of rehydration, these smaller proteins disappeared while the larger form was again expressed. Thus, it appears that factors surrounding dehydration lead to the breakdown of tenascin. We speculate that the disappearance of tenascin is also permissive for the reorientation of the SON-VGL astrocytes that occurs with dehydration.

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2.9. Activity dependent plasticity of microglia in the activated HNS? A second population of nonneuronal cells that has thus far received only minor attention in the SON is the microglia. Mander and Morris (1995) investigated the presence of microglia in the SON with a panel of antibodies including OX-42, F4/80, ED2, OX-6 and OX-18 to demonstrate that, indeed, there are ramified and perivascular microglia in the SON. However, these antibodies only labeled relatively few microglia in the SON. This was our experience as well, therefore we used the isolectin-B4 method (Streit, 1990) for visualizing microglia in the SON. By this method, we can visualize a substantial population of microglia in the SON. They are predominantly found directly apposed to, or very near, the capillaries in the SON proper. Their numbers are small in the SON-VGL where they only appear at the pial surface (Ayoub and Salm, 2003; Fig. 7). We have recently investigated the impact of the dehydration stimulus on these cells. With normal hydration, the SON is populated by predominantly ramified microglia, and many of these appear to be astride the numerous capillaries in the nucleus (Fig. 8). With 2 days of dehydration, an additional population of hypertrophied microglia are seen in the nucleus. By 7 days, there are again significant numbers of hypertrophied microglia, but also a significant number of yet a third population of ameboid cells (Fig. 8). With rehydration, the population of hypertrophied and ameboid microglia disappear, and overall numbers return to normal levels. The role of microglia in the SON under differing activation states is unknown. They may play a role in removing breakdown products of tenascin or other components of the basal lamina. Pow et al. (1989) have shown with OX-42 antibodies and electron microscopy that there is a substantial (19%) population of microglia in the posterior pituitary. Here microglia appear to phagocytose peptidergic axon terminals, and thus may play a role in the sculpting of neurosecretory terminals under normal conditions. The source of the hypertrophied and ameboid microglia population is currently unknown, as there is no commensurate decrease in numbers of ramified microglia, and overall numbers of microglia increase significantly by D7. Thus, it is not clear whether individual cells are undergoing structural plasticity, or whether the new population of hypertrophied and ameboid microglia are being attracted to the nucleus by factors surrounding remodeling. The breakdown of extracellular matrix may be one such factor. It is tempting to speculate that at least some of the proliferation of nonneuronal cells previously reported in the SON (Murray, 1968; Paterson and LeBlond, 1977) includes microglia. However, we have only seen the occasional microglial cell in the SON in the act of dividing. The appearance of hypertrophied microglia at D2, prior to the finding of ameboid cells in the SON at D7 suggests that at least some cells undergo morphological transformation in the activated

Fig. 7. Low-magnification micrographs of the SONs of Control and 2-day dehydrated rats stained with isolectin B-4 to visualize microglia. The SON of the control rat exhibits light staining of ramified cells. The D2 section exhibit darker staining reflective of increased numbers of hypertrophied microglia. Thin arrows point to ramified microglia. Thicker arrows point to hypertrophied cells. OC: optic chiasm. p denotes capillaries. Stars indicate regions where MNCs are located.

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Fig. 8. High-magnification micrographs depicting three microglial phenotypes in the SON. (A) Ramified microglia: the thin arrow points to a ramified cell associated with a capillary. (B) The thick arrows point to hypertrophied microglia, the thinner arrow points to a ramified cell. (C) The thick arrow points to an ameboid microglial cell. Note the numerous filopodia emanating from this cell (after Ayoub and Salm, 2003).

SON. Further work is needed to establish that this is indeed an example of activitydependent plasticity of microglia under nonpathological conditions. 3. Concluding remarks Structural plasticity of astrocytes has been documented in normal brains in the hippocampus with LTP induction, in the suprachiasmatic nucleus across the diurnal cycle, in the cerebellar cortex with motor learning, in the arcuate nucleus of the rat and monkey, across changing estrogen levels, and extensively, in the HNS in response to diverse conditions leading to the synthesis and release of peptide hormones. A feature common to all of these examples is that the neurons in the vicinity are vigorously challenged to perform their specific functions. Given this, one might predict that similar changes would occur in any brain region where neurons can be selectively challenged. It is therefore of key interest to explore factors surrounding structural remodeling. Factors that we have investigated so far in the SON of the hypothalamus include reorientation of astrocytes, reentry of glial cells into the cell cycle, downregulation of morphoregulatory molecules, dissolution of the basal lamina, changes in the GFAP, concomitant changes in expression of GFAP message and changes in the population of resident microglia. Acknowledgements This work was supported by NSF IBN 9109827 and 951457. The authors thank Patricia Dickerson for critically reading the manuscript.

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Glial– neuronal – endothelial interactions and the neuroendocrine control of GnRH secretion Vincent Prevotp, Sandrine De Seranno and Cecilia Estrella p

INSERM U422, IFR 124, Place de Verdun, 59045 Lille cedex, France Correspondence address: Tel.: þ33-3-20-62-20-64; fax: þ33-3-20-62-20-61 E-mail: [email protected](V.P.)

Contents 1. 2. 3. 4. 5.

Introduction: astrocytic – neuronal and endothelial – neuronal interactions Glial – neuronal interactions are involved in the control of GnRH secretion Endothelial – neuronal signaling and GnRH release Endothelial-to-glial communication processes may promote morphological plasticity Concluding remarks

It is becoming increasingly clear that nonneuronal cells, such as glial and endothelial cells, are dynamic signaling components with the potential to modulate the way information is generated and disseminated within the brain. In the hypothalamus, neurons that secrete gonadotropin-releasing hormone (GnRH) offer an attractive model system to study astroglial –neuronal – endothelial interactions and the influence that steroid hormones may have on this process. GnRH is the neuropeptide that controls both sexual maturation and adult reproductive function. During the reproductive cycle, GnRH release into the pituitary portal vessels is modulated by dynamic alterations of the anatomical relationship that exists between GnRH nerve endings and glial cell processes in the median eminence of the hypothalamus. These plastic rearrangements and GnRH secretory activity itself appears to be modulated, at least in part, by specific cell – cell signaling molecules secreted by glial and endothelial cells. An increasing body of evidence suggests that among the different factors that may be involved, glial cells use growth factor members of the epidermal growth factor (EGF) family, acting via receptors endowed with tyrosine kinase activity, to establish this modulatory control, whereas endothelial cells of the median eminence employ nitric oxide to facilitate GnRH release. 1. Introduction: astrocytic –neuronal and endothelial – neuronal interactions Astrocytes, unlike neurons, do not generate action potentials but are capable of propagating cellular signals as waves of intracellular calcium changes that spread from Advances in Molecular and Cell Biology, Vol. 31, pages 199–214 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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astrocyte to astrocyte either via gap junctions or via the release of intercellular messengers (see for review Haydon, 2001 and chapters by Shuai et al. and by Scemes and Spray). Astroglial cells that respond to a variety of synaptically released transmitters, wrap themselves around synapses and affect in a feedback manner the neurotransmitter release that flows at the synaptic gap through the calcium-dependent release of glutamate (Bezzi et al., 1998; Kang et al., 1998; Robitaille, 1998). Exciting new data also suggest that the presence of astrocytes profoundly increases the number of synapses and are required for their maintenance both in vitro (Pfrieger and Barres, 1997) and in vivo (Ullian et al., 2001). Because tissue culture medium ‘conditioned’ by astrocytes is capable of promoting synapse formation (Pfrieger and Barres, 1997; Ullian et al., 2001), astrocytes may secrete soluble neurotrophic factors able to enhance pre- and postsynaptic differentiation, and synaptic efficacy. Cholesterol contained in lipoproteins was identified as one of the synaptogenesis-promoting signal present in glia-conditioned medium (Mauch et al., 2001). In addition, because astrocytes control glutamate clearance and diffusion in the extracellular space (see chapter by Schousboe and Waagepetersen), dynamic changes in glial coverage of neurons also contribute to the regulation of synaptic efficacy (Oliet et al., 2001). Overall, these studies suggest that astrocyte –neuron intercellular signaling is widespread throughout the central nervous system, and that astrocytes play an important active role in modulating synaptic communication between neurons. Analogously, cell –cell communication involving vascular endothelial cells was suggested to be critical in determining the structure of certain brain areas, such as the retina and the optic nerve, during development (see for review Wechsler-Reya and Barres, 1997). More recently, under the regulation of gonadal steroids, brain endothelial cells were shown to secrete neurotrophic factors capable of promoting neuronal differentiation, migration and survival in vitro (Leventhal et al., 1999; Louissaint et al., 2002) as well as in the adult brain in vivo (Louissaint et al., 2002).

2. Glial – neuronal interactions are involved in the control of GnRH secretion GnRH (LHRH) is the neurohormone controlling sexual maturation and adult reproductive function (see for Review Ojeda and Terasawa, 2002). In rodents, the cell bodies of GnRH neurons are diffusely distributed in the preoptic region; in primates, they are also present in the mediobasal hypothalamus. The neuroendocrine fraction of GnRH neurons sends axons to the median eminence of the hypothalamus (Fig. 1), where they release their secretory product into the portal vasculature. Upon reaching the anterior pituitary GnRH elicits the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn stimulate gametogenesis and gonadal hormone secretion. Because GnRH neurons are the final common pathway for the central control of reproduction, their activity is regulated by a complex array of excitatory and inhibitory transsynaptic inputs (Levine et al., 1995; Brann and Mahesh, 1997; Gore, 2001; Ojeda et al., 2001; Terasawa, 2001). Noticeably, both GnRH neurons and the multiple neuronal networks involved in the control of GnRH secretion can be subjected to the direct modulatory influence of gonadal steroids (Herbison, 1998; Herbison and Pape, 2001). In addition to this transsynaptic control, GnRH neuronal function is also regulated by

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Fig. 1. Close association of GnRH nerve terminals with the processes of modified ependymoglial cells (tanycytes) in the median eminence. (A) Light microphotograph of a frontal section of the median eminence from a female rat showing a dense GnRH-immunoreactivity (green) in the external zone of the median eminence. The tanycytic processes are visualized by their immunoreactivity for vimentin (red). 3V, third ventricle; Inf S, infundibular stem. (B) Light microphotograph of a greater magnification of the median eminence. The passing GnRH fibers (green) are seen to travel in the internal zone of the median eminence (white arrowheads). To reach the pericapillary space the GnRH axons abruptly turn ventrally and are guided towards the external zone of the median eminence by tanycytic processes (red, arrows). Scale bar: 100 mm (A), 50 mm (B). Reproduced from Prevot (2002) with permission.

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glial-derived signals (Ojeda et al., 2000, 2001; Prevot, 2002) and by structural changes in glial cell morphology (Prevot, 2002). GnRH neurons are abundantly apposed by astroglial processes (Silverman et al., 1994), especially at the level of the median eminence (Fig. 1), where GnRH nerve terminals are in close contact with both astrocytes and modified ependymoglial cells, known as tanycytes (Kozlowski and Coates, 1985; King and Letourneau, 1994; Prevot et al., 1998). Tanycytes line the ventral portion of the third ventricle and send their processes to the external zone of the median eminence, where they contact the vascular wall of the portal vessels via ‘end-feet’ specializations (Fig. 1B). Interestingly, the direct access of GnRH neuroendocrine terminals to the pericapillary space in the external zone of the median eminence is highly regulated by the tanycytic processes that enwrap GnRH nerve endings (Fig. 2A). Using electron microscopy, we demonstrated that direct neuro-haemal junction for GnRH neurons (Fig. 2B,C) only occurs at times when increased GnRH release takes place, i.e., at proestrus, the time of the occurrence of the preovulatory LH surge (Prevot et al., 1998, 1999a). Tanycytic processes could thus be acting as a brake on the release of GnRH by creating a diffusion barrier for passage of GnRH from the GnRH nerve terminals into pituitary portal blood vessels. The formation of neuro-haemal junctions for GnRH neurons on the day of proestrus is the consequence of a dynamic process that leads to the morphological remodeling of the external zone of the median eminence (Prevot et al., 1999a). On one hand, some GnRH nerve terminals generate filopodial extensions to contact the pericapillary space (Fig. 2B). On the other hand, tanycytes that are anchored by hemidesmosomes to the basal lamina (which delineates the pericapillary space) may, through remodeling of their cytoskeleton, ‘bring’ the pericapillary space to the GnRH nerve endings (Fig. 2C). What are the molecular determinants of such a functional plasticity? It is becoming increasingly clear with the studies that have been undertaken in Ojeda’s lab during the last decade that astroglial cells regulate GnRH secretion through the activation of specific glia-to-glia and glia-to-neuron signaling pathways (for review see Ojeda et al., 2000). One of the communication pathways operates by release of the epidermal growth factor (EGF)-related peptide transforming growth factor alpha (TGFa) and neuregulins. As illustrated in Fig. 3, these glial growth factors stimulate GnRH release via receptors located not on GnRH neurons, but on the glial cells themselves (Junier et al., 1993; Ma et al., 1999). Upon the binding of TGFa and neuregulin, astroglial erbB-1 (HER-1) and erbB-4 (HER-4) receptors, respectively, heterodimerize with their co-receptor erbB-2 (for a review on erbB signaling see Yarden and Sliwkowski, 2001). The signal transduction leads to the release of bioactive substances (e.g., prostaglandin E2 (PGE2)) that stimulate GnRH release by a direct action on GnRH neurons (Ma et al., 1997; Rage et al., 1997). In addition to being involved in communication processes between astrocytes and GnRH neuron, glial erbB signaling may also account for part of the plastic remodeling taking place in the external zone of the median eminence during the estrous cycle. Supporting this hypothesis, recent studies using primary cultures of tanycytes from the median eminence as a model system showed that these cells express erbB-1 and erbB-2 receptors (Fig. 3), but not erbB-4 receptors in vitro (Prevot et al., 2002), as previously shown in vivo (Ma et al., 1994, 1999). Cultured tanycytes respond to TGFa with release of both PGE2 and transforming growth factor beta 1 (TGFb1). TGFb1 is a glial growth factor shown to

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stimulate GnRH release from GnRH secreting cell lines via the activation of specific receptors present in these cells (Melcangi et al., 1995; Buchanan et al., 2000). Interestingly, morphometric studies revealed that TGFa and TGFb1 (which belong to two different growth factor families) had dramatically opposite effects on tanycytic plasticity (Prevot et al., 2002). After a 12h-treatment, TGFb1 induced retraction of tanycytic processes, whereas TGFa promoted tanycytic outgrowth. Noticeably, longer exposure of tanycytes to TGFa caused tanycytic retraction in 60% of the cultures, an effect that was abolished by immunoneutralization of TGFb1 action, indicating that the retraction was due to TGFb1 release induced by TGFa. Thus, the ability of TGFa to stimulate GnRH secretion (Ojeda et al., 1990) may not only involve stimulation of PGE2 release, but also sequential changes in tanycytic morphology. As mentioned earlier, such morphological plasticity may be required for appropriate delivery of GnRH to the pituitary during different stages of the estrous cycle. Because circulating levels of estrogen rise dramatically at the time of the onset of the preovulatory GnRH/LH surge (Smith et al., 1975), and because glial cells of the median eminence have been shown to express estrogen (E2 in Fig. 3) receptors (De Seranno and Prevot, unpublished results; Langub and Watson, 1992), estradiol appears to be a key humoral factor involved in the orchestration of the glia-to-neuron communication processes that allow GnRH neurons to contact directly the pituitary portal blood vessels on proestrus. This sex steroid has indeed been shown in vivo to activate erbB-mediated signaling events at the median eminence-arcuate nucleus level (see for review Ojeda et al., 2000), and also, to up-regulate TGFb1 mRNA levels in the hypothalamus (Galbiati et al., 2001). In addition, estradiol has been shown in vitro to stimulate TGFb1 release from hypothalamic astrocytes (Buchanan et al., 2000). We can anticipate that the involvement of glial cells of the median eminence in the control of GnRH secretion would be as important as the role played by astrocytes in the modulation of synaptic transmission and synaptic plasticity in other areas of the hypothalamus (Oliet, 2002) or of the central nervous system (Haydon, 2001; Pfrieger, 2002). Supporting this view is our recent study demonstrating that the transgenic disruption of a single astrocytic component involved in this glia-to-neuron signaling, the astroglial erbB-4 receptor, a receptor that is neither expressed by GnRH neurons nor by tanycytes (Ma et al., 1999), impairs GnRH release at the median eminence (Prevot et al., 2003). In these transgenic mice, the defect in GnRH release is associated with delayed puberty and compromised fertility (Prevot et al., 2003) providing, for the first time, compelling evidence for the view that astrocytes are key components of the central regulatory system that controls GnRH secretion in mammals. Juxtacrine and/or paracrine communication between astrocytes and tanycytes in the median eminence may play a major role in the integration of the great diversity of neuronal and nonneuronal stimuli that these cells receive under varying physiological situations. Cross-communication between glial cells of the median eminence may be modulated, at least in part, through the control of erbB signaling. It is in agreement with this hypothesis that we recently observed that concomitant activation of metabotropic and AMPA glutamate receptors on hypothalamic astrocytes results in activation of erbB receptors and recruitment of their ligands to the glial cell membrane (Dziedzic et al., 2003). We further demonstrated that metabotropic and AMPA glutamate receptor agonists together induce the

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phosphorylation of both erbB-1 and erbB-4 via a transactivation mechanism (see chapter by Peng), requiring proteolytic activity (Dziedzic et al., 2003) and presumably leading to the release of both an EGF receptor ligand and an erbB-4 ligand from their membrane-bound precursors. These results suggest that the availability of EGF-like peptides in the extracellular matrix may be a key regulatory point for glial –cell-to-glial – cell interactions. EGF-like peptides are membrane-anchored and are released upon cleavage of the ectodomain (Pandiella and Massague, 1991; Montero et al., 2000). The shedding of the ectodomain of these factors is controlled by a class of cell surface proteolytic enzymes, termed metalloproteinases (Gearing et al., 1994; McGeehan et al., 1994; Mohler et al., 1994). Matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinase) are two subfamilies of zinc-dependent-metalloproteinases involved in extracellular proteolysis (Yong et al., 2001). While MMPs, by their capacity to degrade the components of the extracellular matrix, play a pivotal role in modulating interactions between cells and their microenvironment (Damsky and Werb, 1992), ADAM proteins, which mediate both adhesive interactions and proteolysis (Schlondorff and Blobel, 1999), have been shown to participate in the cleavage of transmembrane proteins. Interestingly, ADAM17, also known as TACE (tumor-necrosis-factor-alpha-converting enzyme), which is one of the molecules involved in the shedding of both TGFa (Peschon et al., 1998) and neuregulin (Montero et al., 2000)—the main ligands of erbB receptors in the brain, has recently been shown to be expressed in astrocytes in the human brain (Goddard et al., 2001). From preliminary results, it appears that TACE is expressed in both hypothalamic astrocytes and tanycytes and its biological activity is enhanced by coactivation of metabotropic and AMPA receptors (A. Lomnisczi and S.R. Ojeda, personal communication). Taken together, these studies give strong support to the concept that trophic factors of the EGF family are key components of the glia-to-neuron communication pathways used by glial cells to facilitate GnRH release. Recent studies demonstrating that glial TGFa –erbB-1 signaling is also involved in the control of the circadian clock in the

Fig. 2. Electron micrographs illustrating the dynamic changes occurring in the external zone of the median eminence that control the direct access of GnRH nerve terminals to the pericapillary space during the reproductive cycle in the rat. (A) Electron micrograph of GnRH-immunoreactive terminals (large arrowhead) in the external zone of the median eminence in close proximity of the fenestrated capillaries (Cap) of the portal vasculature. At most stages of the reproductive cycle, GnRH nerve terminals (labeled with 15-nm gold particles) are entirely embedded in tanycytic endfeets (Tan), which prevent them from contacting the pericapillary space (p.s.) delineated by the parenchymatous basal lamina (arrow). Arrowhead, endothelial basal lamina; short arrows, fenestration of the endothelium. Scale bar: 0.5 mm. (B,C) On proestrus, the time of the occurrence of the preovulatory GnRH/LH surge, a significant fraction of GnRH nerve endings (large arrowhead) directly contact the pericapillary space (p.s.) either through filopodial extension of the nerve terminal (arrows) (B) or (C) by evaginations of the parenchymatous basal lamina (small black arrowheads) that allows the pericapillary space (p.s., asterisk) to penetrate into the nervous parenchyma. In (C) note the presence of numerous small clear synaptic vesicles (white vesicles of small size) and the fusion of secretory granules (large-sized black vesicles) with the axo-plasmic membrane of the GnRH nerve terminal in direct apposition with the parenchymatous basal lamina (small arrows). The penetration of the pericapillary space into the nervous parenchyma on the day of proestrus may result from the morphological remodeling of tanycytic end-feets (tan) anchored to the parenchymatous basal lamina through hemidesmosomes seen as dark thickenings within the tanycytic processes in apposition with the basal lamina, small white arrowhead. Scale bar: 0.5 mm. From Prevot et al. (1998, 1999a,b) with permission.

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Fig. 3. Schematic representation of glia-to-neuron and endothelial–cell-to-neuron communication processes involved in the control of GnRH neurosecretion in the median eminence. Glial–neuronal interactions in the median eminence involve the production of EGF-related peptides, TGFa and neuregulins (NRG), by tanycytes and astrocytes. The binding of TGFa to tanycytic and/or astrocytic erbB-1 receptors, as well as the binding of NRGs to astrocytic erbB-4 receptors result in recruitment of the erbB-2 co-receptors and signal transduction. The downstream signaling of the erbB receptors leads to the secretion of bioactive molecules such as prostaglandin E2 (PGE2) able to directly stimulate the releasing activity of GnRH nerve endings. The additional involvement of TGFb1 as part of the glia-to-neuron signaling pathways directly controlling GnRH release within the median

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suprachiasmatic nucleus (Kramer et al., 2001; Li et al., 2002) suggest that astrocyte– neuron intercellular signaling involving EGF-like peptides and their receptors may be widespread throughout the central nervous system (see also chapter by Peng). 3. Endothelial –neuronal signaling and GnRH release The involvement of endothelial cells of the median eminence in the control of neurohormone secretion is a new concept that emerged from our recent studies investigating the effect of nitric oxide (NO) produced in the median eminence on GnRH release (for review see Prevot et al., 2001). In the central nervous system, NO is a gaseous neurotransmitter that is generated under physiological conditions by two different isozymes of NO synthase (NOS)—neuronal NOS (nNOS) and endothelial NOS (eNOS)— that have very different spatial distributions. Within the median eminence, nNOS is confined to neuronal fibers that are segregated from GnRH axonal processes (Herbison et al., 1996), whereas eNOS is expressed in endothelial cells of the portal blood vessels to which GnRH neurons abut (Fig. 4C) (Prevot et al., 2000). It has been known since the early 90s that intracerebroventricular administration of NO donors or NOS inhibitors influence GnRH/LH secretion (see for review Prevot et al., 2000). However, it is only the recent development of amperometric methods to selectively measure NO release in real time that has made it possible to demonstrate that NO is spontaneously produced in medianeminence explants (Knauf et al., 2001a). In female rats, NO secretory pattern appears to be both pulsatile and cyclic in nature (Fig. 4A). The pulse frequency of spontaneous NO efflux (one pulse every 32 ^ 1 min) is strikingly similar to that of pulsatile GnRH release from median-eminence explants (Bourguignon et al., 1993). The amplitude of NO pulses varies across the estrous cycle, reaching peak values on proestrus (Knauf et al., 2001a), concomitantly with the increase in GnRH pulse amplitude observed in vivo (Sarkar and Minami, 1995). These observations together with the finding that GnRH release at the time of the onset of the preovulatory GnRH surge on the afternoon of proestrus can be blocked with L-NIO (Fig. 4B), an NOS inhibitor, demonstrate that NO secretion and GnRH release are causally related in the median eminence during the estrous cycle (Knauf et al., 2001a). In addition, because in the latter experiment L-NIO was used at concentrations that selectively inhibited eNOS (Fig. 4B) and leaves nNOS intact, the results suggest that the major source of NO modulating GnRH release in the median eminence is of endothelial origin. Western blot analysis showing that eNOS protein is maximally elevated on the day of proestrus (Knauf et al., 2001b), whereas nNOS protein levels remain unchanged

eminence is still an issue that needs to be resolved. For that reason TGFb1 is not included in the Figure, but there is evidence that both astrocytes and tanycytes release TGFb1 in vitro. Endothelial –neuronal interactions in the median eminence involve the production of nitric oxide (NO) by the endothelial cells of the fenestrated capillaries of the portal blood vessels. Upon its secretion, this gaseous signaling molecule diffuses from its source and stimulates the release of GnRH from the neighboring GnRH neuroendocrine terminals. Estradiol (E2) appears to facilitate both TGFa/erbB-1 and NRG/erbB-4-dependent events in glial cells and NO production by endothelial cells in the median eminence. ER, estrogen receptor; eNOS, endothelial nitric oxide synthase. From Prevot (2002) with permission.

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Fig. 4. In the median eminence of the hypothalamus, endothelial nitric oxide (NO) secretion may represent one of the synchronizing cues that by coordinating GnRH release from the scattered GnRH neuroendocrine terminals allows the occurrence of functionally meaningful episodes of GnRH secretion. (A) Real time amperometric measurement of spontaneous NO release from median eminence explants at different stages of the rat estrous cycle. DiII, diestrus II; PRO, proestrus; E, estrus. (B) On the afternoon of proestrus, the preovulatory GnRH/NO release is blocked with L-NIO, an NOS inhibitor selective for eNOS at 5 £ 1027M. p and a, significantly different from treated samples, p , 0:05: AUC: area under the curve during a 30 min period. (C) Photomicrograph showing GnRH axonal fibers in the external zone of the median eminence (green fluorescence, arrows) in close apposition to eNOS-immunoreactive portal vasculature (red fluorescence, arrowheads). 3V, third ventricle. The dotted lines underline the third ventricle. Scale bar: 75 mm. (A, B) Reproduced with permission from Knauf et al. (2001a,b); (C) reproduced with permission from Prevot et al. (2000).

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(Lamar et al., 1999), further support the involvement of endothelial NO in the control of GnRH release at the median eminence during the estrous cycle. The importance of eNOS in the modulation of GnRH secretion is further emphasized by the fact that gonadal-steroid induced GnRH/LH surge in ovariectomized rats is inhibited by intracerebroventricular injection of antisense oligonucleotides to eNOS (Aguan et al., 1996). Because treatment of ovariectomized rats with estradiol-benzoate for 48 h results in dramatic increase in both NOS activity and GnRH release from median-eminence explants (Knauf et al., 2001a), it can be concluded that estrogen probably is the main gonadal steroid modulating NO/GnRH release at the median eminence during the estrous cycle. Estrogen is likely to act directly on endothelial cells of the median eminence (Fig. 3) that have been shown to express estrogen receptors (Langub and Watson, 1992). The long-term stimulatory effect of the steroid on NO release during the estrous cycle (Knauf et al., 2001a) appears to involve up-regulation of eNOS gene expression and down-regulation of the synthesis of caveolin-1 protein (Knauf et al., 2001b), a specific endogenous inhibitor of eNOS (Feron et al., 2001). Interestingly, estrogen was also shown to have an acute stimulatory effect on NO release that appears to be mediated by the activation of a cellsurface estrogen receptor located on endothelial cells of the median eminence and does not involve a genomic effect on NOS gene expression (Prevot et al., 1999b). This acute estrogen-stimulated NO release from median-eminence explants is also able to elicit GnRH release (Prevot et al., 1999b). These findings let us anticipate a more universal existence of NO-mediated endotheliato-neuron communication processes, which modulate neuronal action and/or recruitment and provide a unique regulatory mechanism, capable of conveying peripheral information to the central nervous system. In agreement with this point of view, recent studies suggest that vascular – neuronal signaling in the nucleus tractus solitari (NTS), the brainstem termination site for baroreceptor afferents, may be responsible for inhibition of the angiotensin-II-induced baroreceptor reflex by NO (for review, see Paton et al., 2002). Here, the activation of angiotensin-type-1 by circulating angiotensin II may stimulate eNOS activity and elicit NO release from the capillary endothelium. This NO may, in turn, diffuse to nearby GABAergic NTS interneurons to enhance inhibition of neurons mediating the baroreceptor reflex (Paton et al., 2002).

4. Endothelial-to-glial communication processes may promote morphological plasticity The active participation of endothelial cells in the control of morphological plasticity in the external zone of the median eminence has been suggested by our in vitro studies undertaken to determine whether the modulation of GnRH release by NO was purely biochemical or required cellular plasticity (De Seranno et al., 2002). Physiological concentrations of NO induced rapid reorganization of actin cytoskeleton in cultured tanycytes. It is noteworthy that treatment of primary cultures of tanycytes with nanomolar concentrations of the NO donor sodium nitroprusside (SNP) caused a loss of the cortical actin present at the edge of the cells, and considered as submembranous actin, together with the appearance of heavy and long stress fibers, which traversed the cells, could be

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detected as early as after 15 min, and reached a maximum by 30 min. After the treatment, the cells remained attached to the substrate, showed no signs of apoptosis and partially regained their untreated phenotype, when incubated overnight in fresh medium. Interestingly, blockade of either prostaglandin or cGMP synthesis in isolated tanycytes independently prevented the stimulatory effect of SNP on the reorganization of filamentous actin, suggesting that the activity of both cyclooxygenase and soluble guanylyl cyclase, which are the two main intracellular targets of NO (Garthwaite and Boulton, 1995—see also chapter by Garcia and Baltrons) are required for the process. This finding provokes the suggestion that the ability of endothelial NO to stimulate GnRH secretion does not only involve a direct stimulatory effect of this gaseous neuromodulator on GnRH nerve terminals, but it also includes changes in tanycyte morphology that may be required to regulate the access of GnRH nerve terminals to the portal vasculature on the day of the preovulatory GnRH/LH surge. The physiological relevance of endothelial – glial interactions for brain development and function has recently been pointed out by Barres and colleagues (Mi et al., 2001). Using the optic nerve as a model system and their ability to purify in vitro both endothelial cells and astrocyte precursors, the authors demonstrated that endothelial-to-glial signaling was necessary and sufficient to induce astrocyte differentiation. Purified endothelial cells appeared to induce astrocyte differentiation, at least in part, by secreting Leukemia inhibitor factor (LIF), a growth factor previously shown to promote differentiation of neural stem cells and astrocyte precursor cells into astrocytes (Nakagaito et al., 1995; Johe et al., 1996; Mi and Barres, 1999). Because astrocyte differentiation in developing brain (Marin-Padilla, 1995; Zerlin et al., 1995; Zerlin and Goldman, 1997) and retina (see chapter by Stone) is closely linked, both spatially and temporally, to vascularization, endothelial cells may also induce the differentiation of astrocytes in other regions of the central nervous system (see Mi et al., 2001). 5. Concluding remarks Although it is clear that transsynaptic influences are important for the control of GnRH neuron function, the studies we have reviewed here prompt us to acknowledge that glial and endothelial cells of the median eminence of the hypothalamus are dynamic signaling components with the potential to modulate activity and/or recruitment of GnRH neurons and release of GnRH. Fig. 3 summarizes part of the Glial – neuronal – endothelial interactions that may be involved in the neuroendocrine control of GnRH secretion. These forms of cell-to-cell communication processes involving nonneuronal cells are not restricted to the GnRH neuroendocrine system, since similar interactions have been documented in other systems of the developing and/or the adult brain in response to a variety of stimuli (see, e.g., Kramer et al., 2001; Mi et al., 2001; Li et al., 2002; Louissaint et al., 2002; Paton et al., 2002; Theodosis, 2002—see also chapters by Salm et al. and by Mercier and Hatton). Our ability to work on the median eminence, a model system in which nerve terminals, glial cells and endothelial cells readily interact to modulate neuronal secretion, gives us the unique opportunity to further decipher the mechanisms underlying glial – neuronal – endothelial interactions.

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Meninges and perivasculature as mediators of CNS plasticity Frederic Merciera,* and Glenn I. Hattonb a

Laboratory of Matrix Pathobiology, Pacific Biomedical Research Center, University of Hawaii, 1993 East-West Rd., Honolulu, HI 96822, USA p Correspondence address: E-mail: [email protected] b Department of Cell Biology and Neuroscience, University of California Riverside, CA 92521, USA

Contents 1. 2.

3.

4.

5. 6.

Introduction Brain extraparenchyma 2.1. Extraparenchymal cells 2.2. Basal lamina 2.3. Brain cytoarchitectonics 2.4. Communication among neurons, glia and meningeal and vascular cells Cytokines and growth factors 3.1. Cytokine and GF in brain extraparenchymal cells 3.2. Cytokines and growth factors in brain parenchymal cells The hypothalamo– hypophysial systems 4.1. Anatomy 4.2. Cellular interactions 4.3. Effects of cytokines The meningeal hypothesis Concluding remarks

Fibroblasts, perivascular and meningeal macrophages, pericytes, smooth muscle cells, and endothelial cells are non-neural cells that are found in brain but often are not considered to be active players in brain function. Together with extracellular matrix molecules and fluids, these cells form the brain extraparenchyma (connective tissue plus vasculature). A basal lamina is ubiquitously found to separate extraparenchyma from parenchyma (i.e., the neuronal and glial cells). Here, we present the hypothesis that extraparenchymal cells, which produce and release numerous growth factors and cytokines, communicate with glial cells and neurons, across the basal laminae. Basal laminae are known to bind, activate, and present growth factors and cytokines to adjacent cells. Considering the hypothalamo-neurohypophysial system as exemplary, we speculate that meningeal Advances in Molecular and Cell Biology, Vol. 31, pages 215–253 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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vascular, and perivascular cells intervene in neuroendocrine regulations. Generalizing our hypothesis, we speculate that extraparenchymal cells, ideally located at the fluid/neural cell interface, drive and promote neural plasticity. Supporting examples are presented. 1. Introduction During embryogenesis, morphogenic events take place that are termed mesodermoneuroectodermal or mesodermo-epithelial induction, and involve interactions between mesoderm and neuroectoderm (Gilbert, 1994, 2001). The extracellular matrix plays an important role in these interactions by providing a suitable microenvironment for the cells and cell constituents (see chapter by Gomes and Rehen). Special mats of extracellular matrix, the basal laminae (BL), also termed basement membranes, are sites of binding and activation of signaling molecules such as growth factors (GF) and cytokines (Gordon et al., 1987; Roberts et al., 1988). The cytokines and GF bound in BL become activated to influence proliferation, differentiation and migration of stem cells or progenitor cells that are in contact with BL. The BL persist at interfaces between different tissues throughout adulthood (Farquhar, 1991), but the functions of BL in the adult central nervous system (CNS) are poorly understood. Neurons and glial cells together constitute the parenchyma of the CNS. The term glial cells include astrocytes, oligodendrocytes, microglia, the specialized astrocytes in the pituitary and retinal Mu¨ller cells. It is now well established that both neurons and glial cells are important players in brain functions. However, the question arises whether neurons throughout adulthood interact exclusively with glial cells rather than also with other cell types present in the brain. In addition to neurons and glial cells, the brain consists of vascular and connective tissue cells. These cells, which form the extraparenchyma, include fibroblasts, resident meningeal and perivascular macrophages (not to be confused with microglial cells which reside in the parenchyma), mast cells, pericytes, smooth muscle cells (SMCs), and endothelial cells. The extraparenchymal cells are present in the meninges, in the projections of the meninges within the brain, in the whole vasculature of the brain, and in the choroid plexus stroma (Fig. 1A,B). Vascular and connective tissue cells are present in every brain structure and substructure, flanking neural cells at different levels of neural tissue organization. To date, there has been no demonstration of the existence of a functional barrier between the parenchymal neural cells (neurons and glial cells) and extraparenchymal cells. On the contrary, the BL, present at all boundaries between neural cells and meningeal or vascular cells, may represent an interface of communication between parenchymal and extraparenchymal cells. The purpose of the present review is to examine evidence that meningeal and vascular cells, together with neurons and glial cells, form a cooperative system that drives structural and functional plasticity of the CNS throughout adulthood. In order to establish the basis for this hypothesis, we will first describe the anatomy of the brain extraparenchyma and BL (Section 2), introducing the latter as a potential interface of communication between the parenchyma and the extraparenchyma. Then, we will discuss the expression of cytokines and GF and their receptors in extraparenchymal and parenchymal brain tissue and evidence of their involvement in cell proliferation,

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Fig. 1. Models of the brain cytoarchitectonics. (A) Anatomy of the meninges. Meninges (white color) cover the brain, but also enter it by projecting between substructures. A major meningeal projection is represented underneath the hippocampal formation (left drawing). This meningeal projection is continuous with the choroid plexus stroma. Small meningeal projections, such as sheaths of blood vessels are not represented, but the blood vessels are (red). SON: supraoptic nucleus. (B) Schematic representation of brain boundaries. The BBB, which is localized at the level of the endothelium (red line), is a barrier restricting the passage of nonlipid soluble or specifically transported molecules. In contrast, the BL delineating the boundary between neural cells and meningeal/perivascular cells (BL1) does not have barrier properties. In the choroid plexus, the BBB is located at the level of the epithelium. (C) Brain cytoarchitectonics. The respective location of brain cell types is always identical at every level of the brain organization. Fibroblasts and macrophages are always located at the interface between neural and vascular cells. CSF: cerebrospinal fluid.

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differentiation and function in adult brain, where they may interact most directly with glial cells (Section 3). Since glial plasticity has been thoroughly examined in the neuroendocrine hypothalamo – neurohypophysial system, and cytokines play a major role in regulation of both this system and of the hypothalamo – adenohypophysial system, we will review neuronal – glial interactions in neurosecretion and literature supporting the view that not only glial cells but also extraparenchymal cells intervene in neuroendocrine functions. We will highlight the anatomical links between neuroendocrine, vascular and meningeal tissues, and the means by which these tissues may functionally interact, with glial cells functioning as mediators between meningeal cells and neurosecretory cells (Section 4). Finally we will end the chapter by outlining a ‘meningeal hypothesis’ (Section 5) and concluding remarks (Section 6). 2. Brain extraparenchyma 2.1. Extraparenchymal cells The parenchyma of any tissue is defined as ‘the essential elements’ of the tissue and the extraparenchyma as connective and vascular tissue. The ‘essential elements’ of the tissue is a vague notion. Therefore, it seems better to define the parenchyma as whatever is not vascular or connective tissue. The BL, which is interposed between the parenchyma and extraparenchyma of every organ and tissue, is found wherever parenchymal cells abut the connective and vascular tissues (Fig. 1A,B; see also Fahrquar, 1991). Neurons, glial cells, ependymocytes, epithelial cells of the choroid plexus, the extracellular space between all these cells, including the ventricles, constitute the brain parenchyma. One may add supraependymal cells and Kolmer cells (both macrophages, and possibly the same cell type at two different locations) on the list of parenchymal cells, because these cells are found within the ventricular lumen (VL) (which is part of the parenchyma, see below). The brain extraparenchyma consists of endothelial cells, SMCs, macrophages, pericytes (phagocytic and pulsatile cells surrounding capillaries), fibroblasts, and rare connective tissue cells such as mast cells, and the extracellular space in which all these cells bathe, including the subarachnoid space and cisterns. Immune cells, such as T-lymphocytes, are difficult to classify because they cross BL and travel within the brain, disregarding borders. The lineage of extraparenchymal cells (vascular and perivascular/meningeal cells), as well as parenchymal suprapendymal cells, Kolmer cells and microglial cells has not been clarified to date (Thomas, 1999; Korn et al., 2002). Within the extraparenchyma, one can differentiate the vascular cells (endothelial cells, SMCs and pericytes) from the meningeal and perivascular cells (fibroblasts and macrophages). Meninges are primarily known as coverings of the brain, subdivided into pia, arachnoid and dura, with the arachnoid apposed to dura, and separated from pia by the subarachnoid space. In fact, the arachnoid is linked to pia by arachnoid trabeculae that span the subarachnoid space and merge with the pia so subtly that it is impossible to decide where the arachnoid ends and the pia begins. Most recent authors prefer to refer pia and arachnoid as forming a single tissue (pia –arachnoid). In addition, we have found that fibroblasts and macrophages contact one another to form a gap junction-expressing cellular network throughout pia, arachnoid and dura (unpublished data, and partly shown

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in Mercier and Hatton, 2001; Mercier et al., 2002). The meninges also project within the brain as sheaths of blood vessels (Mercier et al., 2002), stroma of choroid plexus and as connective tissue border zones between major brain structures. The meninges also project around and within the spinal cord, and within the cranial nerves (Mercier and Hatton, 2001). Finally, the meninges project around and within circumventricular organs, separating for example the lobes of the pituitary gland, and delineating lobules within these lobes (unpublished data, and see Fig. 7). Thus, the fibroblast/macrophage network extends up to the deepest structures of the nervous system. The meninges and their major projections within the brain are represented in Fig. 1A in two schematized coronal sections. The hippocampal formation is delineated by a large meningeal projection, which itself projects in the lateral ventricle as the stroma of the choroid plexus (Fig. 1A, left drawing). The meningeal projections (adventitia) that surround each blood vessel (Mercier et al., 2002) are not represented in this simple drawing. However, the meningeal projections surrounding and penetrating the pituitary (capsule and pituitary connective tissue) and cranial nerves (epineurium/perineurium/endoneurium) and containing the fibroblast/macrophage network are represented (Fig. 1A, right drawing). The perivasculature (adventitia) consists of vimentin-positive fibroblasts (Mercier and Hatton, 2000) and macrophages (Mercier et al., 2002, 2003), this layer representing a projection of the meninges into brain parenchyma (meningeal sheath of blood vessels). As each vessel enters or leaves the brain, it carries with it a sleeve of perivascular space (Virchow – Robin space). In the meninges, the adventitia is undistinguishable from the arachnoid. The choroid plexus stroma contains both vascular cells and meningeal cells (Fig. 2A). The network formed by fibroblasts and macrophages throughout the meninges and their projections within the brain as adventitia and stroma of the choroid plexus is represented in Fig. 1B (white dashed line). This network is covered by the BL (BL1) delineating the brain parenchymal/extraparenchymal interface in the choroid plexus, at the surface of blood vessels, at the interface connective tissue/neural tissue between major brain structures, and at the brain surface (Fig. 1B, yellow line). The parenchymal cells facing BL1 consist of vimentin-positive (Mercier and Hatton, 2000) and GFAP-positive astrocytes (forming a multicellular layer, the glia limitans, adjacent to BL1; Mercier and Hatton, 2000, 2001), microglial cells or epithelial cells of the choroid plexus. The cytoarchitectonics in the brain and pituitary neural lobe (NL) is represented in Fig. 1C. Both BL1 and the vascular BL, located between pericytes, SMCs and endothelial cells, are represented in this figure (yellow lines). The parenchymal side of the BL of the microvessels is to a large extent covered by astrocytic endfeet (Mercier and Hatton, 2001; Mercier et al., 2002, 2003), but there are ‘holes’ between the endfeet, and at these locations other cellular constituents, including astrocyte cell bodies, oligodendrocytes and microglia, may appose the BL (Ambrosi et al., 1995). There is a potential for long distance intercellular communication via gap junctions (see chapter by Spray) throughout the meninges and their extensions into the brain, including perivascular sheaths. Meningeal gap junctions couple individual fibroblasts and macrophages (Mercier and Hatton, 2001; Mercier et al., 2002), and appear to be functional, as reflected by the propagation of Lucifer yellow from cell to cell (Spray et al., 1991). In the mature CNS, most gap junctions are expressed by glial, meningeal and perivascular cells (Fig. 2B –D). This is extremely important in the context of this review,

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Fig. 2. (A) Choroid plexus BL, and (B–E) confocal mapping of gap junctions in the glial and meningeal network. (A) The stroma (extraparenchyma) of the choroid plexus, which contain vimentin-ir fibroblasts (red) is enclosed between two BL (laminin-ir, green), one of which is indicated by a large arrowhead. The ependymal cells bordering the ventricle wall are also vimentin-ir. Some subependymal fractones are indicated by arrowheads. (B) Glial network in the lateral preoptic nucleus. Astrocytes (GFAP, green) are interconnected (arrow) by connexin (Cx) 43 immunoreactive gap junctions (red puncta). (C) Glial network in the brain border (periamygdaloid cortex). Cell bodies and processes of astrocytes are interconnected by Cx43 immunoreactive gap junctions (red puncta) (arrow). The arrowhead indicates an astrocyte process with numerous gap junctions. (D) Fibroblast network in the meninges (underneath the interpeduncular fossa) around an artery of the circle of Willis (art). Fibroblasts are immunoreactive for vimentin (red). The field displays an important amount of Cx26-ir puncta (green). (E) Magnified field showing vimentin-ir (red) fibroblasts with numerous Cx26-ir (green) gap junctions (arrow). Scale bars. A –D: 20 mm; E: 10 mm.

because these cells may eventually cooperate with each other (gap junctions exist among glial cells and among extraparenchymal cells, but the BL prevents glial cells and extraparenchymal cells to be connected to each other by gap junctions) and transfer information over a long distance to modulate the expression and release of GF, cytokines

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and extracellular matrix proteins that interact with these signaling factors. In astrocytes, connexin (Cx) 43-mediated gap junction connectivity appears to form the basis of a large intercellular communication network driven by transient, regenerative increases in intracellular calcium concentrations, which are at least partly mediated by intercellular transport of inositol trisphosphate (IP3) (see chapter by Cornell-Bell et al.). Astrocytes also express, but to a lesser extent, Cx30 (Kunzelman et al., 1999) and Cx26 (Mercier and Hatton, 2001) gap junctions, but the functional implication of these gap junctions in terms of interastrocytic communication is unknown. The functional implications of gap junction connectivity in meningeal cells have not been determined, but it is thoughtprovoking that astrocytes express the largest number of gap junctions in zones that are proximal to the meninges, blood vessels and ventricles (Mercier and Hatton, 2001). Furthermore, the distribution of Cx 26 immunoreactivity (an indication of gap junctions) in the extraparenchyma suggests the presence of a network of cells that courses throughout the three layers of meninges, the perineurium of cranial nerves, and the projections of the meninges into the pituitary and brain, including perivascular sheaths and stroma of the choroid plexus. A blood –brain barrier (BBB) separates the parenchymal cells from the systemic circulation. Although inter-astrocytic (Nicholson et al., 2000), inter-vascular and astrocytic – vascular cell interactions may intervene in modulating the diffusion of molecules that have crossed the endothelium by transcytosis (transcellular movement of molecules), it appears that the BBB is primarily due to the connection of the endothelial cells to each other via tight junctions (Rubin and Staddon, 1999). Thus, the BBB is located on the luminal side of the endothelium. All cells, parenchymal and extraparenchymal, including the abluminal side of endothelial cells, are on the brain side of the BBB. This is important, as we will see in further sections, because signaling molecules, once produced by any of the brain cell types may reach all parenchymal and extraparenchymal cell types. This is in keeping with the concept that both parenchyma and extraparenchyma may form the functional brain. In this context, it becomes important to establish whether meningeal capillaries have permeability characteristics that are similar to capillaries that directly contact the neural tissue, despite the fact that only the latter are surrounded by astrocytic endfeet. Although similarities do exist in the ultrastructural features and permeability to tracers in brain and meningeal capillaries, tight junctions between cerebral endothelial cells consist of a uniform population, whereas tight junctions in pial microvessels consist of two populations. In one of these, the tight junctions resemble those between cerebral endothelial cells, with fusion of adjacent cell membranes, but in the second population discernible gaps occur between adjacent cell membranes. Also, an endothelial barrier antigen (EBA) is uniformly distributed between endothelial cells of cerebral microvessels, whereas its expression in endothelial cells of pial microvessels is related to the proximity of the cells to the astrocytic glia limitans (Allt and Lawrenson, 1997). Since the capillaries in the stroma of the choroid plexus are fenestrated, circulating molecules have free access to the rest of the choroid plexus stroma, which consists of cerebrospinal fluid (CSF), fibroblasts and macrophages. These cells produce cytokines and GF (Stylianopoulou et al., 1988; Falk and Frisen, 2002), which may in turn modulate the secretion by choroid epithelial cells. The choroid epithelial cells produce numerous cytokines and GF by activation of processes that may occur within the

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Fig. 3. The BL delineate the brain parenchymal/extraparenchymal interface. Confocal mapping of laminin-ir. (A) Coronal section showing the distribution of BL in the anterior portion of the third ventricle wall, medial preoptic area, and underlying meninges. A BL delineates the brain surface (BS) at the meninges/neural tissue interface. Most of the large laminin-ir structures located within the brain are vascular BL, which also delineate the parenchymal/extraparenchymal interface. Fractones (specialized BL projections) of the subependymal layer of the third ventricle (3V) are indicated by an arrow. Additional BL are found within the meninges, particularly in the adventitia of the arteries (Art) (here Circle of Willis). A magnified view of the subependymal layer is shown in the inset. BV: blood vessel; OC: optic chiasm. (B) More posterior coronal section showing the distribution of

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BL that is interposed between epithelial cells and stromal cells. This is consistent with the developing view that the choroid plexus may be a frequent port of entry for inflammatory processes to the brain (see chapter by Couraud et al.). Processing by ‘cytokine cascades’ may be a major mean by which the inflammatory processes originating from the circulation or from the meninges (via the network of fibroblasts and macrophages coursing in choroid plexus stroma) propagate to the brain parenchyma. Therefore, such cascades are able to ‘bypass’ the tight junction barrier that exists between the epithelial cells of the choroid plexus. These tight junctions form a barrier for the passage of large molecules (Brightman, 1968), preventing most of the blood-borne and meningeal-borne cytokines and GF to enter the VL. This barrier is commonly referred as CSF –blood barrier, a term that seems inappropriate because the barrier is located between the CSF bathing the choroid plexus stromal cells and the CSF bathing both the VL and the general intercellular space of the neural tissue. It should also be noted that the choroid plexus is the main site of synthesis of transthyretin, a molecule involved in the transport of hormones by transcytosis through the choroid plexus epithelium. Therefore, there are possible alternatives to the transmission mechanisms of cytokine cascades mentioned above for transporting signaling molecules from the blood or choroid plexus stroma to the VL and associated intercellular space bathing neurons and glial cells. 2.2. Basal lamina Basal laminae consist of extracellular matrix molecules, the principal ones being laminins, fibronectin, collagens IV and heparan sulfate proteoglycans (HSPG) (reviewed in Farquhar, 1991), and the recently characterized collagen XV and XVIII. All these molecules, which form mats of extracellular matrix among vascular and perivascular cells, and wherever connective tissue cells contact parenchymal tissue, interact with one another, leading to binding and activation of numerous GF and cytokines (Gordon et al., 1987; Roberts et al., 1988; Gilbert, 1994, 2001) that may be blood-borne (passing through fenestrated capillaries), meningeal-borne, or even produced by the neural cells on the parenchymal side of the BL. The adult brain possesses vascular BL and a BL delineating the parenchymal/extraparenchymal interface within the choroid plexus, at the surface of every blood vessel, at the external surfaces of the CNS, and wherever connective tissue contact the neural tissue within the CNS (for example at the interface between major brain structures). There are additional BL within the arachnoid and dura (unpublished data), and in the subependymal layer of ventricles, where a network of specialized BL (fractones) is particularly well developed (Mercier et al., 2002, 2003; and Fig. 1A, arrowheads). The distribution of BL, labeled with an antibody against Laminin (Mercier and Hatton, 2000, 2001; Mercier et al., 2002, 2003), in the adult brain is illustrated in Fig. 3. Fig. 3A shows the BL at the brain surface, the perivascular BL, arachnoid BL as well as the fractones BL in the SON and its ventral border (arrowhead). The thin arrowheads indicate the BL that delineate the perivascular wall. The regular arrowheads indicate special BL that run from blood vessel to blood vessel, covering fibroblasts or macrophages (Mercier et al., 2003), therefore also delineating the parenchymal/extraparenchymal interface. The inset shows the location of the confocal field at lower magnification. Scale bars. A, B: 50 mm; A inset: 10 mm.

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located in the subependymal layer of the third ventricle (3V). Fig. 3B shows a particularly high concentration of blood vessels, detected by their outer BL, in the supraoptic nucleus (SON) of the hypothalamus, and the BL delineating the ventral surface of this nucleus, at the interface between pia and the glia limitans. Additional thin BL connect transversely some blood vessels of the SON capillary plexus (see Fig. 3B, and Mercier et al. (2003) for further information regarding these BL). Although not visible in the two-dimensional figure, each perivascular BL is continuous with the BL delineating the pia/glia limitans interface (Mercier and Hatton, 2001). The fractones (specialized BL localized in the subependymal layer of ventricles) enclose neural stem cells and progenitor cells (Mercier et al., 2002), and form a network, which is continuous with the perivascular BL (Mercier et al., 2002, 2003). Finally, the BL delineating the interface on the choroid plexus between parenchyma (epithelial cells) and extraparenchyma (stroma) is continuous with the BL delineating the pia/glia limitans interface. This continuum is illustrated in Fig. 1B, and the confocal images shown in Fig. 4 represent maps of the interface between the connective perivascular tissue and neural tissue in brain. 2.3. Brain cytoarchitectonics Figures 4– 6 show ultramicrographs that illustrate brain cytoarchitectonics, and the schema in Fig. 1C summarizes the layering shown in the ultramicrographs. Similar cytoarchitectonics exists wherever one observes brain ultrastructure. Glial cells are intercalated between neurons and perivascular/vascular cells, although astrocytes may be replaced by microglial cells and at some places even by oligodendrocytes. In proximity to a small blood vessel, the order (linearly depicted) is as follows: neuron –astrocyte – macrophage – pericyte –endothelial cell (Fig. 4). However, macrophages and pericytes do not form a complete layer, and astrocytes may directly face endothelial cells. In proximity to a larger blood vessel, pericytes are replaced by SMCs (the ultrastructure of these cells is difficult to distinguish in capillary– arteriole transition zones (Mercier et al., 2002), and fibroblasts appear in addition to macrophages in the perivascular layer (adventitia) (Fig. 5A). The order becomes as follows: neuron – astrocyte – (macrophage and/or fibroblast) –smooth muscle cell – endothelial cell. In the meninges, in proximity to an artery, the order becomes: (fibroblast and/or macrophage) – smooth muscle cell – endothelial cell (Fig. 5B). In the choroid plexus, the organization is similar and the order becomes: epithelial cell – (fibroblast and/or macrophage) – endothelial cell (Fig. 5C). At the brain surface, the order is at most places neuron – astrocyte – (macrophage and/or fibroblast) (Fig. 6). The BL delineating the parenchymal/extraparenchymal interface (BL1) is always found between the glial cells and perivascular fibroblasts and macrophages (Figs. 4 –6). The vascular BL are found between all vascular cell layers (Figs. 4 and 5). 2.4. Communication among neurons, glia and meningeal and vascular cells Because of the existence of the BBB, the composition of the extracellular fluid of the brain differs from that of the other organs and of blood plasma (see, for example,

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Fig. 4. Brain cytoarchitectonics. Both ultramicrographs show neural and vascular tissues in the periventricular nucleus. (A) The orderly arrangement consists of neurons, astrocytes (Ast), perivascular macrophages (Mac), pericytes (Per) and endothelial cells (End). Intermediate filaments (IF) identify astrocytes. The BL are located between the vascular cells and at the periphery of perivascular macrophages. The latter BL typically displays fibrils of collagen (col). Mag: magnocellular cell body; Ax: axon; D: dendrite. (B) Other field showing the same cytoarchitectonics. The perivasculature (adventitia) consists here of two layers of macrophages. One macrophage shows typical large lysosomes (lys). Scale bars: 500 nm.

chapter by Walz). Many molecules, including proteins and some amino acids, cannot cross the blood vessel wall at all, whereas others, like lipid-soluble substances, can cross the endothelium with ease. Some nonlipid soluble substances cross the endothelium by transcytosis, and a few compounds, like glucose and to a lesser extent lactate and several amino acids are transferred by special transporters. Cytokines and GF are large proteins

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Fig. 5. Brain cytoarchitectonics. Vasculature and choroid plexus. (A) Brain arteriole showing a typical monolayer of smooth muscle cells (SMC). Fib: fibroblast in the perivascular layer. End: endothelial cell. Perivasc BL: perivascular BL. (B) Artery of the Circle of Willis in the meninges. The media consists of multiple layers of SMC. A BL surrounds all SMC. The perivascular layer (adventitia), which is indistinguishable from the arachnoid, comprises numerous fibroblasts (Fib) and collagen fibers. SAS: subarachnoid space. (C) Choroid plexus. The orderly arrangement consists of epithelial cells (Ep), Fib (no macrophage is visible on this field), and endothelial cells (End). A BL (arrow) separates the epithelium from the stroma. A thin BL (arrowhead) covers the fenestrated endothelium (see in the 3 £ magnified field). Fenestrations are indicated by arrows in the magnified field. VL: ventricular lumen. Scale bars: A, C: 1 mm; B: 2 mm.

(average Mw 15 –30 kDa) that normally do not pass the BBB, the choroid plexus epithelial barrier (mentioned above), and the tight junction-barrier located at the arachnoid/dura interface (most large molecules injected into the dura do not enter into the arachnoid). Therefore, peripherally circulating cytokines and GF cannot easily reach parenchymal brain. These molecules cannot reach the pia – arachnoid or the pial sheath of blood vessels either, because of the tight junction barrier located in the endothelium of blood vessels penetrating the brain or the pia –arachnoid. However, the cytokines usually reach the dura,

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Fig. 6. Brain cytoarchitectonics. Ultrastructure of the brain parenchyma/meningeal border (SON). The cytoarchitecture consists of astrocytes (Ast) of the ventral glia limitans, macrophages (Mac), and endothelial-like cells (End) bordering a venous sinus. A circumvoluted BL (arrow) separates the astrocyte processes (Ast P) from the pial Mac. Col: collagen fibrils. Scale bar: 1 mm.

being stopped at the dura –arachnoid interface (arachnoid barrier). Whether or not the blood-borne signaling molecules enter the dura via the local vasculature or leaks from the head connective tissue, which possesses fenestrated capillaries, is unknown, but this remains a possibility due to the differences in tight junctions in blood vessels penetrating the meningeal and neural tissues (Allt and Lawrenson, 1997) mentioned above. Although not mentioned in the literature (to our knowledge), the circulating cytokines and GF may alternatively reach the meninges via the connective tissue of the circumventricular organs and the stroma of the choroid plexus (in both cases, the capillaries are fenestrated).

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Once released in any part of the connective tissue system, it is likely that the signaling molecules may reach the entire extraparenchyma, and eventually the brain parenchyma. Cytokines and GF that are produced at the level of the brain, whether by parenchymal or extraparenchymal cells of the pia – arachnoid, vasculature and perivasculature within the pia –arachnoid and meningeal projections within the brain, are free to diffuse throughout the extracellular space that bathes glial cells, neurons, vascular cells and pia – arachnoid cells, because there is no barrier between the pia – arachnoid and the brain proper, and no barrier between the brain vascular/perivascular cells and the neural cells. Horseradish peroxidase (Mw 48 kDa) penetrates the brain tissue when injected into the meninges (Brightman, 1968). This is possible because neither the BL between meningeal or vascular cells nor the astrocytic endfeet of the glial limitans form a true barrier for these molecules. Collagen IV, laminins and fibronectin, main components of BL, elevate endothelial transcellular resistance (Tilling et al., 1998), but allow the passage of lipid-soluble substances, carbohydrates and proteins. Astrocytes of the glial limitans do not possess tight junctions (Wagner et al., 1983). This is probably the reason why these cells do not obstruct the movement of even large molecules in the intercellular spaces. Nevertheless, the densely packed astrocytic processes of the glial limitans, which increase the local tortuosity factor (Nicholson et al., 2000), may slow the diffusion of molecules (Wagner et al., 1983). Thus, there is no significant barrier separating the space that is occupied by the walls of the vasculature, the subarachnoid (and generally speaking pia – arachnoid) space and the intercellular space of the brain parenchyma. It is, however, worth mentioning that while the CSF that bathes pia – arachnoid cells is in free communication with the extracellular space of the neural tissue, this may not be the case for CSF in the dural sinuses. The presence of tight junctions between the arachnoid cells delineating the dura – arachnoid interface and between cells delineating the venous drainage system, probably reflects the presence of a selective barrier for the passage of proteins. These cells resemble endothelial cells in their morphology (Fig. 6) and ultrastructural characteristics. The presence of the BBB at the level of the endothelium, and not at the level of the glial limitans, implies that GF and cytokines may circulate among meningeal/perivascular macrophages and fibroblasts, vascular cells, glial cells and neurons. GF and cytokines produced by endothelial cells, SMCs, pericytes, fibroblasts and resident meningeal/perivascular macrophages may enter the extracellular space between neurons and glial cells. Similarly, GF and cytokines produced by neurons and glial cells may enter the extraparenchyma and influence the functions of meningeal and vascular cells. They may also reach the lymphatic system of the head and neck via perivascular spaces (Cserr and Knopf, 1992). Endothelial cells may communicate with the other brain cell types via cytokines and GF, because cytokines and GF may be released from the abluminal side, which is located outside of the BBB. Since one surface of the endothelial cells is on the systemic side of the BBB and the other on the brain side, they might even register signals (e.g., the presence of a cytokine) in blood and translate this information to the meninges and neural tissue via endothelial-borne cytokines (cytokine cascade). Passage of protein signaling molecules within the entire extracellular space bathing all CNS cell types does not necessarily mean that these molecules, once released in some precise area of the brain or meninges, reach the entire brain. It has been shown that GF diffuse slowly through brain tissue (Krewson et al., 1995), and that GF and cytokines bind

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to extracellular matrix proteoglycans, particularly in the BL, a privileged site for their action. In addition, cellular uptake by any brain cell type may contribute to the restriction of distribution of specific molecules, leading to further possibilities of local actions of GF and cytokines. 3. Cytokines and growth factors 3.1. Cytokine and GF in brain extraparenchymal cells 3.1.1. Roles of cytokines and growth factors Cytokines and GF (see chapter by Nakagawa and Schwartz) are signaling protein molecules that participate in the immune defense and response after injury (inflammatory processes). They also induce as well as regulate different cell functions, such as proliferation, differentiation and migration, during both development and throughout adulthood. In the adult brain, cytokine and GF are directly involved in the induction and regulation of neural stem cell and progenitor cell proliferation, differentiation and migration. They also intervene in specific cell functions such as neuroendocrine secretion (Turnbull and Rivier, 1999). Almost all cells produce cytokines and GF (but not always the same factors) and these molecules can be presynthesized, and stored in cytoplasmic granules or in the adjacent extracellular matrix (Kinnunen et al., 1999). Therefore, responsiveness to GF and cytokines may be regulated not only by production and receptor expression but also by storage or alteration (activation/deactivation following interactions with extracelllar matrix proteins) in the BL or extracellular space in general, or by competition with other cytokines and GF that possess affinity for the same receptors (reviewed in Hopkins and Rothwell, 1995b). Moreover, truncated forms of the receptors are present extracellularly and may either neutralize or enhance the effect of some of these factors, depending upon their identity (Turnbull and Rivier, 1999). It is particularly interesting that these factors intervene early in plasticity events, stimulating other cells and initiating cascades of other cytokines and GF (Hopkins and Rothwell, 1995a,b). Thus, it is possible that effects mediated by astrocytes or other glial cells, including microglial cells, can be triggered themselves by meningeal or vascular cells. 3.1.2. Cytokine and GF production In Table 1, we have summarized recent literature showing that extraparenchymal cells in the adult brain are both producers (indicated by the expression of mRNA and presented in all columns A) and targets (indicated by binding or physiological effects and presented in all columns B) of several GF and cytokines. The different cell types represented in Table 1 may be interacting at the BL of their interfaces (valid for all cell types besides neuron– glia interfaces). Endothelial cells, SMCs and/or pericytes serve as important sources of GF and cytokines, producing interleukin 1b (IL-1b) (Wong et al., 1996), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and IGF1 (Biro et al., 1994), and brain derived neurotrophic factor (BDNF) (Leventhal et al., 1999). Although meninges have been poorly investigated, it is known that meningeal cells produce bFGF (Gonzalez et al., 1995), a potent agent involved in adult neural stem

Extraparenchyma

Pituit. gliaa Neurons

Smooth M.C.

Pericytes

Macrophages

Fibroblasts

Astrocytes

A

A

A

A

A

A

B

22

24 5, 13, 26, 39, 40, 57 38 57

B

41 30, 55 30 30 30 2, 9 41 30 30 30 30 30 30

B

B

B

30 23

30

9 30

46 30 30 30 30 30, 49 36 30, 35 50 20, 23, 49

54

32 15, 31 49, 43 57 6

37 16

54

54 54

38

45 47

37, 51 8 4, 7, 11, 39, 44

6, 10, 57

54 19

28

B

34

9

51

34 42 56

52 58 53 32, 51

46

30 48, 54

A

A

B

3 31 6 31 30 31

30 30 30 35

B

55 55

42 2

A

End. Cellsb

1

4 13 25 45 27

38 38 38 34 38 38

12 49

27, 38 27 38 28 3 38 16 18 38 14, 16, 18, 56 16, 17, 33 16, 38 21, 25 25, 43 16 28,52

A: synthesized by- upon physiological conditions or after injury; B: effect on- (mitosis, differentiation, morphological change or presence of receptors for-) a Pituitary glia ¼ folliculostellate cells, pituicytes and stellate cells. b Endocrine cells of the pituitary anterior lobe.

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VEGF PDGF bFGF aFGF HB-EGF EGF CNTF IGF1 IGF2 TGFa TGFb (c) TGFb1 NGF IL1 (c) IL1b IL6 PTHrP LIF

Parenchyma

Endothelial cells B

230

Table 1 Intercellular communication via GF and cytokines bound in the BL. Extraparenchymal cells (vascular and meningeal cells) produce and release GF and cytokines that bind (or alternatively cross) BL where they become activated and may exert their influence on neural cells. Neurons and glial cells also produce GF and cytokines, which participate in neuroglial interactions but also in neural cells– extraparenchymal cells interactions after eventual binding in the pial or perivascular BL. This table is based on mRNA in situ hybridization experiments (columns A), and different functional tests or presence of plasma membrane or nuclear receptors for a given signaling molecule (columns B)

c

Meninges and Perivasculature as Mediators of CNS Plasticity

Subtype not defined or other subtype. 1, Banner et al. (1997); 2, Biro et al. (1994); 3, Calza et al. (2000); 4, da Cunha et al. (1993); 5, Eclancher et al. (1996); 6, Frisen et al. (1998); 7, Gagelin et al. (1995); 8, Gomes et al. (1999); 9, Gonzalez et al. (1995); 10, Goss et al. (1998); 11, Han et al. (2000); 12, Hentges et al. (2000); 13, Hinkle et al. (1997); 14, Honegger et al. (1991); 15, Ignotz and Massague (1986); 16, Jones and Kennedy (1993); 17, Kahn et al. (1997); 18, Katsuura et al. (1988); 19, Lafortune et al. (1996); 20, Lehrman et al. (1998); 21, Li et al. (2000); 22, Lippold et al. (1996); 23, Logan et al. (1992); 24, Luo and Miller (1995); 25, Marz et al. (1999); 26, Meiners et al. (1993); 27, Messi et al. (1999); 28, Mi et al. (2001); 29, Minami and Sarkar (1997); 30, Moses et al. (1995); 31, Motohashi et al. (1995); 32, Ojeda and Ma (1998); 33, Parsadaniantz et al. (1997); 34, Perez-Castro et al. (2000); 35, Pollman et al. (1999); 36, Powell et al. (1998); 37, Rabchevsky et al. (1998); 38, Ray and Melmed (1997); 39, Reilly et al. (1998); 40, Reuss and Unsicker (1998); 41, Seghezzi et al. (1998); 42, Seniuk-Tatton et al. (1995); 43, Spinedi et al. (1992); 44, Struckhoff (1995); 45, Struckhoff and Turzynski (1995); 46, Stylianopoulou et al. (1988); 47, Subang and Richardson (2001); 48, Turnbull and Rivier (1999); 49, Unsicker et al. (1991); 50, Verbeek et al. (1994); 51, Voigt et al. (1996); 52, Vutskits et al. (1998); 53, Wilczak et al. (2000); 54, Wong et al. (1996); 55, Yamagishi et al. (1999); 56, Yasin et al. (1994); 57, Yoshida and Gage (1991); 58, Zhou et al. (1999).

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cell proliferation, numerous members of the transforming growth factor family including amphiregulin, TGFa and TGFb1 (Rappolee et al., 1988; Unsicker et al., 1991), IGF2 (Stylianopoulou et al., 1988), and IL-1b (Wong et al., 1996). These GF and cytokines are also expressed during development, and they are particularly active in inducing morphogenetic events. A number of in situ hybridization and immunocytochemical studies have investigated the distribution of IL-1b mRNA or IL-1b protein in both brain parenchyma and extraparenchyma after systemic (intravenous or intraperitoneal) administration of bacterial lipopolysaccharides (LPS). Expression of IL-1b is especially marked in the meninges, choroid plexus, and circumventricular organs (organum vasculosum of the lamina terminalis (OVTL), subfornical organ (SFO), median eminence, area postrema, pineal gland, and pituitary). The cell types expressing IL-1b in these regions include macrophages, microglia and perivascular cells (Turnbull and Rivier, 1999). These circumventricular organs are not separated from the systemic circulation by any BBB (capillaries are fenestrated in the circumventricular organs). Within these structures, circulating substances can directly access neural and meningeal tissues, playing a pivotal role in blood – brain communication. Why the circumventricular organs do not provide access to brain parenchyma at large is, to our knowledge, unknown. Although some of the circumventricular organs possess continuous tight junctions barriers on their ependymal walls (Proescholdt et al., 2002), it is not yet explained why the blood-borne molecules do not enter the main neural tissue and the meninges after penetrating into circumventricular organs. For example, after intravenous administration, horse radish peroxidase invades the median eminence, but it remains restricted to this tissue despite its direct contacts with the pia –arachnoid and hypothalamic neural tissue (Broadwell et al., 1987).

3.1.3. Cytokine and GF receptors All types of extraparenchymal cells are also targeted by GF and cytokines, and possess receptors for mediating intracellular actions (Table 1, column B). For example, a multitude of studies agree that vascular and perivascular elements are the most prominent loci of receptor expression for IL-1b. Intense IL-1 receptor expression has been described in endothelial cells throughout the brain (Turnbull and Rivier, 1999). It is thus possible that meningeal and brain vascular tissues represent a vast field for production and activation of these molecules, and that they exert both autocrine and paracrine effects, leading to a cascade of events. In addition, intercellular communication through the network of fibroblasts and macrophages may occur to induce effects at long distance. The possibility also exists that BL located at the parenchymal/extraparenchymal interfaces (BL1) may serve as an interface of communication, storing and activating signaling molecules for the interaction between macrophages/fibroblasts and glial cells. However, BL and the glial compartment are not necessarily an obligatory interface for targeting neurons. GF and cytokines produced by vascular and meningeal cells may alternatively cross the BL and reach the general intercellular space bathing all neural-derived cells.

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3.1.4. Role of cytokines and GF in cell proliferation Previous authors have suggested that pericytes and perivascular macrophages (sometimes the two cell types have been confused in the literature, see in Mercier et al., 2002—ultrastructural criteria for distinguishing the two cell types; in addition the macrophages express specifically the CD163 antigen) may be vascular stem cells, i.e., potential precursors for smooth muscle and endothelial cells (Thomas, 1999), as well as potential precursors for microglial cells (which are sometimes referred as parenchymal macrophages). That they produce GF that are responsible for cell proliferation and cell differentiation (Table 1) supports this idea. A recent paper has also suggested that brain endothelial cells, via VEGF and BDNF production, are involved in adult neurogenesis (Louissaint et al., 2002). We recently characterized BL projections (fractones) that may transport and activate GF and cytokines produced by perivascular macrophages to induce neural stem cell proliferation and differentiation in the neurogenic zones of the adult brain (Mercier et al., 2002). The anatomical correlations between neurogenesis and angiogenesis in these zones (Palmer et al., 2000) may reflect that the resident connective tissue perivascular macrophages, by means of the signaling molecules they produce and release in the BL, may be responsible for inducing cytogenesis in general (Mercier et al., 2002, 2003). The literature supports the hypothesis that meningeal cells and their overlying BL are involved in regeneration after injury. After immune, chemical, or mechanical challenge of the CNS production of proinflammatory cytokines by fibroblasts and macrophages is the first reaction (Basu et al., 2002). Astrocytic (astrogliosis and/or release of cytokines and GF) and neuronal responses occur after the response of the connective tissue/immune cells (see also chapter by Dezawa). Also, meningeal tissues are the first to regenerate and serve as scaffolds after section of the tail (spinal cord) of the lizard (Alibardi, 1996). Similarly, invasion by connective tissue cells and formation of BL is the first detected morphological event after section of peripheral nerves (Ide, 1983). Regeneration processes ultimately involve migration of meningeal cells and astrocytes, astrocyte proliferation (reactive gliosis), angiogenesis, formation of new BL, the release of new extracellular matrix molecules and upregulation of neurotrophins (Choi, 1994; Miyoshi et al., 1995; Goss et al., 1998). Meningeal cells and BL are usually considered detrimental to axonal regrowth, since meningeal cells and BL form a molecular barrier that stops growth cones of regenerative axons (Davies et al., 1999). However, these tissues can also be beneficial for axonal regrowth, as seen by the fact that grafting of neonate meningeal tissue significantly increases axonal regeneration after injury in the adult (Ishikawa et al., 1995). There is no reason to believe that fundamental differences exist between the mechanisms occurring during development and throughout adult life (see, however, chapter by Kalman). It is well established that morphogenetic events during development are dependent on connective tissue inductions, and that the BL serve as scaffolds for guiding newly generated tissues (Gilbert, 1994). There are clear demonstrations that during development, the BL forming an interface between connective tissue and parenchymal tissue (epithelium or any other parenchymal cells directly facing the BL) is a major site of interactions between extracellular matrix proteins, cytokines and GF. These interactions serve to activate the signaling molecules that control fate and function of overlying cells (Gordon et al., 1987; Roberts et al., 1988). That specialized BL (fractones) and meningeal/perivascular tissues are present and particularly developed in adult

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neurogenic zones may indicate that extraparenchymal tissues are still active throughout adulthood to control stem cell proliferation and differentiation (Mercier et al., 2002). The fact that stem cells are systematically apposed to a BL that itself overlies connective tissue cells (Grisham and Thorgeirsonn, 1997; Mercier et al., 2002) reinforces this hypothesis. The particular anatomical relationships between meningeal tissues, fractones of the third ventricle and neuroendocrine tissues (Mercier et al., 2003) may also reflect the functional importance of meninges and BL, the former as site of production of signaling molecules, the latter as site of activation of these signaling molecules.

3.2. Cytokines and growth factors in brain parenchymal cells Many cytokines and GF produced in brain parenchyma serve the function of autocrine and paracrine communication with the parenchyma itself and will not be reviewed in the present chapter (see, however, chapter by Nakagawa and Schwartz regarding cytokines and GF in astrocytes). In addition, brain parenchyma reacts to systemic stresses, e.g., infection or even psychological stress, with induction of cytokine expression. One way of investigating such induction is to administer endotoxin, e.g., as purified preparations of bacterial LPS and monitor the induced cytokine expression in brain parenchyma. Marked induction of IL-1b in microglia throughout the entire brain has been observed, although this tended to require larger doses of LPS than corresponding IL-1b induction in perivascular spaces and meninges (Turnbull and Rivier, 1999). The time course of IL-1b mRNA expression after i.p. administration of LPS was followed by Quan et al. (1998). In an initial wave of labeling, which began after 0.5 h, peaked at 2 h and declined after 4 h, the labeled cells were concentrated in OVLT, SFO, median eminence and area postrema as well as in extraparenchymal cells. In a second wave, which began after 8 –12 h, small cells identified as glia became labeled throughout the brain and the pituitary, whereas no neurons became labeled as a result of LPS injection. Two waves were similarly demonstrated in c-fos mRNA induction after i.v. administration of IL-1b (Herkenham et al., 1998). During the first wave, c-fos was induced at 0.5 h in cells of the outer meninges (mainly outside the arachnoid– dural border), blood vessels (arteries, veins and capillaries), choroid plexus and vascular and connective tissue cells of circumventricular organs. After 3 h, a second activation pattern appeared in cells located just inside the now quiescent barrier cells, with colocalization of c-fos and GFAP mRNAs in the arcuate nucleus, median eminence and glia limitans. At the same time, various cell types in the circumventricular organs showed a characteristic labeling pattern. The authors proposed that the first wave of activation represented the effects of blood-borne immune signals, the second wave propagation by aid of molecules generated within the first set of activated cells to neighboring receptive cells. In the retina, it has also been shown that production of a proinflammatory chemokine, monocyte chemotactic protein-1 (MCP-1), in response to intraocularly injected IL-1b occurs both in extraparenchymal cells (endothelial cells and macrophages) and in microglia and astrocytes of the glia limitans (Cuff et al., 2000). Conversely, intracerebroventricularly injected IL-1b rapidly penetrated into periventricular tissue, spread along white matter fiber bundles and blood vessels in the caudate/ putamen, hypothalamus and amygdala, and induced transcription factor nuclear factor

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kappa B translocation and/or c-fos expression in choroid plexus, ependymal cells, cerebral vasculature and meninges, supraoptic and paraventricular hypothalamus, and central amygdala as well as widespread vascular-mediated leukocyte infiltration and brain-wide glial activation (Konsman et al., 2000; Proescholdt et al., 2002). Cytokine- and GF-induction in astrocytes of the hypothalamic nuclei secreting vasopressin (VP), oxytocin (OT) and pituitary hypophysioptrophic hormones (see below) as well as in specialized glial-like cells in the pituitary (pituicytes, stellate cells and folliculostellate cells in the NL, the intermediate lobe and the anterior lobe, respectively) are of special interest, because systemic cytokine production leads to enhanced or reduced (depending on cytokine and type of hormone) secretion of several of these hormones and because neuroendocrine secretion in this system to a large extent is influenced by glial cells, as will be discussed in Section 4. However, first the anatomy and the physiology of the hypothalamo – hypophysial systems will be described. 4. The hypothalamo – hypophysial systems 4.1. Anatomy 4.1.1. Magnocellular and parvocellular hypothalamo –hypophysial systems Two different neuroendocrine systems exist in the hypothalamus, the magnocellular system, producing VP and OT, and the parvocellular system producing hypophysiotropic hormones, which are releasing and inhibitory factors, e.g., gonadotrophin-releasing hormone (GnRH), also called luteinizing hormone releasing hormone (LHRH), and corticotropin-releasing hormone (CRF). The magnocellular system is functionally associated with the posterior, NL of the pituitary, the parvocellular system with the anterior lobe, the adenohypophysis. 4.1.2. The magnocellular hypothalamo –neurohypophysial system (mHNS) One of the earliest indications that nonneuronal cells might play significant, dynamic roles in mammalian CNS function came to light in relation to neuroendocrine regulation (Tweedle and Hatton, 1976, 1977). The discovery that physiological activation of the mHNS induced dramatically altered anatomical relationships between the neurons and the astrocytes of this system made it obvious that these glial cells were active participants in neuroendocrine function (for reviews, see Hatton, 1990, 1997, 1999; Miyata and Hatton, 2002; Theodosis and Poulain, 1993; Theodosis et al., 1998; Theodosis, 2002—see also chapter by Salm et al.). This active role was in rather stark contrast to the passive, supportive participation previously attributed to the astrocytes of not only this system but, indeed, to those of the brain in general. The mHNS (Fig. 7), a main focus of this review, is a model for dynamic glial – neuronal interactions (Miyata and Hatton, 2002) and a continuing source of enlightenment regarding interactions between neural and nonneural cell types (Mercier and Hatton, 2000, 2001; Mercier et al., 2002, 2003). The nonapeptides oxytocin or vasopressin synthesized in the cell bodies and dendrites of mHNS neurons and either released from the dendrites or transported by axoplasmic flow down their principal axons to the pituitary neural lobe

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Fig. 7. (A) The magnocellular hypothalamo–neurohypophysial system. Cell bodies, dendrites and proximal portion of axons of magnocellular neurons are located in the hypothalamus. The axons project into the NL of the pituitary. SON: supraoptic nucleus; PVN: paraventricular nucleus. (B) Brain and pituitary BL. The BL are identified by laminin-immunoreactivity (white in the main image, green in the magnified fields). Brain BL are primarily located at the blood vessel surface (see magnified image, at top) and at the pia/glia limitans interface (arrowhead in the main image). Pituitary NL and anterior lobe (AL) BL delineate connective tissue labyrinths (see left magnified field, arrowhead). Colored surfaces have been added on this image to indicate the location of pia, arachnoid and dura. IL: intermediate lobe. (C) Cytoarchitectonics of a cord in the AL. Endocrine cells release their hormones by crossing two BL. BL1 operates as an extended surface of contact between folliculostellate cells and fibroblasts. The vascular BL overlies the fenestrated endothelium. (D) Anatomical relationships among the cell types of the hypothalamo–pituitary system. Note the key location of fibroblast/macrophage network at the interface between neural cells and fluids compartments.

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(NL in Fig. 7B). There, under appropriate stimulus conditions, the fully processed hormones are released, and must cross both the BL located at the parenchymal/ extraparenchymal interface (BL1) and the periendothelial BL to enter the NL fenestrated capillaries, with eventual access to the general circulation. Considering this particular aspect, the organization of NL is similar to the endocrine anterior lobe (AL in Fig. 7B), where the hormones also have to cross two BL to reach the blood within the fenestrated capillaries (Fig. 7C). Both tissues also have a similar connective tissue and BL organization (compare the distribution of BL in the two lobes in Fig. 7B). One has only to replace endocrine cells by neurosecretory terminals (which of course have different morphologies) and folliculostellate cells by pituicytes in Fig. 7C to obtain a schema of the cytoarchitectonics of the NL. The mHNS system is ideal for investigations of interactions between neurons, astrocytes and extraparenchymal cells, both because a great deal is known about the stimulus conditions (e.g., dehydration, hemorrhage, suckling of the young, etc.) that activate the mHNS cells, and about the neural inputs involved, and because the downstream consequences of the neuronal outputs are well established, i.e., release of specific peptides with known physiological effects (VP: vasoconstriction in certain vascular beds and water reabsorption by the kidneys; OT: sodium excretion in the kidney, uterine contractions during parturition, and contraction of the mammary gland myoepithelial cells during lactation, resulting in the milk ejection reflex). The hypothalamic components of the mHNS include the supraoptic nuclei (SON), paraventricular nuclei (PVN) and several so-called accessory neurosecretory nuclei. The SON, which is highly vascularized, but does possess a BBB, lies above the meninges (Fig. 3B, insert; Figs. 7– 9). In the rat, it lies on either side of the optic chiasm/optic tracts and directly dorsal to the large blood vessels of the Circle of Willis. The meninges that are located between the hypothalamus and the pituitary gland contain, besides the Circle of Willis, also veins, and large CSF cisterns, providing an opportunity to integrate information from peripheral organs and the brain itself. Large meningeal projections enter with the optic tracts on the lateral sides of the hypothalamus (see Fig. 1). The PVN is in contact with the walls of the third ventricle and faces the ependyma of the third ventricle. Two accessory neurosecretory nuclei, nucleus circularis and lateral hypothalamic perivascular nucleus both encircle blood vessels (Duan and Ju, 1998). Thus, magnocellular nuclei have a privileged relationship with the ependyma, meninges and their projections as vascular sheaths. The location of the nuclei projecting into the magnocellular and parvocellular systems is also interesting, because these nuclei are all associated with meninges and ependyma. Among them are: (1) the OVLT, inserted between the anterior side of the third ventricle and meninges; (2) the SFO, located in the roof of the third ventricle, in the proximity of meningeal projections that ultimately form the stroma of the choroid plexus; and (3) the tuberomammillary nuclei, which lie above the meninges in the posterior hypothalamus. In Fig. 8, the salient features of the rat SON in the coronal plane are diagrammed. Dorsally lying cell bodies receive input from a variety of extrinsic sources (see Hatton and Yang, 2002, and Tables 1 and 2 in Hatton, 1990). Nonchemical synaptic interactions between mHNS neurons also have been studied. Electrical synapses that support dye transfer occur between OT neurons and between VP neurons, but not between individual

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Fig. 8. Diagram of the SON and the pial–glial limitans in coronal plane. Profiles representing somata are lateral to the myelinated fibers of the optic chiasm (OC) in the somatic zone (SZ). Ventral to the SZ are the parallelprojecting dendrites (unfilled small circles), depicted in cross section, and constituting the dendritic zone (DZ). Mingling with only the most ventral dendrites are nuclei of the astroglial cells (larger filled circles), whose ventrally projecting processes (shown in Fig. 6) fill the clear space between the BL (small arrows) and the most ventral dendrites. These glial cell bodies and processes constitute the ventral glial lamina (VGL). Dorsally projecting processes from these glia fill most of the space not occupied by the somata and dendrites. Ventrolaterally projecting dendrites are not included here.

neurons of the two cell types (for review see Hatton, 1998). There is a tendency to think of all of these neural inputs to the SON as synaptic, but this would be an oversimplification. Some terminals (e.g., noradrenergic, histaminergic and perhaps others) appear to engage in paracrine release, which affects all nearby cells expressing receptors for the transmitter in question. Each magnocellular neuron in the SON projects one to three dendrites ventrally, which then turn to course rostrocaudally in the dendritic zone. A dorsally projecting dendrite from the soma (not shown) generally gives rise to the axon, which exits the nucleus dorsally on its way to the NL via the internal zone of the median eminence. Immediately subjacent to the overlying dendritic zone is a bed of astrocytes (seen here twodimensionally as a line), whose cell bodies and ventrally projecting processes constitute the SON’s ventral glial lamina (VGL). These same astrocytes also give rise to extremely long processes that project dorsally between adjacent dendrites in the dendritic zone and continue between adjacent somata in the somatic zone to reach the most dorsal aspects of the nucleus (see Fig. 9 for examples). Both extensive and tortuous, the astrocytic cell processes of the VGL fill the space between the astrocytic cell bodies and the pia (Fig. 6). This glial membrane is in direct contact with the BL that separates this portion of the parenchymal compartment from the pia, across from which lie the arachnoid and the large blood vessels supplying the anterior brain areas. Arterioles from these vessels, along with their accompanying pial membrane, perforate the SON and extensively ramify, decreasing in size to form capillaries ranging from 6 to 20 mm in diameter, all except the smallest with

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Fig. 9. Brain cytoarchitectonics. Confocal laser scanning microscopy of the same region as depicted in Fig. 8. (A) Immunocytochemical distribution of GFAP (green) and vimentin (red) in the SON. GFAP is a specific marker of astrocytes. Vimentin is primarily expressed by meningeal (M) and perivascular cells. A subset of astrocytes also express vimentin (arrows). Astrocyte processes are extensively intermingled in the ventral glia limitans (vgl). ACA: anterior cerebral artery (Circle of Willis). BV: blood vessel; OC: optic chiasm. (B) Distribution of GFAP astrocytes (green) and oxytocinergic neurons (OT, red) in the SON. Vasopressin neurons (VP) are not stained. (C) Diagrammatic illustration of the spatial relationship between Fig. A and Fig. B. Scale bars: 50 mm.

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perivascular sheaths (see Fig. 2B in Mercier and Hatton, 2001). As a result, the SON has one of, if not the highest capillary density in the entire mammalian brain (Ambach and Palkovits, 1979). Thus, in addition to inputs from both local circuit and more distant neurons, SON neurons and astrocytes, both individually and in the aggregate, are highly likely to be directly exposed to blood-borne information, and there are vast opportunities for interactions between brain parenchyma and extraparenchyma. The other major component of the mHNS is the pituitary NL. The NL is in direct contact with meninges (via the pituitary capsule) and is invested by an extensive lattice of connective tissue connected to the capsule (Fig. 7B). The parenchyma of NL is largely made up of axons, axonal swellings (known as Herring Bodies), axon terminals of the hypothalamic neurosecretory and other neurons and the pituicytes. Pituicytes are a type of astrocytes (Salm et al., 1982), and they are the only resident cell type in the NL parenchyma. Underneath the hypothalamic dura, which is shared by both brain and pituitary, the pituitary meninges consist of a capsule and projections of the capsule between the lobes of the pituitary (Fig. 7B). These projections are connected to labyrinths of connective tissue coursing throughout the neural and anterior lobes (Fig. 7B,C), and between the lobules of the intermediate lobe (Fig. 7B). A diagram summarizes the anatomical relationships between the different cell types present in both pituitary and parvocellular/magnocellular hypothalamic systems (Fig. 7D). The association of both the SON and pituitary NL with the Circle of Willis deserves emphasis. The Circle of Willis is an anastomotic system of arteries coursing in the subarachnoidal space at the base of the hypothalamus (Ambach and Palkovits, 1979). Basilar artery and carotids supply the Circle. Arteries arising from the Circle supply most of the brain. In cat, dog, monkey and rat, the supraoptic nuclei are located just dorsal to the anterior cerebral arteries, the latter forming the front of the Circle (Christ, 1969). This is particularly interesting when one considers that the location of the SON, relative to other brain structures, is not consistent in these species (Christ, 1969). The pituitary gland is closely associated with the posterior portion of the Circle (see arteries above the pituitary in Fig. 1A, right drawing), which courses just above the dorsal surface of intermediate lobe. The anatomical evidence that extraparenchymal cells and the BL are closely associated with both neurons and glial cells of the mHNS suggests that interactions between parenchymal and extraparenchymal components play important roles in neuroendocrine modulation and control (Mercier and Hatton, 2000, 2001; Mercier et al., 2003). The extraparenchymal cell types include both vascular and perivascular elements: endothelial cells, pericytes, macrophages, as well as fibroblasts of the meninges that penetrate the brain along with the blood vessels. Furthermore, the presence of specialized BL (fractones) in the neurogenic zones of the adult brain (Mercier et al., 2002) strongly suggests that BL now have to be accepted as active players in brain function. Like other known functional elements, they vary in composition and extent from one brain area to another, dynamically varying within certain brain areas, including the SON, as physiological conditions change (Salm et al., 1998; see chapter by Salm et al.), and they can serve as sites for delivery of chemical signals.

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4.1.3. The parvocellular hypothalamo –adenohypophysial system The second important neuroendocrine system of the hypothalamus is the parvocellular neurosecretory system. These neurons are responsible for the production and release of the hypophysiotropic hormones, which besides LHRH and CRF are thyrotropin releasing hormone (TRH), growth hormone releasing hormone (GHRH) and somatostatin. The parvocellular neurosecretory system consists of tubero-infundibular neurons that are mainly located in the arcuate nuclei and neurons projecting through the lateral retrochiasmatic area. The arcuate nuclei surround the ventral portion of the third ventricle, which form an extended chamber at this level, and contact the meninges in their ventro-posterior portion. Medial preoptic nuclei, periventricular nuclei in the anterior hypothalamus, and parvocellular neurons of the PVN also belong to this system, and they are all located in the vicinity of the third ventricle. These nuclei receive input from many sources, including the limbic system, and they project to the external zone of the median eminence, where the hormones are stored until they are released into the perivascular spaces surrounding the capillaries of the pituitary portal plexus following appropriate stimulation. The parvocellular hypothalamic system is also astrocyte-rich (see chapter by Prevot). The anterior pituitary contains different cell types (e.g., gonadotrophs, corticotrophs, thyrotrophs, somatotrophs, lactotrophs), which release hormones (e.g., follicle-stimulating hormone [FSH]; luteinizing hormone [LH]; adrenocorticorticotropic hormone [ACTH]; thyrotropin; growth hormone [GH]; and prolactin [PRL]), when exposed to the appropriate releasing hormones, or in the case of PRL, to dopamine. Moreover the release of ACTH is also stimulated by AVP (see chapter by Chen). Conversely, the secretion of many of these hormones is inhibited by somatostatin. The portal blood vessels containing the hypophysiotropic hormones distributes the releasing hormones slowly to the endocrine cells. In turn, the endocrine cells release their hormones through the fenestrated capillaries, after the hormones have crossed two BL (Fig. 7C). The organization of the endocrine units, termed cords, in the anterior lobe and their visualization by immunocytochemistry using laminin immunoreactivity are illustrated in Fig. 7B,C. It should be noted that a very important network of connective tissue, delineated by BL, exists in both anterior and NL of the pituitary (Fig. 7B). This gives important opportunities for the macrophages/fibroblast network to interact with the vascular and parenchymal cells (see the cytoarchitectonics in Fig. 7C). In addition, the parenchyma of the anterior lobe contains a gap junction-coupled network of folliculostellate cells, which based upon morphology, location in the parenchyma, and staining for vimentin (Kameda, 1996), S100b (unpublished data) and occasionally for GFAP can be classified as glial-like cells. This network forms a functional circuitry carrying Ca2þ signals and small molecules across the entire anterior pituitary (Fauquier et al., 2001).

4.2. Cellular interactions 4.2.1. Interactions between neuroendocrine neurons and glia The SON component is the best example of the hypothalamic magnocellular system. Since neuronal – astrocytic interactions in this system are described in detail in the chapter

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by Salm et al., and have been extensively reviewed (see above), we will only highlight a few, functionally important aspects. To the extent that they have been studied, the other hypothalamic components have yielded similar and equivalent data. When physiological conditions such as dehydration or lactation arouse the mHNS, readily observable changes occur in SON morphology and function. As different from one another as these two stimulus conditions are, the alterations in SON morphology that they produce are remarkably similar. These changes are extensive and appear to be completely reversible upon return to basal conditions, apparently in response to b-adrenergic receptor activation (Lafarga et al., 1992; McNeill and Sladek, 1980). The ventrally located SON astrocytes retract their processes to an extent large enough to allow adjacent somata and adjacent dendrites, whose membranes had previously been in direct apposition to glial membrane, to now be juxtaposed to membrane of neighboring neurons. Continuation of the stimulus produces further glial retraction and results in dramatically increased numbers of multiple synapses (one synaptic terminal contacting two or more postsynaptic elements) in both the somatic and dendritic zones. Astrocytic process retraction from among adjacent dendrites also appears to play a permissive role in formation of dendrodendritic gap junctions, as dye-coupling increases are observed. Several functional consequences of these morphological changes are apparent. Under basal conditions, when the astrocytic processes are interposed between neighboring neuronal membranes, the extracellular Kþ that is released during firing activity is rapidly removed by glial uptake (see chapter by Walz). Removal of transmitters, such as glutamate, that escape or spill over from the synaptic clefts would also be efficient (see chapter by Schousboe and Waagepetersen). Both of these factors limit potentially depolarizing influences and contribute to the relative quiescence of the neurons. During excitatory synaptic drive, glial retraction reduces the effectiveness of the astrocytes to remove either Kþ or escaped transmitters from the extracellular space. In the SON, there is also an astrocyte-based tonic inhibitory influence operative under basal conditions. Taurine, a GABA-like amino acid is prominently expressed in SON astrocytes (Decavel and Hatton, 1995) and released under normo- or hypo-osmotic conditions (Deleuze et al., 1998), providing for tonic inhibition of at least VP neurons via a glycine receptor-mediated chloride conductance increase (Hussy et al., 1997). Dehydration-induced activation of the system results in osmotic inhibition of taurine release. Reduction in taurine’s influence would be abetted by glial process retraction. In concert, inhibition of taurine release and astrocyte retraction summate to insure heightened excitability of SON neurons. Parallel, although distinct, morphological plasticity characterizes the elements of the NL. Under basal conditions, the NL pituicytes wrap around the axons in a manner similar to the way the SON astrocytes wrap the dendrites. Also, they engulf many of the neurosecretory terminals, some of which make synaptoid contacts with the astrocytes, and the glia occupy a relatively large proportion of the BL abutting the perivascular spaces. NL pituicytes are even richer in taurine than are those in the SON, and they, too, selectively release this amino acid in response to low osmolality (Miyata et al., 1997). Thus the terminals are also potential sites of tonic inhibition by glial-released taurine. Arousal of the mHNS results in release of the engulfed axons and a retraction of the NL astrocytes, probably mediated by catecholamine input (Luckman and Bicknell, 1990;

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Smithson et al., 1990), as in the SON. In this case, however, one possible source of these transmitters is the blood, since the NL is outside of the BBB. Glial retraction then allows the neurosecretory terminals to occupy a greater proportion of the BL. Such repositioning reduces the glial influence and expands the extracellular space in the NL. These rearrangements are consistent with facilitation of release and delivery of peptide across the perivascular space and into the fenestrated capillaries. That the signaling between neurons and glia in the NL is bi-directional is indicated by the finding that VP can stimulate intracellular calcium release in the NL astrocytes (Hatton et al., 1992). This has the potential to facilitate peptide release since raising intracellular calcium in astrocytes has been shown to cause the release of glutamate (Parpura et al., 1994—see also chapter by Cornell-Bell). Nonneuronal cells in the hypophysial – parvocellular secretory system carry out functions that are similar or parallel to those seen in the mHNS (see chapter by Melcangi et al., 2002). Thus, in the arcuate nucleus, astrocytes appear to regulate the number of synaptic inputs in contact with LHRH-releasing neurons, and tanycytes and astrocytes encapsulating LHRH terminals in the arcuate nucleus and median eminence modulate LHRH secretion, probably mainly by releasing GF and cytokines (Garcia-Segura et al., 1996; Voigt et al., 1996; Messi et al., 1999), as will be described in Section 4.3

4.3. Effects of cytokines There are numerous papers relating to the effects of cytokines and GF on the hypothalamo – hypophysial systems (see reviews by Jones and Kennedy, 1993; Ray and Melmed, 1997). Cytokines and GF have marked effects on the magnocellular hypothalamo – hypophysial system. Principal cytokines and GF that are known to be involved in the regulation of hormone secretion from the hypothalamo – hypophysial system are presented in Table 1 (see last column). Among these, IL-1b was shown to stimulate the transcription of AVP mRNA (Lee and Rivier, 1994) and the release of AVP from rat pituitary NL (Nakatsuru et al., 1991) and from hypothalamo – hypophysial explants (Christensen et al., 1989) in a dose dependent fashion. IL-1b also excites SON neurons in slices (Li et al., 1993) and stimulates the release of both AVP and oxytocin (OT) from hypothalamic explants (Yasin et al., 1994). Consistent with these findings, IL-1 receptors have been localized in the SON and PVN (Diana et al., 1999). Magnocellular vasopressinergic neurons in explant cultures are rescued from cell death by ciliary neurotrophic factor (CNTF) (Vutskits et al., 1998), and implantation of polymer rods containing IGF-1 in the adult SON induces outgrowth response of OT fibers (Zhou et al., 1999). Astrocytes secrete several GF that are able to stimulate LHRH secretion. This topic is discussed in the chapter by Prevot, so only a few points will be mentioned here. Hypothalamic astrocytes produce TGFa and neuregulins (NRGs), which can elicit LHRH secretion indirectly, via activation of receptor complexes formed by three members of the EGF receptor family, also located on astrocytes. Activation of these receptors results in the production of at least one neuroactive substance, prostaglandin E2 (PGE2), which stimulates LHRH secretion upon binding to specific receptors on LHRH neurons

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(Ojeda et al., 2000). Moreover, TGFa and IGF1, expressed by astrocytes and tanycytes, stimulate the release of LHRH from the median eminence, and they inhibit cell proliferation in neural cell lines producing LHRH, an effect shared by EGF (Ochoa et al., 1997; Voigt et al., 1996). The effect of TGFa appears to be mediated via astrocytes, since it can be evoked by conditioned medium from astrocytes treated with TGFa but not by direct exposure of the neuronal cell line to the GF (Voigt et al., 1996). A temporal correlation between variations of astroglial release of TGF-a and IGF-1 and of plasma steroid levels during the estrous cycle also suggests that these factors may be involved as mediators in the regulation of LHRH release. Other authors have found that TGFb2 is able to modify mRNA levels and release of LHRH in a LHRH-producing cell line (GT1-1) (Messi et al., 1999). Moreover, bFGF increases the rate of cell proliferation and neurite outgrowth, and chronic administration of IL-1b disrupts the estrous cycle by inhibiting the synthesis and release of LHRH (Garcia-Segura et al., 1996). The release of somatostatin and GHRH from hypothalamic explants is stimulated by IL-1b (Honegger et al., 1991). At low doses of IL-1b, the release of GH is also increased, but at higher doses GH release is suppressed (Payne et al., 1992), perhaps suggesting an overriding effect on somatostatin. IL-2 potently stimulates somatostatin release (Karanth et al., 1993). TGF-b1 and IL-6 (Jin et al., 2001). Expression of TRH is suppressed by IL1b, and interactions with astrocytes appear to enhance TRH expression, which is increased by conditioned medium from cultures enriched in hypothalamic glia (Charli et al., 1995). Regulation of the hypothalamic – pituitary – adrenal axis by cytokines has been reviewed by Turnbull and Rivier (1999). A multitude of different studies have shown that several different cytokines stimulate CRF secretion from hypothalamus in vitro. It may be of special interest that IL-1b, IL-6 and TNFa, the three major proinflammatory cytokines, all have potent stimulatory effect, and that available data suggest that this effect is mainly exerted at the hypothalamic level. The effects of cytokines on the different hypophysiotrophic hormones (e.g., an increase in CRH but a decrease in TRH) are likely to play a major role in the response of the organism to infection or stress, e.g., an increase in secretion of ACTH and thus of glucocorticoid and a decrease in secretion of thyrotropin and thus of thyroid hormones. These responses occur in conjunction with cytokine-induced effects on other centrally regulated parameters, e.g., development of fever. Effects of GF and cytokines have also been extensively studied in the pituitary anterior lobe. Briefly, it has been shown that the cytokines IL-1a, IL-1b, IL-2, IL-6, interferon g, and the GF bFGF, IGF1, IGF2, TGFa and TGFb can all affect hormone secretion. Some of these factors specifically modulate one or the other secretory cell types. But IL-6, for example, broadly affects endocrine cell types of the anterior pituitary (Ray and Melmed, 1997), perhaps reflecting that it is a cytokine released from the network of folliculostellate cells, which express both TGF-b1 and IL-6 (Jin et al., 2001). These cells also release VEGF (which can increase vascular permeability) and bFGF, when challenged with TGFb (Hentges et al., 2000; Renner et al., 2002). The folliculostellate cells are responsive to additional cytokines, e.g., TNFa and IL-6, which stimulates release of VEGF (Gloddek et al., 1999). They might accordingly play a major role in modulation of hormone release from anterior pituitary cells and across endothelial cells.

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5. The meningeal hypothesis We have presented evidence that meninges and their projections within the brain, including perivascular sheaths, stroma of the choroid plexus, and large meningeal projections located between the major substructures, are ideally located and have a great potential (producers of potent signaling molecules) to be actively involved in brain functions throughout adulthood. It is likely that due to their privileged (but not exclusive) anatomical relationship with neurogenic zones (ventricle walls), hypothalamic neuroendocrine zones and pituitary/circumventricular organs, their functions may at least pertain to cell proliferation, differentiation, migration, phenomena of morphological/functional plasticity in general, and regulation of neurosecretion (including the endocrine pituitary system at large). Extraparenchymal cells, namely macrophages, fibroblasts, pericytes, SMCs and endothelial cells are good candidates to play important, if not a leading role, in brain functions, particularly for all aspects which concern neural tissue maintenance, cell renewal (including neurons), response after injury, and all functional aspects which necessitate a structural reorganization (such as neuroendocrine regulation) or more discrete microreorganization (perhaps including learning and memory at the level of the hippocampus). All the extraparenchymal cells may be active players in virtually all brain functions because they release numerous signaling molecules that can reach the entire neural tissue. The goal of this review was to highlight the possibility that extraparenchymal cells and neural cells may form a functional syncytium, and that all cells without exclusion may participate in brain functions. There are several reasons to think that this may be the case. First, the BBB is located at the level of the endothelium. Therefore, all brain cells types, including vascular and meningeal cells, are located beyond the BBB. Extraparenchymal and neural cells may interact and each of them can influence other’s fate and function via large signaling molecules such as GF and cytokines. Second, the meningeal – vascular tissue system, is a large provider of GF and cytokines, which once released from the cells can bind within the BL, interact with extracellular matrix proteins, and become activated to ultimately influence the morphology, cell proliferation, cell differentiation, and function of the neural tissue that is in contact with or close to the BL. These mechanisms would be analogous to well-known mesodermoectodermal inductions during development. Third, the anatomy of the brain connective tissue is particularly well suited to participate in functional interactions. The meninges cover and penetrate the brain deeply at every level of its organization as large projections between major brain structures, as sheaths of blood vessels, and as stroma of the choroid plexus. The BL covering the brain connective tissue may represent not the border of the functional brain, but an enormous interface of communication between connective and neural tissues. This may be particularly important in the neurogenic and gliogenic zones of the adult brain, the subependymal layer of the ventricles, where the BL network (fractones) is maximally expanded, interfacing a large number of neural progenitor cells (neuroblasts and glioblasts) and potential neural stem cells (Mercier et al., 2002). The hypothalamo – pituitary system, and circumventricular organs, also represent areas that are especially intimately connected with extraparenchymal tissue. Although plasticity is not restricted to these regions, the extent of cytoarchitectural

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changes during functional activity and cell renewal (for the endocrine cells of pituitary anterior and intermediate lobes) may be greater than in most other regions. Fourth, gap-junction-connected fibroblasts and macrophages in brain connective tissue form a potential long distance communication network throughout the meninges and their projections in brain. This may be important for transferring information through a meningeal network, and ultimately influence the production and release of GF and cytokines at every level of the parenchymal/extraparenchymal interface. Gap junction expression in brain is highest, wherever meninges and astrocytes interface, i.e., at the pia/glia limitans border, at the surface of blood vessels, and in the subependymal layer of the ventricles, which all are potential zones of plasticity. Fifth, brain connective tissue cells are ideally located to pick up information originating from the blood or CSF. The perivascular fibroblasts and macrophages bathe in the Virchow – Robin space (extension of the subarachnoid space), and contact the vascular cells. The meningeal fibroblasts and macrophages directly bathe in the subarachnoid space. The meninges may also receive information from the blood originating in the arteries of the Circle of Willis and associated veins. Choroid plexus macrophages and fibroblasts bathe in CSF, in close contact with thin blood vessels. The brain connective tissue cells are thus ideally located to receive and transfer information originating from both the brain and peripheral organs.

6. Concluding remarks We have suggested that the meninges and their multiple projections within the brain serve to drive and promote neural plasticity. Neural plasticity encompasses events as different as neural stem cell proliferation, differentiation and migration, morphological retraction/elongation of astrocytes, synapse formation, growth cone guidance, and neuroendocrine regulations. The brain, like every organ, is a plastic structure that regenerates its cells (although normally to a modest extent in the case of neurons), evolves and creates new neuronal connections, responds to homeostatic challenges, and has the potential (although to a limited extent) to repair itself after injury, throughout adulthood. We have suggested that the fibroblasts and resident macrophages of the meninges and their projections within the brain are crucial actors initiating and regulating plasticity. This is not to neglect the role of other extraparenchymal cells (vascular cells) and neural cells as regulators of plasticity, but to highlight a potential function for a network of cells, highly interconnected by gap junctions, producing GF, cytokines and extracellular matrix proteins that are activators for these signaling molecules. Moreover, this network has an extensive surface of contact with the neural tissue via the meninges and their diverse projections within the brain. The signaling mechanisms initiated from the network of fibroblasts and macrophages may primarily occur in the BL interfacing parenchyma and extraparenchyma, but may also occur at every level of the neural tissue, because GF and cytokines can cross the BL and travel in the extracellular space. It is reasonable to speculate that cytokines bound and activated in the BL induce the production and release of other cytokines by the cells overlying the BL, i.e., astrocytes, epithelial cells of the choroid plexus, ependymal cells,

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microglial cells, oligodendrocytes, and glial-like cells of the pituitary, which influence both fate and function of neurons (and/or endocrine cells). In view of this possibility, the glial network (including all sorts of glia) may be considered as a gigantic interface of communication between connective tissue network and neurons. Thus, neuronal – glial interactions may represent one facet of general intercellular communication among all brain cell types. Because neurons also produce signaling molecules, which potentially influence the fate and function of glial, vascular and connective tissues, communication among all brain cell types may be infinitely complex.

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Mechanisms of infiltration of immune cells, bacteria and viruses through brain endothelium P.O. Couraud,a,* X. Nassif b and S. Bourdoulousa a

De´partement de Biologie cellulaire, Institut Cochin, CNRS UMR8104, INSERM U567, Universite Paris V, 22, rue Me´chain, 75014 Paris, France p Correspondence address: E-mail: [email protected] b INSERM U570, Faculte´ de Me´decine Necker-Enfants Malades, 75015 Paris, France

Contents 1. 2.

3.

4.

5.

6.

Introduction The blood– brain barrier 2.1. Biology of the Blood – Brain Barrier 2.2. In vitro models for blood – brain barrier studies Mechanisms of infiltration of immune cells through brain endothelium 3.1. Pathophysiology of leukocyte infiltration through brain endothelium 3.2. Signaling coupled to adhesion molecules in brain endothelial cells Viral entry into the CNS 4.1. HIV-1 entry into the CNS 4.2. HTLV-1 infection of the CNS Mechanisms of infiltration of bacteria through brain endothelium 5.1. Common mechanisms 5.2. Infiltration of S. pneumoniae into the CNS 5.3. Mechanisms of transendothelial migration of N. meningitidis Concluding remarks

Brain endothelium exhibits a unique phenotype, based on the expression of highly impermeable intercellular tight junctions, which physiologically control the infiltration of blood-borne nutrients or cells into the central nervous system. However, several diseases are characterized by the cerebral infiltration of activated leukocytes, bacteria or viruses, respectively, multiple sclerosis, meningitis and HIV infection. This chapter will present a brief up-date of the most recent in vitro and in vivo studies, which contributed to our present understanding of the molecular mechanisms (adhesion molecules, signaling pathways) involved in the infiltration of immune cells or microbial pathogens through brain endothelium. Advances in Molecular and Cell Biology, Vol. 31, pages 255–267 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction Homeostasis of brain interstitial fluid is maintained by two physiological barriers between blood and the central nervous system (CNS): (i) the blood – brain barrier (BBB), constituted by brain parenchyma microvascular endothelial cells; and (ii) the blood – cerebrospinal fluid barrier (BCSFB), found only at the level of the choroid plexus epithelium. Under physiological circumstances, these CNS barriers strictly limit the passage of nutrients or immune cells from the blood to the brain and protect the CNS against circulating bacteria or toxins. However, under pathological conditions such as multiple sclerosis (MS) or virus (HIV, HTLV-1) infection, activated leukocytes infiltrate into brain parenchyma, whereas, encephalitis or meningitis is caused by bacteria that cross the CNS barriers. The subject of this chapter is to provide a short overview of the recent progress in our understanding of the mechanisms of infiltration of leukocytes, bacteria or viruses through brain endothelium. 2. The blood – brain barrier 2.1. Biology of the blood – brain barrier 2.1.1. Cellular components of the blood – brain barrier The structural basis of the BBB is the endothelium of the brain capillaries, which differs from that of other capillaries in the organism by the expression of intercellular tight junctions, the presence of pericytes within the capillary basement membrane, and the proximity of astrocyte processes that ensheath most of the abluminal surface of brain capillaries (Goldstein and Betz, 1986; Wolburg and Lippoldt, 2002). This structure provides the dual feature of low-rate fluid phase endocytosis and low-paracellular permeability due to highly impermeable intercellular tight junctions. Under physiological conditions, BBB permeability is regulated by its microenvironment, especially by astrocytes, although the astrocyte-derived factors involved in that process still remain largely unknown. Choroid plexuses are highly differentiated structures lining the ventricles that secrete the cerebrospinal fluid (CSF) (see chapter by Mercier and Hatton). In contrast to endothelium within brain parenchyma, the microvascular endothelium of choroid plexuses is permeable (so-called ‘fenestrated’), quite similar to non-brain microvascular beds: indeed, the BCSFB is localized at the level of choroid plexus epithelial cells, which express intercellular tight junctions (Strazielle and Ghersi-Egea, 2000). 2.1.2. Molecular structure of tight junctions The molecular composition of tight junctions at the CNS barriers has been the subject of intensive investigations over the past few years (Tsukita and Furuse, 1999; Wolburg and Lippoldt, 2002). Briefly, multiple integral membrane proteins localized in brain endothelial tight junctions have been identified: occludin, claudin-5, -3 and -12, expressed by adjacent endothelial cells are engaged in tight homophilic interactions and are responsible for the formation of the tight junctions, whereas JAM-1, the prototypical

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member of a small group of proteins of the immunoglobulin superfamily, rather appears localized at the periphery of the tight junctions (Bazzoni et al., 2000). Several membrane-associated cytosolic proteins, including the three proteins, ZO-1, ZO-2 and ZO-3, have been localized to the tight junctions, where they interact with the cytoplasmic domains of occludin, claudins and JAMs through several interaction domains (three PDZ domains, one SH3 domain, and one guanylate kinase-like (GUK) domain) (Tsukita and Furuse, 1999); ZO-1 and ZO-2 associate with actin filaments, thus forming a molecular link between the plasma membrane and the actin cytoskeleton. Moreover, ZO-1 is able to shuttle, at least in epithelial cells, from the membrane to the nucleus where it behaves as a transcription factor and may participate in the control of cell proliferation and barrier permeability (Balda and Matter, 2000). In addition, recent reports have identified other scaffolding proteins that associate with the cytoplasmic domains of integral membrane proteins of the tight junctions (Itoh et al., 2001; Hamazaki et al., 2002). 2.2. In vitro models for blood –brain barrier studies 2.2.1. Primary cultures of brain endothelial cells Several groups have reported the isolation and in vitro pharmacological characterization of primary cultures of brain microvascular endothelial cells from various species (Bowman et al., 1983; Dorovini-Zis et al., 1991; Abbott et al., 1992). These cells retain an endothelial phenotype reminiscent of that expressed by brain endothelium, although they usually fail to develop functional tight junctions and rapidly undergo senescence and dedifferentiation, even upon limited passaging (Joo, 1993). In order to mimic the anatomical proximity and functional relationship between brain endothelial cells and astrocyte processes, a coculture system was developed, where brain endothelial cells are grown on microporous filter membrane inserts, most often in the presence of primary cultures Sˇ of astrocytes or astrocyte-conditioned medium (Rubin et al., 1991; Cecchelli et al., 1999; Engelbertz et al., 2000). Measures of in vitro permeability of a large number of standard molecules, over a wide range of hydrophilicity, correlate with the in vivo cerebral bioavailability of the same molecules. Such validated models are of great value for both drug screening and basic studies on BBB biology. However, with the availability of these models so far being limited to bovine or porcine brain endothelial cells, their use for immunological studies remains difficult because of lack of appropriate molecular tools. Moreover, the absence of a human model hampers any investigations on the mechanisms of infiltration of human immune cells or human-specific viral or bacterial pathogens. 2.2.2. Immortalized brain endothelial cell lines Numerous efforts have been made during the last decade to establish continuous, immortalized endothelial cell lines, which would retain a stable phenotype in culture and would allow the production of standardized models. The rat brain endothelial RBE4 and GP8 cell lines have been extensively characterized and shown to maintain most of the differentiated phenotype of brain endothelium (Durieu-Trautmann et al., 1993; Roux et al., 1994; Greenwood et al., 1996). These cells, together with the more recent GP8-derived GPNT cell line (Regina et al., 1999) are now widely used as validated models of brain

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endothelium for biochemical, pharmacological, toxicological or immunological purposes. However, these cells appear to display a reduced complexity of tight junctions associated with a moderate permeability barrier capacity, which limits their use in permeability regulation studies or drug screening. Immortalization of human brain endothelial cells might in the future constitute a valuable approach towards the production of an in vitro model of human BBB. 3. Mechanisms of infiltration of immune cells through brain endothelium 3.1. Pathophysiology of leukocyte infiltration through brain endothelium 3.1.1. Leukocyte infiltration in multiple sclerosis Although the CNS has often been regarded as an immunologically privileged site, mostly because of the existence of the BBB and the absence of lymphatic vessels, it is now well established that immunosurveillance does take place in the CNS under physiological conditions (Hickey et al., 1991). Moreover, during CNS inflammation, large numbers of immune cells are recruited into the CNS through brain endothelium and possibly also through choroid plexus epithelium (Couraud, 1998; Engelhardt et al., 2001). Multiple sclerosis is associated with perivascular and parenchymal infiltration of the brain and spinal cord by mononuclear cells, predominantly activated T-cells and monocyte/macrophages (Raine, 1994). The MS animal model, experimental allergic encephalomyelitis (EAE), can be induced in some susceptible rodent strains by injection of various myelin antigens, such as myelin basic protein, or by adoptive transfer of activated-T-lymphocytes directed against these myelin antigens. Altogether, these observations strongly suggest that lymphocyte and monocyte recruitment to the CNS is a critical step in the pathogenesis of MS and EAE; however, the precise role of brain microvascular endothelial cells, or possibly choroid plexus epithelial cells, in the onset of the pathology has not yet been clarified. 3.1.2. Adhesion molecules expressed by brain endothelium Activated T-lymphocytes can enter the CNS irrespective of their antigen specificity, following interaction with brain endothelium: the adhesion molecules of the immunoglobulin superfamily expressed by brain endothelial cells, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1), interact with the leucocyte-expressed integrins LFA-1 (aLb2), Mac-1 (aMb2) and PECAM-1, respectively. These adhesion molecules are differentially involved in the firm adhesion and migration of distinct subsets of leukocytes: whereas ICAM-1 and PECAM-1 are intimately involved in lymphocyte recruitment to the CNS (Reiss et al., 1998; Wong et al., 1999), the recruitment of monocytes is principally mediated by VCAM-1, PECAM-1 and possibly other adhesion molecules, such as CD47 (de Vries et al., 2002). ICAM-1 or VCAM-1 expression by brain endothelium is dramatically up-regulated during inflammatory conditions like MS and EAE or following various cerebral insults such as stroke, traumatic brain injury, or Alzheimer’s disease (Sobel et al., 1990). Although migrating leukocytes are generally considered to traverse

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the vascular endothelium through the intercellular endothelial junctions, one cannot exclude a transendothelial cell route, as was recently reported for neutrophils migrating through dermal venules (Feng et al., 1998). In addition, enhanced expression of ICAM-1 and VCAM-1 was observed during EAE in choroid plexus epithelial cells, suggesting a previously unappreciated function of the choroid plexus in the immunosurveillance of the CNS (Engelhardt et al., 2001). 3.1.3. Cytokines and chemokines at the BBB Local release of multiple cytokines and chemokines at the BBB level and within cerebral parenchyma has been observed during CNS inflammation, and it likely contributes to leukocyte recruitment to the brain parenchyma or the CSF. Astrocytes and microglia are generally considered as the major sources of cytokines in the CNS (see chapters by Nakagawa and Schwartz and by Benveniste), for instance IL-1b and TNF-a, which appear to be directly associated with the development of EAE. Also, chemokines, such as CSF-1, GM-CSF or IP-10 have been detected in astrocytes during EAE (Ransohoff, 2002). Brain microvascular endothelial cells, and more recently choroid plexus epithelial cells, now appear as alternative sources of cytokines and chemokines, including IL-1, IL6, GM-CSF, IL-8 or MIG (Ghersa et al., 2002). In addition, these cells express a number of chemokine receptors, including CCR2b, CCR5, CXCR3 (Andjelkovic and Pachter, 2000). ICAM-1 or VCAM-1 expression on cultured brain endothelial cells can be enhanced in response to pro-inflammatory cytokines such as TNF-a, IL-1b and IFN-g in parallel to an increase in transendothelial leukocyte migration and down-regulated by IFNb, an antiinflammatory agent clinically used in MS, which reduces monocyte infiltration into the CNS (Floris et al., 2002); in vivo, extensive breakdown of the BBB is observed in transgenic mice expressing IL-6 in astrocytes (Brett et al., 1995). Taken together, these observations indicate that, during CNS inflammation, perivascular astrocytes and microglial cells, as well as brain endothelial cells, may secrete locally, at the level of the BBB, pro-inflammatory cytokines and chemokines, which will act in concert to recruit activated leukocytes into the CNS. Again, although the capacity of choroid plexus epithelial cells to secrete cytokines or chemokines has been less investigated, they appear to release matrix metalloproteases and their inhibitors, which might contribute to leukocyte infiltration (Pagenstecher et al., 1998). 3.2. Signaling coupled to adhesion molecules in brain endothelial cells 3.2.1. ICAM-1-coupled signaling An important concept for understanding [of] the molecular mechanisms involved in the infiltration of immune cells through vascular endothelium is that beyond their ability to provide docking sites for circulating leukocytes, adhesion molecules expressed by endothelial cells behave as signal transducers (Hubbard and Rothlein, 2000; Greenwood et al., 2002). Indeed, in response to leukocyte adhesion, a number of intracellular transduction pathways have been shown to be activated in brain endothelial cells and to participate in the regulation of leukocyte migration into the CNS.

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Coculture of brain endothelial cells with activated T-lymphocytes was shown to increase the activity of the nonreceptor tyrosine kinase Src in the endothelial cells, as well as the phosphorylation of the Src substrate, the actin-binding protein cortactin (Durieu-Trautmann et al., 1994). This process of T-lymphocyte attachment could be mimicked by cross-linking of endothelial ICAM-1 with specific antibodies, pointing to ICAM-1 as a key signal transducer in brain endothelial cells during leukocyte infiltration. Subsequent studies further demonstrated the capacity of ICAM-1 (i) to evoke a rapid increase of intracellular calcium concentration (Etienne-Manneville et al., 2000); (ii) to induce tyrosine phosphorylation of several cytoskeleton-associated proteins (focal adhesion kinase, paxillin and p130Cas) (Adamson et al., 1999); (iii) to stimulate the MAP kinases JNK and p38/MAPK (Etienne et al., 1998); (iv) to initiate cytoskeletal rearrangements in brain endothelial cells through activation of Rho GTPases (Etienne et al., 1998). Indeed, inhibition of Rho GTPases in brain endothelial cells, by treatment with posttranslational prenylation inhibitors, resulted in a significant reduction in transendothelial T-lymphocyte migration (Walters et al., 2002). The potential clinical importance of this finding is obvious. Interestingly, ICAM-1 engagement in astrocytes stimulates distinct signaling pathways, both cAMP and ERK pathways, which are responsible for the activation of the transcription factor CREB and for the secretion of the inflammatory cytokines TNFa, IL1b and IL-6 (Etienne-Manneville et al., 1999; Lee et al., 2000). Since these cytokines are implicated in BBB disruption in various pathophysiological situations, the data reported here suggest that ICAM-1 engagement on astrocytes by infiltrated leukocytes may play a significant role in the development and exacerbation of inflammatory processes within the CNS by facilitating further waves of leukocyte infiltration. 3.2.2. A complex network of signaling pathways Together with ICAM-1, other adhesion molecules including VCAM-1 (Lorenzon et al., 1998) and PECAM-1 (Newton-Nash and Newman, 1999) have been demonstrated to activate intracellular signaling in endothelial cells. The cytoplasmic domain of PECAM-1 contains several tyrosine residues within an ‘immunoreceptor tyrosine-based inhibitory motif’ (ITIM), which can be phosphorylated by cytosolic tyrosine kinases belonging to the Src and Csk families. Tyrosine phosphatases, such as SHP-1, SHP-2 and SHIP, that contain a phosphotyrosine binding SH2 domain, can thus be recruited to the phosphorylated ITIM motif of PECAM-1 and activated, leading to a de-phosphorylation of downstream effector substrates (Newton-Nash and Newman, 1999). These observations suggest that PECAM-1 may function as an inhibitory receptor, limiting activating signals triggered by other membrane receptors or adhesion molecules (Newman et al., 2001). Since leukocyte migration through the BBB is mediated by multiple interactions between pairs of adhesion molecules, we can hypothesize that the signaling pathways activated by the engagement of each of these molecules in brain endothelial cells, especially ICAM-1, PECAM-1 and VCAM-1, contribute to this process. Future research will aim at understanding of the functional integration of these signals within brain endothelial cells.

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4. Viral entry into the CNS 4.1. HIV-1 entry into the CNS Although it is well established that HIV-1 can enter the CNS very early post-infection, the involved mechanism still remains uncertain. It has been proposed that HIV-1 enters the brain covertly, through infected monocytes that migrate through brain endothelium in response to inflammatory signals (Bussolino et al., 2001). This ‘Trojan horse’ hypothesis is supported by observations that several chemokines are significantly elevated in brains of demented AIDS patients and that, in vitro, interactions between HIV-1-infected macrophages and brain endothelial cells result in increased expression of chemokines, such as MCP-1 or RANTES, by both cell types, that may contribute to the recruitment of infected monocytes into the brain (Boven et al., 2000). Along the same lines, other investigators, using coculture of primary human brain endothelial cells and astrocytes, pointed out the role of inflammatory cytokines in the activation of BBB endothelium and the opening of intercellular junctions, leading to enhanced migration of HIV-infected monocytes (Persidsky et al., 1999). Whether brain endothelial cells can be productively infected by HIV-1 constitutes a highly controversial issue (Bussolino et al., 2001). Whereas only sporadic evidence of brain endothelium infection in situ was reported, cultured brain endothelial cells could be selectively infected by HIV strains with T-cell tropism, but not macrophage tropism, suggesting that T-cell tropism is important for HIV entry through the BBB. The productive infection of brain endothelial cells may alter their expression of adhesion molecules or BBB permeability or integrity and thus favor the infiltration of infected cells or of HIV-1 particles (Bussolino et al., 2001).

4.2. HTLV-1 infection of the CNS Human T-cell leukemia virus type I (HTLV-I) is the causative agent of a chronic neurological syndrome called tropical spastic paraparesis or HTLV-I-associated myelopathy (TSP/HAM) (Gessain et al., 1985). Although pathogenesis of HTLV-I associated neurological disease is still poorly understood, it is well established that the major target cells of HTLV-1 in vivo are the CD4 þ lymphocytes (Richardson et al., 1990), and chronic CNS inflammation with perivascular cuffing and parenchymal lymphocytic infiltrates have been reported. Astrocytes are a major source of inflammatory cytokines following HTLV-I infection (Szymocha et al., 2000). A recent study suggested that HTLV-I might cross the brain endothelial barrier by several mechanisms: adhesion of HTLV-I-infected lymphocytes onto brain endothelial cells in vitro not only enhanced the paracellular permeability of the endothelial monolayer through a TNFa -mediated mechanism, but it also resulted in the internalization of virions by brain endothelial cells and their apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway (Romero et al., 2000). Moreover, transient HTLV-I infection of brain endothelial cells was observed in the same study. Other reports indicated that matrix metalloproteinases (MMP-1, -9) could be detected in

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the CSF of HTLV-I-infected patients (Lezin et al., 2000), suggesting the choroid plexus as an alternative route for the infiltration of infected T-lymphocytes. From these sets of data, we can conclude that CNS infection by retroviral vectors may involve several nonexclusive mechanisms including (i) infiltration of infected lymphocytes through brain endothelium and perhaps through choroid plexus epithelial cells; (ii) transcytosis of viral particles through brain endothelial cells; and (iii) infection of brain endothelial cells and alteration of their functions. 5. Mechanisms of infiltration of bacteria through brain endothelium 5.1. Common mechanisms Among the invasive bacterial pathogens, a few are capable of invading the brain to cause meningitis, which suggests that they have developed special attributes capable of circumventing the BBB. The majority of bacterial meningitis cases are caused by Streptococcus pneumoniae, Neisseria meningitidis(or meningococcus), Haemophilus influenzae and, in the newborn, K1 Escherichia coli and Group B Streptococcus (Zhang and Tuomanen, 1999; Kim, 2000). All these pathogens multiply in the extracellular compartment and can possibly interact directly with the components of the BBB to invade the meninges. Alternatively, some intracellular pathogens, like Mycobacterium tuberculosis and Listeria monocytogenes, may enter the CSF within infected leukocytes (Drevets and Leenen, 2000) and can cause meningitis, often associated with an inflammatory process of the parenchyma. Extracellular pathogens that have invaded the bloodstream from their port-of-entry, usually the throat, multiply to high densities in the blood, the level of bacteremia correlating with the risk of meningeal invasion. Although the precise entry site into the CNS remains unknown for most bacteria, H. influenzae is suggested to enter the CSF at the choroid plexus, whereas N. meningitidis appears to interact with endothelial cells of the choroid plexuses as well as the meninges (Pron et al., 1997). In physiological conditions, the BBB and BCSFB barriers protect the brain efficiently against any pathogen infections. Pathogenic bacteria may by-pass these barriers by using different mechanisms: (i) bacterial toxins like lipopolysaccharide (LPS) or the release of host inflammatory cytokines induced by bacterial components can drastically alter the paracellular permeability of brain barriers, presumably by disturbing the structural organization of tight junction proteins and the associated actin cytoskeleton (Perry et al., 1997); (ii) a direct interaction between bacteria and components of the BBB can trigger a cascade of intracellular events by subverting host cell processes, leading to disruption of intercellular junctions and/or transcytosis of the bacteria (Zhang and Tuomanen, 1999). 5.2. Infiltration of S. pneumoniae into the CNS In the case of S. pneumoniae, all bacteria are capable of adhering to the brain endothelial cells in vitro, although only those expressing a specific adhesin, the choline-binding protein, can be efficiently internalized and trancystosed through cell monolayers (Ring and

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Tuomanen, 2000). Furthermore, efficient internalization requires the expression of the platelet activating factor receptor by endothelial cells (Cundell et al., 1995), and more recent observations indicate that Toll-like receptor-2 and CD14, two essential components of the innate immune response to Gram-negative bacteria, are also involved in the adhesion and internalization of S. pneumoniae (Yoshimura et al., 1999). Following internalization, bacteria are either killed within the cell, recycled in vacuoles back to the luminal surface, or traffic through the cells to the basolateral surface.

5.3. Mechanisms of transendothelial migration of N. meningitidis Two virulence factors are essential for meningeal invasion by N. meningitidis: (i) the capsular polysaccharide, which is required for bacterial survival in extracellular fluids; and (ii) the multimeric structures type IV pili, which are required for bacterial adhesion to host cells (Rudel et al., 1995; Pron et al., 1997; Nassif et al., 1999). However, following the initial interaction, pili retract, suggesting the involvement of unidentified bacterial components in the interaction with host cells. The recent availability of meningococcal genome sequence should allow the precise identification of additional factors required for bacterial survival and invasion. A series of evidence suggests that N. meningitidis crosses human physiological barriers using the transcellular route, without altering the organization of intercellular tight junctions (Nassif et al., 2002). In the case of human endothelial cells, recent in vitro observations contribute to a better understanding of the molecular mechanisms involved in the adhesion and internalization of these bacteria (Hoffmann et al., 2001; Eugene et al., 2002). Adhesion of capsulated N. meningitidis onto human vascular endothelial cells is associated with the formation of microvilli-like cell membrane protrusions underneath bacterial colonies (Eugene et al., 2002), resulting from the localized polymerization of cortical actin associated with the clustering of integral membrane proteins, such as ICAM1, CD44, as well as ezrin and moesin, two related actin-binding proteins that link these transmembrane proteins to the actin cytoskeleton. Following adhesion of N. meningitidis, actin cytoskeleton rearrangements require the activation of the GTPase Rho and its downstream effector Rho-kinase, as well as Cdc42, likely involved in the formation of microvilli-like structures. The mechanism of activation of these GTPases remains unknown: indeed, whereas other bacterial pathogens responsible for dramatic cytoskeletal modifications inject bacterial proteins with GTPase stimulating activity into the cytosol via a type III secretion system (Knodler et al., 2001), analysis of the genome content of N. meningitidis did not reveal any type III secretion system (Eugene et al., 2002). Recently, an additional signaling event associated with the internalization of N. meningitidis into human endothelial cells has been identified. ErbB2, a receptortyrosine kinase of the EGF receptor family, is specifically recruited underneath bacterial colonies following pili-mediated adhesion (Hoffmann et al., 2001). ErbB2, which seems to be activated by homo-dimerization, is responsible for the downstream activation of Src tyrosine kinase activity and for the tyrosine phosphorylation of the actin binding protein cortactin. Activation of this signaling pathway is required for the efficient internalization of N. meningitidis.

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6. Concluding remarks In summary, brain endothelium and choroid plexus epithelium are the two faces (the BBB and BCSFB, respectively) of the physiological CNS barriers, which control the infiltration of immune cells and protect the brain against viral or bacterial pathogens. The understanding of the molecular organization of the cell – cell junction complexes expressed in these barriers has dramatically increased over the last few years, while in vitro cultures of brain endothelial cells and more recently choroı¨d plexus epithelial cells allowed recent progress in our understanding of the molecular mechanisms of infiltration of immune cells, viruses and bacteria through brain endothelium and choroid plexus. It is anticipated that these in vitro experimental approaches will pave the way for the identification of new therapeutic targets.

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Hydrocephalus disorders: their biophysical and neuroendocrine impact on the choroid plexus epithelium Charles E. Weaver, Paul N. McMillan, John A. Duncan, Edward G. Stopa and Conrad E. Johansonp Department of Clinical Neuroscience, Brown Medical School, Providence, RI 02903 USA p Correspondence address: E-mail: [email protected]

Contents 1. 2.

3.

4.

Introduction: choroid epithelium as the generator of brain extracellular fluid The CP– CSF – brain nexus in health and hydrocephalus 2.1 Normal CP– ventricular relationships 2.2 Hydrocephalus effects on the CP– CSF system Working model: there is a neuroendocrine system within the brain that helps to regulate CSF formation and intraventricular pressure 3.1 Is hydrocephalus-damaged tissue capable of responding to neuropeptide regulation? 3.2 Plasticity of neuropeptide receptors in CP 3.3 Central source of neuropeptides that target the CP 3.4 Relationship between intracranial pressure and CSF neuropeptide levels 3.5 Do peptides downregulate CSF formation in hydrocephalus? 3.6 AVP-induced changes in choroid epithelial ultrastructure and function 3.7 Putative role of nNOS in CP reabsorption Recapitulation and looking forward

Choroid plexus (CP) epithelial cells carry out a wide variety of secretory and reabsorptive functions that, by way of cerebrospinal fluid (CSF) volume transmission, contribute to the stability of the neuronal extracellular environment. During fetal development, the brain critically depends upon the CP – CSF nexus for a steady supply of micronutrients and trophic factors to support normal growth. In adulthood, the CP reacts to biochemical and physical perturbations, associated with disease and trauma, by secreting into CSF a wide array of growth factors, transport (carriage) proteins and neuropeptides. Arginine vasopressin (AVP) and basic fibroblast growth factor (FGF –2) colocalize in the choroid epithelium. Release of these peptides at the blood – CSF barrier, in response to ischemia and augmented intracranial pressure, help to repair injured tissue and adjust CSF Advances in Molecular and Cell Biology, Vol. 31, pages 269–293 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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formation and pressure. Although high-pressure hydrocephalus, or dehydration, physically distort the choroid epithelium, nevertheless these cells remain capable of upregulating AVP and growth factors for the apparent purpose of controlling fluid transfer in and out of the CNS. CSF levels of AVP and atrial natriuretic peptide (ANP) increase in some forms of hydrocephalus; consequently, these peptides regulate CP blood flow, ion transport and CSF formation. As part of homeostatic mechanisms to modulate extracellular fluid parameters, in the face of CSF distortions such as ventriculomegaly, there is plasticity in regard to CP receptor ‘targets’ for AVP and ANP. Functional and ultrastructural evidence is offered to support the model of a CSF neuroendocrine system, the histological substrate of which is the so-called ‘dark’ epithelium in CP. The response of the AVP, ANP and nitrergic (nNOS) systems to hydrocephalus will likely provide insight on the fluid dynamics in the basolateral (interstitial) space between choroid epithelial cells. 1. Introduction: choroid epithelium as the generator of brain extracellular fluid Serving the brain with numerous secretory and reabsorptive capacities, the epithelial cells of the kidney-like choroid plexus (CP) sustain a wide spectrum of metabolic, transport and secretory activities (Spector and Johanson, 1989). Choroidal functions are markedly altered by the augmented ventricular volume and pressure that attend fluid overloading in hydrocephalus. In health, the cerebrospinal fluid (CSF) continually streams from the plexuses, supplying proteins and micronutrients as well as ions and water to the delicate neuronal microenvironment (Johanson, 1993, 2003; Chodobski and Szmydynger-Chodobska, 2001). Simultaneously, in the opposite direction, many catabolites and protein breakdown products in brain extracellular fluid diffuse into CSF from which clearance is needed to assure optimal neuronal status. Such active removal of metabolites from CSF occurs by organic ion reabsorptive mechanisms in the choroid cells. Severe hydrocephalus disrupts the extensive, balanced bidirectional transport across the epithelium of the plexus, thereby imparting untoward effects, e.g., edema, downstream on the brain interstitium (Milhorat, 1987). Given the dynamic functions of the CSF, originating mainly from the plexuses, it is clear that functional breaches in the CP – CSF system place the brain at risk in both early development (congenital hydrocephalus) and in later life when neurodegenerative processes set in (normal pressure hydrocephalus). The interior and exterior surfaces of the brain are both bathed by CSF (Johanson, 1988, 1998). Therefore, alterations in the volume, pressure and composition of the ventricular as well as the subarachnoid fluids can impact the brain detrimentally. More attention needs to be focused on how the choroid epithelial cell secretions, biochemically and hydrodynamically, ultimately modulate neuropil targets throughout the CNS. 2. The CP – CSF – brain nexus in health and hydrocephalus This review examines: the structure and function of the choroid epithelium in regard to its normal role in CSF formation and volume transmission (Johanson et al., 2001); how the configuration and operation of the plexuses are altered in hydrocephalic states; and how

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choroid tissue pathophysiology interacts with that of the brain in various types of hydrocephalus.

2.1. Normal CP – ventricular relationships 2.1.1. Structural considerations The complex anatomy of the CSF ventriculo –subarachnoid system, especially as it undergoes dynamic changes in development, affects how the CP interacts with specific brain regions (Mashayekhi et al., 2002). Fortunately, in . 99.5% of human births, the ventricular system develops normally. In mammalian species there are four distinct plexuses: one on the floor of each lateral ventricle and one each on the respective roofs of the 3rd and 4th ventricles. Choroid tissue develops on the dorsal aspect of the neural tube following its closure. Hindbrain CP forms first as two parallel folds at the top of the 4th ventricle, and by adulthood exists in sheet form; there are two horizontally oriented branches extending laterally from each foramen of Lushcka into the subarachnoid space of the cerebellopontine angle. Forebrain CP of the lateral ventricle develops next from neuroepithelium lying in the choroidal fissure, i.e., the groove formed by the edge of the 3rd ventricular roof and the medial wall of the cerebral hemisphere. When fully mature, the lateral CP begins at the interventricular Foramen of Monroe and then courses posteriorly within the choroidal fissure. ‘Glomus’ is the term given to CP swelling at the point where it turns laterally and inferiorly in the trigone area. Last to develop is the 3rd ventricle CP. This plexus originates from the neuroepithelium and underlying mesenchyme in the 3rd ventricular roof. By adulthood, there are two parallel tissue portions (suspended from the top of the 3rd ventricle and joined anteriorly), which are contiguous with each lateral plexus near the foramina of Monroe. Third ventricle CP epithelium and its neighboring ependyma, including the fibrous tanycytes, have dynamic molecular exchange with adjacent circumventricular organs (CVO) and hypothalamic nuclei (Johanson, 2003). Grossly, all choroid tissues are delicate frond-like projections into the ventricular CSF. Their pinkish/red color belies a prominent vascular system lying just beneath the thin epithelial layer. Collectively, the human CPs in the four ventricles weigh about a gram and contain 108 epithelial cells that impart a total surface area of about 200 cm2 (Voetmann, 1949). Figure 1 depicts the major anatomical compartments within each CP villus: (i) an inner vascular core, (ii) an intermediate interstitial space (which houses extracellular matrix as well as plasma ultrafiltrate), and (iii) an outer ring of epithelial cells that cap each choroidal villus (Johanson, 2002). Ultrastructurally, the CP parenchymal cells resemble proximal tubule epithelium. The choroid epithelium typically presents with a lush microvillus surface at the cellular apex, a high density of mitochondria and Golgi apparati, and an extensive basal labyrinth network on the plasma-facing membrane (Keep and Jones, 1990). The organelle profile for the choroid epithelial cells is consistent with that of a high-capacity transport interface, similar to that of the kidney which is also engaged in the movement of a great flux of water, ions and organic solutes (Fig. 2).

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Fig. 1. Sagittal view of CP villi from the lateral ventricle of an adult rat. Note the inner core of blood vessels (VS, vascular space) and the outer ring of CP epithelium (CPE) on the apical pole facing the CSF. Mv, microvilli N, nucleus CSF, cerebrospinal fluid. The epithelial cells are crowned with a lush brush border membrane that maximizes surface area for transport and secretion.

2.1.2. Functional considerations Due to relatively high rates of metabolism, protein synthesis and fluid output by CP, it is not surprising that blood flow measurements reveal a choroid perfusion rate of 4– 5 ml/min/g, i.e., about an order-of-magnitude greater than the average cerebral blood flow (Faraci et al., 1994; Szmydynger-Chodobska et al., 1994; Yacavone et al., 1994). Marked arterial pulsations transmitted from the plexus to CSF have long been recognized (Bering, 1955) as a factor in setting the ventricular volume (Egnor et al., 2002). Choroidal and cerebellar arteries, respectively, feed the telencephalic and myencephalic tissues. Venous drainage from the plexuses eventually empties into the veins of Galen. Hemodynamics in the plexus can be a significant factor in matching (or not) epithelial transport capacity and intimately connected CSF hydrodynamics. Autoregulation of blood flow normally stabilizes the supply of ions and water to the basolateral (plasma-facing) membrane of the epithelium. In very severe hydrocephalus, however, there can be markedly compromised choroidal and cerebral hemodynamics (Higashi et al., 1986; Jones et al., 1993; Klinge et al., 2003). It is pertinent to analyze how vascular parameters affect, and are affected by, the resulting ventriculomegaly and altered intracranial pressure. CSF is formed by most mammalian species, including human, at the relatively constant rate of 0.4 ml/min/g choroid tissue (Johanson, 2002). The main uptake process in fluid elaboration is the parallel operation of Na –H and Cl – HCO3 antiporters at the basolateral membrane (Fig. 2). Such cation and anion exchangers carry Na and Cl (the predominant ions of CSF) from the interstitium into the epithelial cells in exchange, respectively, for the intracellularly formed H and HCO3. To sustain high levels of CSF output into the ventricles, the transport enzyme carbonic anhydrase rapidly generates the ‘labile’ protons and

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Fig. 2. Schematic diagram of ion transporters and channels in a typical choroid epithelial cell in an adult mammal. The plasma-facing (basolateral) membrane has the NaZH and ClZHCO3 antiporters (exchangers) which move Na and Cl from interstitium (plasma ultrafiltrate) into the choroid cell (depicted as a box) as the initiating step in CSF secretion. HCO3 generated in the cell from carbonic anhydrase (c.a.) activity, along with Na and Cl in the cytoplasm, are extruded into the ventricular CSF by primary (Na pump) and secondary (cotransport) active transport mechanisms, as well as via apical membrane channels. Other aspects of the secretory process are discussed in the text.

bicarbonate ions from the catalyzed CO2-hydration reaction. At the apical membrane, the constituent ‘fixed’ ions of the active secretion are vectorially extruded from the choroid cells by the Na pump (Na – K – ATPase) and Na –K – 2Cl cotransport. Also, Cl and HCO3 diffuse down electrochemical gradients, via specific channels, from cytoplasm to ventricular fluid. A cardinal element in the fluid transfer is the movement of Cl, which attains a concentration in CSF that is 20% greater than in plasma. To complete the secretory process, water follows the translocated ions ‘osmotically’ via facilitation through protein structures in the membrane, i.e., aquaporin pores and cotransporters. What about modulation? Biogenic amines, peptides and growth factors can alter ion transport and consequently CSF formation, usually in an inhibitory manner (Nilsson et al., 1992). Although CSF production apparently is not neurohumorally sensitive to a sudden rise in intracranial pressure, the fluid output by the CP seems to be responsive chronically to ‘feedback’ regulatory mechanisms involving growth factors and neuropeptides (Johanson et al., 1999b; Hakvoort and Johanson, 2000; Chodobski and Szmydynger-Chodobska, 2001). Transport at the choroid epithelial aspect of the blood – CSF barrier has distinctive features that set it apart from the cerebral endothelium of the blood –brain barrier. Vitamin C (ascorbate), for example, can be viewed as a prototype molecule that gains access to the CNS by way of the CP – CSF system rather than by transport across the blood – brain interface (Rice, 2000; Johanson, 2003). To regulate the CSF concentration, there is both

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C.E. Weaver et al. Table 1 Substances transported or secreted into CSF mainly by choroid plexus Ions

Micronutrients

Peptides/hormones

Proteins

Naþ Cl2 HCO2 3 Caþ þ

ascorbate folate thymidine

prolactin insulin leptin AVP

transthyretin cystatin C ceruloplasmin transferrin

facilitated diffusion and active transport of ascorbate, respectively, in the basolateral and apical membranes of the CP epithelium (Spector and Johanson, 1989). Table 1 summarizes several solutes that reach neurons either entirely or predominantly by way of circuitous transport via the CP-to-CSF-to-ependyma route. Moreover, up to 80 –90% of the water (depending upon species) that penetrates the CNS does so by way of the four CP tissues. Consequently, any model that evaluates CSF hydrodynamic parameters (including volume) in hydrocephalic states needs to include the CP as a major component (Egnor et al., 2002). Accordingly, altered transport of substances across the CP, secondary to hydrocephalus-induced disruption, is potentially a key factor in evaluating brain damage.

2.2. Hydrocephalus effects on the CP –CSF system Hydrocephalus disorders span a wide array of CSF malfunctions (Braun, 2000; Johanson and Jones, 2001). In comparing models, it is essential to delineate the type and stage of hydrocephalus, and whether effects are primary or secondary (Hochwald, 1985; McAllister and Chovan, 1998; Del Bigio, 2001). Basically, hydrocephalus is CSF accumulation within the cranial cavity causing ventricular expansion. Numerous terms describing the physical finding of dilated ventricles reflect the complexity of this disorder. Hydrocephalus is usually a ‘downstream’ CSF reabsorption failure rather than an ‘upstream’ CSF formation problem, e.g., hypersecretion by CP (Gudeman et al., 1979). Hydrocephalus induced by subarachnoid bleeding, however, affects not only the function of the nearby arachnoid membrane but also that of the more distant choroid epithelium (Liszczak et al., 1984). Hydrocephalus can be acute or chronic, compensated or uncompensated, normal or high pressure, communicating or noncommunicating, and obstructive or nonobstructive (Herndon and Brumback, 1989). Chronic hydrocephalus has a course of months to years and may be symptomless. Communicating or noncommunicating indicates CSF movement (or not) between ventricles and subarachnoid space (Epstein and Johanson, 1987). Normal- and high-pressure hydrocephalus refer to CSF pressure. Obstructive and nonobstructive indicate physical blockage of, or free, CSF flow (Milhorat, 1987). Inactive, arrested, and compensated is asymptomatic hydrocephalus implying no need for shunting. This chapter mainly treats hydrocephalus models with elevated CSF pressure due to inherited mutations (congenital), intra-CSF hemorrhage, or cisternal injections that block CSF flow (Braun, 2000).

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2.2.1. Structural considerations A dysmorphic CP is an early sign of fetal hydrocephalus (Bronshtein and Ben-Shlomo, 1991). Grossly distorted human CP, revealed as early as 15 week by ultrasonography, predicts later problems with the CSF system (Tabsh et al., 1995). Normally, the CP appears by 9 week and occupies a prominent portion of the intracranial space (Fig. 3); ventriculomegaly during the first trimester can also be normal. Malformation, however, manifests as the early absence of choroid tissue, hypoplasia and shrinkage. A greatly enlarging cerebroventricular system, despite total absence of CP or its gradual attenuation, has prompted the deduction that CSF reabsorption as well as secretion occurs across the apical membrane in normal development (Bronshtein and Ben-Shlomo, 1991). The respective apical membranes of the choroid epithelial and ependymal cells face ventricular CSF (Fig. 4). Elevations in ventricular pressure can physically distort these cell types, in regard to their external limiting membranes and organelles. Sustained force on the choroid and ependyma alters their cytoarchitecture, with the attendant functional implications for these cells at the blood – CSF and CSF –brain interfaces. After birth, both the choroidal and ependymal epithelium in mammalian brain proliferate slowly; the former have a lower turnover than the latter (Chauhan and Lewis, 1979; Kaplan, 1980; Bruni et al., 1985). On the basis of observations of sparse, or lack of, mitotic activity, it has been long known that ependyma in the adult hydrocephalic brain do not significantly regenerate (Russell, 1949). Due to the very low degree of postnatal renewal by these nonneuronal cells lining the ventricles (unlike many extra-CNS epithelial cells having high proliferative capacity in adulthood), it is especially significant clinically to prevent

Fig. 3. Coronal sections through adult and fetal human brains. The comparison is graphically made of the ratio of CP tissue mass to that of brain tissue, to emphasize the point that the plexus occupies a larger proportion of the intracranial space in early development. In adults, the CP mass, relative to brain, is substantially less. Disruption of CP–CSF development by hydrocephalus has severe repercussions for the developing brain. Reprinted with permission from Scientific American.

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their deterioration in hydrocephalic states. Can choroid and ependymal cells be stimulated to proliferate, or undergo restitution, under certain pathologic conditions (Johanson et al., 2000)? It is imperative to analyze the ventricle-bordering cells for morphological changes that ensue from hydrocephalic insults, with the goal of modifying such alterations to improve brain function in the face of fluid retention. With respect to hydrocephalus/ventriculomegaly, the choroid epithelium has not been as extensively investigated as ependyma. Embryologically, the two cell types are closely related (Bradbury, 1975—see also chapter by Wolff and Chao), and so their responses to intracranial pressure elevation are more similar than different. As for detachment of epithelium from underlying substrate, the appearance of both choroid and ependymal cells in infant hydrocephalic CSF implies exfoliation as the ventricles progressively dilate (Wilkins and Odom, 1974). Such cellular sloughing can clog CSF percolation routes and exacerbate problems with fluid drainage. Ependymal damage from hydrocephalus has been thoroughly characterized. A prolonged rise in ventricular pressure wreaks considerable damage on the ependyma overlying white matter; less harm is exerted on ependymal cells covering gray matter, due to physically anatomical differences. Typically, in a variety of mammals analyzed for hydrocephalic damage, the ependyma has been either normal, stretched or torn (Hochwald et al., 1969; Bruni et al., 1985). Damage is worst according to the greatest severity and duration of ventricular expansion and pressure (Del Bigio, 1993). Ependymal disruption can occur as early as a half day following CSF flow interruption. When ventricles expand gradually, there is more likelihood that the ependymal lining will stay intact. Still, the normally cuboidal or columnar ependymal cells are flattened (Page et al., 1979). Upon stretching of the membrane, the surface area of the ependyma is augmented, effectively reducing the density of the cilia (Torvik and Stenwig, 1977). As ependymal damage progresses in certain regions, e.g., over the septum pellucidum, there is loss of the apicallyoriented microvilli and cilia. Occasionally the ependyma is destroyed, with only clusters of cells remaining in the gliotic wall of the ventricle. In response to the disintegrating lining (as the result of high CSF pressure and its consequent ischemia), macrophages appear on the ependymal surface to engulf the debris. Hydrocephalus also stresses the choroidal epithelium, the closest ‘cell relative’ of the ependyma. Neuroanatomical differences between CP tissue and the ependymal wall should be considered when comparing their respective responses to biophysical stimuli.

Fig. 4. (a) Top: Electron micrograph of lateral ventricle choroid epithelial cells from a fetal rat (E 18) Particularly evident is the sparse microvilli system at the pole adjacent to CSF. The paucity of the brush border in the fetal CP is consistent with the much lower capacity at the E 18 stage to actively form CSF. Remarkably, adult-like tight junctions near the microvilli are present at this early age. However, the prenatal organelle profile is generally reflective of a cell not as well equipped as the adult counterpart to manufacture CSF. Congenital high-pressure hydrocephalus retards the maturation of the epithelium; thus, when hydrocephalus/ventriculomegaly is present in the fetus to thwart maturation, this type of cellular ultrastructure (cytoarchitecture) can be seen at even later stages of organism development. (b). Bottom: Ultrastructure of adult CP epithelial cells. The morphology is quite similar to the proximal tubular epithelium. C, centriole ER, endoplasmic reticulum L, lysosome, G, Golgi The mature brush border is much richer than in the fetal plexus in (a) above.

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Grossly, the plexus is a frond-like structure, i.e., a tissue anchored at its narrow base to a small region of the brain wall but having extensive tufts or villi that more or less dangle into the large-cavity ventricular CSF where they float. In contrast, the ependyma exists as a single-cell lining, which is entirely apposed (fixed) to larger surface areas underlying white or gray regions of the brain. Because the plexus is tethered whereas the ependyma is not, one would expect variable responses of these tissues to altered CSF pressure and volume. There are also histological differences. CP epithelial cells apically are joined by tight junctions and basolaterally are set on a true basement membrane; on the other hand, the ependyma in the lateral ventricle have gap junctions between cells and lack a basement membrane. The ability of these different cell types to withstand shearing forces and pressure variations is likely a function of their respective physical structures, both at the ‘macro’ (multi-cellular sheet) and ‘micro’ (ultrastructural) levels. There has been a narrower focus on CP, versus ependyma, in describing structural alterations in hydrocephalic states and their impact on brain. Perhaps this is because the plexus tissue is locationally a step further removed from the CSF – brain nexus, than is the ependyma that intimately hugs the white and gray matter over large ventricular surfaces. Now that it is well known that CP is the major supplier of trophic and micronutritional substances to neurons (Table 1), it seems pertinent to devote more attention to structural changes and damage to the choroid epithelium in a CSF system under pressure and distention. In some hydrocephalus models, CP structure has appeared fairly normal (Hochwald et al., 1969). CSF shunting can either prevent or reverse damage to CP and brain (Dohrmann, 1971; Boillat et al., 1997; Jones et al., 1997, 2000). However, as the nonshunted hydrocephalus insult expands both directionally (larger ventricles) and temporally (longer duration), the deviations from healthy tissue morphology become more evident. Humans with chronic hydrocephalus have a sclerotic stroma and atrophied epithelium in CP, which may be buried in the ventricular wall (Netsky and Shuangshoti, 1975; Di Rocco et al., 1977). Models involving inherited mutations, experimental induction or cisternal injections, and carried out with mice, rats and dogs, have provided consistent information on the altered epithelial layer (Dohrmann, 1971; Lawson and Raimondi, 1973; Go et al., 1976; Miyagami et al., 1976; Liszczak et al., 1984; Shuman and Bryan, 1991); see compiled findings in Table 2. Quite common is the squashing and vacuolarization of epithelial cells (Tennyson, 1960); there are also dilated intercellular spaces. Similar to macrophage responses to traumatized ependyma, there are macrophages (so-called Kolmer cells, normally present in low number) that help to clear debris from the apical surface of choroid epithelial cells compromised by CSF anomalies (Cerda-Nicolas and Peydro Olaya, 1988; Go et al., 1976; Lu et al., 1996). Descriptions of research on animal CP have lined up well with light and electron microscope observations on autopsied tissue from humans with hydrocephalus disorders. Unlike the case for nearly complete ependymal destruction in the most severe hydrocephalus, it is uncommon to find fully obliterated CP; this may indicate that the choroid tissue is more resistant than the ependymal wall to waxing changes in CSF pressure and volume (Nielsen and Gauger, 1974). Choroid cytoarchitectural responses to hydrocephalus, either experimentally-induced or spontaneously occurring, are remarkably similar. Recurring themes, e.g., of expanded

Species

Model of hydrocephalus

Apical membrane

Basolateral membrane

Organelles/Cytoplasm

Nucleus/Cell Shape

Guinea pig (young)

Kaolin injectiona 1 –4 wk

# microvilli surface area

hy-3/hy-3 mutantb

Normal

Less Golgi and RER; # mitochondrial membrane area Normal

Flattened nucleus and epithelium

Mouse (Postnatal) 3 wk Hamster 1 –7 wk

Reovirus Injection; aqueduct stenosisc Kaolin injectiond 1 month 4–5 £ " ICP

Long, thin microvilli

Dilated inter-cellular spaces; Loosely-packed basal infoldings Greatly dilated intercellular space along lateral border Increased cell separation and intercellular space Adjacent choroid cells not separated; junctions intact

Blood injectede in cisterna magna hpy/hpy mutantf

Tight-packed microvilli

Marked dilation of intercellular space 2-fold " in dilated intercellular space

Cytoplasmic vacuoles and inclusions 4-fold " in multivesicular bodies

Dog (adult)

Rabbit (adult) Mouse (adult) a

Madhavi and Jacob, 1990, 1992, 1995 Lawson and Raimondi, 1973 c Nielsen and Gauger, 1974 d Dohrmann, 1971 e Liszczak et al., 1984 f Shuman and Bryan, 1991 b

Normal microvilli

Filiform microvilli

– Compact cytoplasm; multi-vesicular bodies

Normal Cell diameter unchanged Flattened cells; # cell volume by 60 –70% " dark cells " dark cells

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Table 2 Ultrastructural alterations in the mammalian lateral ventricle choroidal epithelium subjected to elevated-pressure hydrocephalus

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intercellular spaces between the basolateral portions of adjacent epithelial cells, suggest a common pattern in choroidal adjustment. One limitation, however, in comparing pressureinduced changes in structure (Table 2) is that intraventricular pressure can vary with brain maturation, stage of disorder, degree of compensation, and other pathological variables such as bleeding. Still, it is instructive to identify ‘strong common denominators’ such as the extensive finding of the markedly dilated intercellular space in CP that occurs in most hydrocephalus analyses (Table 2). Is this a biophysical phenomenon related to pressure gradients within choroidal tissue compartments, and/or is it due to a biochemical modulation of the cell that redistributes water between the cell and its surrounding interstitial fluid? In other words, the observed ultrastructural alterations in the basolateral membrane vicinity might relate to physical factors associated with hydrocephalus; or alternatively, they could be part of a compensatory neurohumoral response, say reduced CSF secretion and/or enhanced fluid reabsorption across the plexus, to restore CNS extracellular fluid homeostasis. Ultrastructure comparisons, control vs. experimental, are thus useful in predicting or corroborating functional insights. Electron micrographs have revealed that some epithelial cells in the CP have a fine structure typical of a secretory mode, while others designated as ‘dark cells’ are reminiscent of a fluid-reabsorbing structure (Schultz et al., 1977; Shuman and Bryan, 1991). An increase in the number of ‘dark cells’, thought to have a neuroendocrine-like function in fluid balance (Johanson et al., 1999a) is intimately associated with hydrocephalic states. On the other hand, a physiological state in which there is a shift in the choroid epithelial cell population to one in which the ‘light cells’ are more numerous than dark ones is consistent with a secretory rather than a reabsorptive mode (Shuman and Bryan, 1991). Such structural delineations by microscopy are helpful in interpreting as well as devising hydrocephalus experiments. 2.2.2. Functional considerations The CP displays remarkable resiliency in the face of traumatic insults to the CNS interior. Even with substantial increments in intracranial pressure in hydrocephalus, the endothelial cells and lumen of the choroid microvessels remain intact (Dohrmann, 1971; Lawson and Raimondi, 1973; Madhavi and Jacob, 1990). In other studies, not involving hydrocephalus, there is similar capillary protection against severe ischemia. Thus, following blood flow interruption/reperfusion, e.g., transient forebrain ischemia, the choroidal endothelium and epithelium either escape serious damage or are replaced by new cells (Palm et al., 1995). Such efficient reparation at the blood – CSF barrier allows the healed choroid epithelial lining, by way of its secretory/reabsorptive activities, to promote recovery in other regions, e.g. hippocampus, greatly hurt by interrupted perfusion (Johanson et al., 2000). The brain is thus well served as choroidal hemodyamics are stabilized acutely by autoregulatory mechanisms and more chronically by recovery/ plasticity abilities; the latter are undergirded by growth factor upregulation in response to hypoxia/ischemia (Johanson et al., 2003) and mechanical stress. In part, as a consequence of elevated CSF pressure exerted on the CP, the blood flow to choroid tissue is reduced in hydrocephalus if the intraventricular pressure rise causes a large fall in the net perfusion pressure to the plexus (Pollay et al., 1983; Nakamura

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and Hochwald, 1983). Biochemical as well as biophysical factors, however, may also contribute to diminished vascular perfusion in chronic adjustments. Arginine vasopressin (AVP), which is centrally released into CSF in response to hypoxia or ICP elevation, can lower choroidal blood flow (Faraci et al., 1994) and reduce Cl efflux from isolated CP (Johanson et al., 1999a). CSF formation rate, which is dependent on blood flow, is also suppressed in hydrocephalus (Sahar et al., 1970, 1971). This inhibitory effect on fluid dynamics is likely due to the combined effects of ICP, hypoxia and attenuated ion transport across CP. Because choroid fluid production is reduced in proportion to the blood-to-CSF transport of Cl (Johanson et al., 1994), it is not surprising that Cl efflux (from choroidal epithelium to artificial CSF) is decreased in plexus tissue removed from kaolin-induced hydrocephalic rats and analyzed in vitro (Knuckey et al., 1993). This points to direct effects on the epithelium, as a reaction to hydrocephalus, in addition to those on blood flow. Collectively there is considerable evidence, from a variety of functional and ultrastructural analyses, for downregulated CSF production in hydrocephalus. 3. Working model: there is a neuroendocrine system within the brain that helps to regulate CSF formation and intraventricular pressure Peptides such as AVP, ANP and Ang II are complexly involved in regulating the volume and osmolality of the plasma, i.e., the main extracellular fluid in the body. Given the physiologic protection afforded the brain ‘behind’ or ‘inside’ its unique blood – brain and blood – CSF barriers, it is not unreasonable to think that the CNS has its own hormonal systems, more or less independent of the periphery, that guard the neurons by stabilizing against perturbations in CSF volume and composition. The triad of the AVP, ANP and Ang II peptides is abundantly expressed and functional in the CP, cerebral endothelium and glia (Liszczak et al., 1984; Nilsson et al., 1992; Doczi, 1993; Chodobski et al., 1997, 1998; Johanson et al., 1999a; Hertz et al., 2000; Vajda et al., 2001). The presence of receptors and mRNA for these neuropeptides at the blood –CSF and blood –brain interfaces was originally considered by Raichle (1981), who proposed a central neuroendocrine system that regulates brain ion homeostasis and volume. It is now known that there is widespread but circumscribed expression of central receptors and transcripts for AVP, ANP and Ang II. Moreover, the presence of these peptides in CSF, where their titers are largely independent of those in plasma, constitutes evidence for their potential modulation of brain fluid balance by controlling fluxes of ions and water at the main transport interfaces in CNS (Doczi et al., 1995; Nilsson et al., 1992). To reiterate, the CP is the site of preponderant movement of Na, Cl and water from blood into the CNS; therefore, when analyzing homeostatic responses to hydrocephalus, or designing agents to control fluid overloads, the choroid epithelium should be a primary target. 3.1. Is hydrocephalus-damaged tissue capable of responding to neuropeptide regulation? Major irreversible damage to the CP from excessively high ICP might preclude this tissue from participating in homeostatic responses to disordered fluid balance. However, evidence has been presented that CP is better able to withstand the mechanical stresses

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imparted by increased CSF volume and pressure than its counterpart ependyma. Choroid epithelial cell compression can occur early in the course of hydrocephalus (Madhavi and Jacob, 1990). Even though microvilli patterns can change from a predominantly bulbous or clavate form to a thinner, filiform configuration (Nielsen and Gauger, 1974), there is otherwise no serious damage or necrosis to the epithelium. Longstanding compression of the choroid epithelium, for up to a month, is reversible after a day of ventriculojugular shunting to relieve pressure and decrease volume (Dohrmann, 1971). Overall, investigations of many hydrocephalus models have revealed minimally damaged epithelium in the CP (Table 2), and perhaps even the ability to repair degenerated cilia (Go et al., 1976). Moreover, other CVOs, like the CP, do not sustain damage in the rat kaolin model (Collins, 1979). Such findings of relatively intact plexus epithelium and other CVOs open the possibility that neuroendocrine systems are able to regulate ventricular fluid transfer across the blood –CSF barrier in severe hydrocephalus. 3.2. Plasticity of neuropeptide receptors in CP There is a plethora of evidence that various nonneuronal cells in the CNS respond to neuropeptides like AVP and ANP, probably to maintain or restore fluid balance among compartments (Doczi, 1993; Hertz et al., 2000—see also chapter by Chen and Spatz). In some cases, AVP and ANP have opposing or counterbalancing effects in these fluid adjustments (Doczi et al., 1988). In both congenital and kaolin-induced hydrocephalus, there is altered binding capacity for ANP in rat CP (Tsutsumi et al., 1988; Tsutsumi, 1990; Mori et al., 1990). This has prompted the idea that CSF production by CP is modified by ANP in these hydrocephalic rats (Mori et al., 1990). Supporting evidence is that intraventricularly-administered ANP decreases both ICP and brain edema in rats with congenital hydrocephalus (Minamikawa et al., 1994). Although AVP receptors in CP have not yet been analyzed in hydrocephalus models, there is the finding of upregulated expression of mRNA for the V1 receptor in CP following dehydration (Zemo and McCabe, 2001). This increase of AVP and V(1b)R mRNAs in the CP further shows the involvement of AVP in regulating brain water content and edema. There is now solid evidence for CNS plasticity in regard to AVP and ANP expression, for peptides as well as their receptors, following changes in CSF pressure and volume. 3.3. Central source of neuropeptides that target the CP The hypothalamo –hypophysial system and plexuses are rich sources of peptides and growth factors in the CNS. AVP, and basic FGF with which it colocalizes (Szmydynger-Chodobska et al., 2002) are secreted from both hypothalamic and extrahypothalamic sources like the CP (Matthews et al., 1993; Chodobski et al., 1997). In need of identification is the source of ANP to stimulate choroidal receptors, of which there is a high density in hydrocephalus (Tsutsumi et al., 1988). ANP interacts with AVP by inhibiting the release of the latter (Gerstberger et al., 1992). Peptides released into CSF can stimulate receptors on the choroidal epithelium (Phillips et al., 1988; Tribollet et al., 1999) either locally in a paracrine fashion or by volume transmission (bulk CSF flow) from more

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distant sites of origin near the ventricles (Nilsson et al., 1992; Johanson, 1993, 2002). The hypoxia/ischemia and elevated ICP that occur in some hydrocephalic states are probable stimuli promoting release of AVP (Faraci et al., 1994) and other CSF-inhibiting peptides. 3.4. Relationship between intracranial pressure and CSF neuropeptide levels When the ICP is elevated, there is a concomitant rise in the titer of certain neuropeptides in the CSF. Human ANP in the CSF increases proportionally to increments in ICP, especially over a threshold value of ICP (Yamasaki et al., 1997); moreover, CSF ANP is independent of levels in serum. A similar situation obtains for AVP. In humans, CSF AVP concentration is insulated from that in plasma (Barreca et al., 1988). CSF AVP does not undergo a circadian rhythm in hydrocephalic patients. In another hydrocephalus study, CSF AVP concentration in benign intracranial hypertension was twice that of controls and patients with normal pressure hydrocephalus (Hammer et al., 1982). Consequently, it appears that an augmentation in ICP, rather than ventriculomegaly, triggers AVP release into CSF. 3.5. Do peptides downregulate CSF formation in hydrocephalus? Intracerebroventricular (i.c.v.) injection of a peptide mimics the endogenous situation in which that peptide is released into CSF from a central region. A small i.c.v. dose of AVP in the rat reduces ICP by 26%, even in the face of simultaneously increased arterial pressure up to 44% (Saladin and Bruni, 1993). In investigations with ANP, it was found that i.c.v. injection (0.2 mg) could decrease ICP and reduce brain edema in rats with congenital hydrocephalus (Minamikawa et al., 1994). The ICP-lowering effect of these centrally administered peptides could be due to enhanced CSF reabsorption or reduced CSF formation. There are substantial data from nonhydrocephalus studies demonstrating that AVP curtails both hemo- and hydrodynamics at the blood –CSF barrier. Intravenous AVP reduced blood flow to rabbit CP by 60%, and CSF production by 35% (Faraci et al., 1988, 1990). In the rat, AVP mediates the inhibitory effect of central Ang II on CSF formation (Chodobski et al., 1998). I.c.v. injection of ANP in rabbits caused a small decrease in CSF formation even in the face of a substantial increase in CP blood flow (Schalk et al., 1992). This uncoupling between blood flow and transport suggests a direct inhibitor effect of ANP on choroid epithelial transport mechanisms. Overall, ANP and AVP are particularly promising test peptides because they alter CP blood flow, ion transport and CSF formation. CSF formation appears to be reduced in chronic hydrocephalus (Silverberg et al., 2002). However, the literature is mixed in regard to findings on the inhibitory effect, or lack thereof, of hydrocephalus on CSF formation (Marlin et al., 1978; Sahar et al., 1970; Shaywitz et al., 1969). In addition to the necessity for controlling many experimental variables, it is technically challenging to accurately quantify CSF formation rate indirectly in humans and by ventriculo-cisternal perfusions in animals. Still, the definitive experimental findings of the AVP inhibitory effects on CP – CSF dynamics, together with the direct measurements of CSF AVP levels in hydrocephalic subjects in whom ICP

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is quantified (discussed above), constitute a promising basis for extending peptide modulation studies to dynamic imaging assessments.

3.6. AVP-induced changes in choroid epithelial ultrastructure and function AVP has the ability to induce ‘dark’ epithelial cells in CP (Dohrmann, 1970; Schultz et al., 1977; Liszczak et al., 1984, 1986; Johanson et al., 1999a). Choroidal dark cells, which are thought to have neuroendocrine-like functions, occur more frequently in hydrocephalic disorders and in other water-imbalance states (Shuman and Bryan, 1991). Typically, in untreated mammals, dark cells comprise about 5% of the total choroid epithelial cell population. The darkness, visible with both light and electron microscopy, is due to the condensed cytoplasm of a shrunken cell that has lost water (Fig. 5). Dark cells are inducible by peptides (e.g., AVP, ANP and FGF-2) both in vitro and in vivo (Johanson et al., 1999b), whether the exposure is from the blood or CSF side of the epithelium. This AVP effect is rapid, e.g., 20 min of incubation with peptide (Fig. 6). Induction involves peptide action on the V1 receptor, the blockade of which with an antagonist prevents dark cell onset. AVP and dark cells have also been intimately associated with dilated basolateral spaces between choroid epithelial cells. Schultz et al. (1977) were apparently the first investigators to point out that the AVP-facilitated dark cell ultrastructure, including the intercellular dilations (Table 2), gives the impression of a reabsorptive state. Due to the greater presence of dark cells in the CSF system subjected to hydrocephalus, interest has been generated about the possibility that these dark cells and their modified basolateral

Fig. 5. Electron micrograph ( £ 15,500) of ‘dark’ (on left) and ‘light’ (right side) epithelium from adult rat lateral CP. Animal was untreated. The dark cell has a greater cytoplasmic electron density; the light cell is more electronlucent. The dark cell typically has filiform microvilli. Where the light cell interdigitates with the dark one at the base (basal labyrinth, BL), note how the interdigitating ‘fingers’ retain their respective electron densities. RER, rough endoplasmic reticulum S, secretory granule. Dark cell is more shrunken.

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Fig. 6. In vitro incubation of adult CP with AVP, to induce dark cells. The graph is a dark cell frequency analysis of CP epithelium exposed in vitro for 20 min to 1 nM AVP, with and without the V1 antagonist to prevent the induction. Y-axis represents % of cells that are dark. Means ^ SEM for N ¼ 5. Statistical analysis: 1-way ANOVA, then Bonferroni. Reprinted with permission from the Am. J. Physiol.

spaces are reflective of stimulated reabsorption of excess CSF (Milhorat et al., 1970). As is the case for gall bladder epithelial dynamics, there can be an alternate opening or closing of the CP basolateral space depending upon the functional status of fluid transfer, i.e., secretory or absorptive. Because ion transport is integral to CSF formation, it has been insightful to analyze Cl transport in the context of AVP regulation (Fig. 7). Cl efflux from the choroidal epithelium is a primary step in the CSF secretory process (Fig. 2), even when analyzed in vitro (Johanson et al., 1999a). AVP inhibits Cl extrusion, a process that involves the V1 receptor in CP; this was deduced by the simultaneous use of the peptide agonist and its receptor antagonist (Fig. 7). It is now clear from a variety of physiologic experiments that the CP epithelium and vasculature are sensitive to regulation by AVP and other neuropeptides. More information is needed to delineate at the molecular level the peptidergic modulation of secretion and reabsorption by CP, the ‘kidney’ of the CNS (Spector and Johanson, 1989).

3.7. Putative role of nNOS in CP reabsorption In a homeostasis context, there are some fascinating comparisons between CP and kidney. A large component of fluid absorption in the proximal tubule is controlled by the neuronal isoform of nitric oxide synthase (nNOS). In knock-out mice that are deficient in nNOS, there is a major defect in ion and fluid absorption in the kidney (Wang et al., 2000).

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Fig. 7. Effect of V1 receptor blockade, in presence or absence of 1 nM AVP, on the efflux coefficient for Cl transport out of choroid cells loaded with radioactive 36Cl. V1 ANTAG ¼ V1 antagonist d(CH2)5Tyr(Me)AVP at 2 £ 1028 M. Means ^ SEM for N ¼ 4–6. Statistical analysis: 1-way ANOVA, then Bonferroni. The antagonist prevented the AVP-induced inhibitory effect on Cl release from the CP epithelium. Reprinted with permission from the Am. J. Physiol.

nNOS also has a predominant localization in the ciliary body of the eye, suggesting involvement of this NO-generating enzyme in balancing the fluid production and reabsorption of the aqueous (Meyer et al., 1999). Given the similar functions of the renal, ciliary and choroidal epithelia, the nNOS knock-out information will likely afford clues about altered fluid phenomena in hydrocephalic and ischemic animals (Rhoden et al., 2002). NOS, which has a key role in epithelial fluid dynamics, is abundant in rat CP (Lin et al., 1996). Immunoreactivity of nNOS has been observed in CP epithelium (Alm et al., 1997). CSF formation rate is inhibited by stimulation of the NO/cGMP pathway in CP (Ellis et al., 2000), which is regulated by peptides like ANP. There is also nitrergic modulation of AVP and ANP secretion (Raber and Bloom, 1994; Cao et al., 1996; Ventura et al., 2002). It is interesting that AVP enhances nNOS expression in the rat renal medulla (Martin et al., 2002). Epithelial transport analyses are thus providing substantial information to indicate a complex interplay among water-regulating peptides, NOS, cGMP, ion transport and fluid movement. Curiously, nNOS is upregulated in hydrocephalus. In adult rats injected with kaolin, there was a significant increase after 2 week in the nNOS activity in hippocampal and cortical neurons (Klinge et al., 2002). Six to eight weeks after kaolin injection, when ventricular size was changing, the immunohistochemical changes in the hypoperfused hippocampus were most prominent (Klinge et al., 2003). Information is needed for CP expression of nNOS as a function of hydrocephalus progression. The nNOS knock-out

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model seems promising for exploring altered fluid dynamics in the CP –CSF system in health and disease. 4. Recapitulation and looking forward Much more than acting as a physical buffer or ‘shock absorber’ for the brain, the CSF has a multiplicity of humoral and cytological functions (Johanson, 2002). Among many physiologic roles, the CP –CSF system can justifiably be regarded as a neuroendocrine ‘transducer’ (Scott et al., 1977; Scott, 2002). As both a source and a target for peptides, the CP plays a key role in CSF homeostasis (Chodobski and Szmydynger-Chodobska, 2001). The cerebroventricular system is more than the mere neuroanatomical core of the brain; it is also the physiologic basis for the control of the internal milieu of the mammalian nervous system. The CP is a unique ‘circumventricular organ’ the duty of which is to maintain a precise and stable environment for the neuropil. As such, the CP is a masterful physiologic ‘servant’ with an amazing repertoire of homeostatic abilities which are mediated by the CSF that it elaborates. Just as the trio of peptides—AVP, ANP and Ang II—integrate the control of systemic (plasma) volume and osmolality, by acting on the kidney and other peripheral organs, so also do these same three peptides regulate central (CSF) fluid volume and pressure via actions at the CP and blood –brain barrier. Moreover, CSF titers of neuropeptides are independent of those in plasma; this is powerful support for any CSF neuroendocrine model. Thus, evidence is steadily building that the CNS has ‘on site’ independent neuroendocrine mechanisms for stabilizing its unique extracellular fluid. Regulation of intracranial pressure is a vital business, as can be attested to by any neurosurgeon challenged with CNS fluid retention problems. CSF shunts have been the mainstay solution. As more understanding is gained about mechanisms underlying fluid movement to and from the brain, it should become more feasible to pharmacologically regulate fluid balance and solute flux among CNS compartments (Johanson, 1989). CSF composition of ions and cerebral metabolites in hydrocephalus disorders has been analyzed (Del Bigio, 1989; Keep and Jones, 1989). However, more information is needed from hydrocephalus models concerning the resultant CSF levels of choroidally secreted micronutrients, elements and proteins when the fluid transfer (secretion or reabsorption) at the CP is substantially modified. It would be desirable pharmacologically to enhance reabsorption of excess CSF from the ventricle into the plexus without deleteriously affecting the CSF content of essential ions and molecules destined for target cells in the brain. Acknowledgments For making these studies possible, the authors gratefully acknowledge support from NIH NS 27601 (C.E.J), Lifespan (Rhode Island Hospital), and the Department of Clinical Neurosciences at the Brown Medical School. We also thank R. Croft and H. Jones for helpful comments on the manuscript.

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Roles of retinal macroglia in maintaining the stability of the retina Jonathan Stonep and Krisztina Valter Department of Anatomy and Histology and Institute for Biomedical Research, University of Sydney Correspondence address: Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra 2601 Australia, E-mail: [email protected]

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Contents 1. 2. 3.

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Introduction: the challenge of lifelong stability Macroglia, microglia, the RPE: A working classification Macroglia stabilize the retinal surfaces: the retinal glia limitans 3.1. The inner limiting membrane (ILM) 3.2. The outer limiting membrane (OLM) Pathology of the ILM and OLM 4.1. Gliosis 4.2. Hypoxia-induced breakdown of astrocytic glia limitans Macroglia guide blood vessel formation 5.1. Astrocytes are required for the surface spread of the retinal vasculature 5.2. Mu¨ller cells and the deeper vessels of the retinal circulation 5.3. Angiogenesis in retina and brain compared Macroglia stabilize adult retinal vasculature 6.1. Maintenance of the blood – retinal barrier (BRB) 6.2. Vessel maintenance 6.3. Physical restraint Macroglia stabilize the photoreceptor population 7.1 Mu¨ller cell processes wrap every retinal neurone 7.2. Macroglia express factors that protect photoreceptors 7.3. FGF-2/FGFR1 colocalization: sites of FGF-2 action 7.4. CNTF, CNTFRa 7.5. Mu¨ller cells provide metabolic protection for photoreceptors Implications for therapy 8.1. Immediate oxygen for patients with retinal detachment 8.2. Neovascularizing diseases of the retina: the key importance of astrocyte survival 8.3 Photoreceptor degenerations: the trade-off between neuronal protection and retinal sensitivity Concluding remarks

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Fig. 1. (A) Section of monkey retina, stained with Masson’s trichrome, to show the tight laminar structure of the retina. The inner surface of the retina is at the top of the panel. Retinal neurons form three layers, the ganglion cell layer (g), the inner nuclear layer (i) and the outer nuclear layer (of photoreceptors) (o). The inner- and outer segments of the photoreceptors stretch outwards and are tightly aligned into layers (is and os). The outer tips of the outer segments abut the RPE (arrow). Adjacent to the outer side of the RPE are capillaries of the

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The retina is a highly specialized extension of the central nervous system (CNS), enclosed into the eyeball as the sensor for vision. Like the rest of the CNS, the retina is highly stable, remaining functional for the lifetime of the organism, in Homo sapiens for many decades. Clinically, many disease results from instability in two elements of the retina, its vasculature and the photoreceptor population. Analysis of this instability (proliferation and degeneration) has given evidence of roles played throughout adult life by the macroglia of the retina (astrocytes and Mu¨ller cells) in maintaining the integrity of retinal structure. Both astrocytes and Mu¨ller cells are involved in the formation of the glia limitans of the retina, define the inner and outer limits of its highly laminated structure. Both forms of macroglia are also involved in guiding the formation of retinal vessels, inducing their barrier properties and maintaining their adult structure. The Mu¨ller cells play important roles in the stability of photoreceptors, upregulating protective proteins and cytokines in response to stress, and providing metabolic substrates for the rapid oxidative metabolism of photoreceptors. Understanding of these mechanisms may contribute to the devising of therapy for the blinding diseases, which result from their breakdown. 1. Introduction: the challenge of lifelong stability A major challenge for the retina is to maintain the stability of its neurones and their detailed, strictly layered (Fig. 1A) structure for the life of the individual, in long-lived species like humans, for many decades. Retinal neurones share the stability of all neurones of the central nervous system (CNS)—they can and do last a lifetime, but they are also subject to unique stresses. For example: . Ganglion cells (located in the cell layer g in Fig. 1A) are vulnerable to damage where their axons pass, while still unmyelinated, from the eye into the optic nerve,

choriocapillaris. (B). The human eye at mid-gestation. A large artery (the hyaloid) extends from the optic disc to supply the forming lens. This artery becomes vestigial around the time of birth. The retinal vessels develop from the hyaloid vessels at the optic disc and spread over the surface of the retina. (C). The optic disc region of the rat retina, seen en face in a whole-mounted retina. Vessels are labeled green (with a vessel specific lectin G. simplicifolia) and astrocytes are labeled red (with an antibody to GFAP). Retinal vessels radiate from the optic nerve head, and are covered with astrocytes, which form their glia limitans and the inner glia limitans of the retina. The stump of the hyaloid artery pokes forward into the cavity of the eye. It is astrocyte-free and appears bright green. (D). Section of rat retina labeled for CNTFRa immunohistochemically (red). CNTFRa is prominent in the layer of outer segments (os). (E,F). Negative control for immunolabeling for CNTFRa: E and F were labeled in the same procedure; the primary antibody to CNTFRa was omitted from F. (G). Anti-CNTFRa labeling is found in granular form along the length of photoreceptor outer segments. (H). CNTFRa þ granules (red) are also found between photoreceptor somas in the ONL. Although not labeled in this panel, the spaces between photoreceptor somas in the ONL are filled by the processes of Mu¨ller cells. (I,J,K). When an antibody was applied which labeled vimentin, an intermediate filament protein prominent in Mu¨ller cells, CNTFRa þ granules (red) appeared to be closely associated with vimentin þ (Mu¨ller cell) processes (green). (L). In this panel, red labeling is specific to CNTF. In a stressed retina (in this case stressed by section of the optic nerve) CNTF is prominent in the processes of Mu¨ller cells stretching radially across the retina, between the somas of neurones. Abbreviations: g, ganglion cell layer; i, INL; o, ONL; is, layer of inner segments; os, layer of outer segments.

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past the stiff collagen of the lamina cribrosa, across the pressure drop from the eyeball to the nerve. Photoreceptors (layers o, is and os in Fig. 1A) are vulnerable to scores of genetic mutations, many of which are photoreceptor-specific and therefore (because they are not lethal to the fetus) survive the attrition of development. The dependence of photoreceptors on the retinal pigment epithelium (RPE) (indicated by the arrow in Fig. 1A) for recycling of breakdown products of rhodopsin, for the disposal of discarded membrane, and for the establishment of a barrier from the highly vascular and permeable choriocapillaris, makes them vulnerable to RPE dysfunction. The unique vascular arrangements of the eye (the supply of photoreceptors by diffusion from the choroidal circulation) make photoreceptors vulnerable to oxidative stress. The high sensitivity of photoreceptors to light, their primary function, makes them susceptible to damage by light.

The macroglial cells of the retina (astrocytes, Mu¨ller cells) play key roles in stabilizing the retina in the face of those stresses, providing structural templates for vessel formation, structural seals (the glia limitans) for the retina’s inner and outer surfaces and a glia limitans to every vessel, secreting factors which induce the growth and maintain the adult structure and barrier properties of blood vessels, and providing trophic factors and cytokines which make neurones resistant to cell death. This review will examine the cellular and molecular mechanisms involved in the stabilization of the two elements of the retina which are the site of most retinal pathology, its blood vessels and photoreceptors.

2. Macroglia, microglia, the RPE: a working classification Several classes of retinal cells have been considered to be glial (Latin glia ¼ glue), in particular astrocytes, Mu¨ller cells and RPE cells. Microglial cells are now considered to be vascular in origin, activatable scavenger cells which arise from monocytes and invade the inner layers of retina (and the rest of the CNS) from its vasculature (Kaur et al., 2001; Cuadros and Navascue´s, 1998). The RPE serves several glial functions in relation to photoreceptors (transmitter recycling, ionic buffering (Steinberg, 1985)), but also has major epithelial and phagocytic functions. This review concerns astrocytes and Mu¨ller cells, collectively termed macroglia, to distinguish them from microglia. The very different morphologies of astrocytes and Mu¨ller cells led early analysts to regard them as two quite different cell types. Ramon y Cajal (Cajal, 1893), translated in (Rodieck, 1973), at p885, described Mu¨ller cells as ‘epithelial cells’, and astrocytes as the ‘spider-like or true neuroglial cells’ of the retina. Subsequent work on the origin of astrocytes (Section 5.1.2) has led to the converse view on this particular point: that the Mu¨ller cells are the intrinsic macroglia of the retina, and astrocytes are immigrants from the optic nerve. When the functional and morphological specializations of astrocytes and Mu¨ller cells are compared in detail (Hollander et al., 1991; Stone et al., 1995b), the view has emerged that these two forms of macroglia share many functions (the ability to form the vascular glia limitans, to ensheath neurones, to buffer and recycle the ionic and

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chemical fluxes associated from neuronal activity), and differ in several, in particular that retinal neurones are ensheathed primarily by the Mu¨ller cells. 3. Macroglia stabilize the retinal surfaces: the retinal glia limitans 3.1. The inner limiting membrane (ILM) The inner and outer surfaces of the retina correspond, respectively, to the pial and ventricular surfaces of the neural tube and adult brain. At each surface, in the retina as in the brain, macroglial cells form a structural seal. At the inner retinal surface, the seal is formed by adherent junctions between the inner end-feet of the Mu¨ller cells (Hollander et al., 1991). In the many retinas in which astrocytes are also present at the inner surface (those species in which a retinal circulation forms (see Section 5.1)), astrocyte and Mu¨ller cell processes join to each other by the same form of junction (Hollander et al., 1991). In this function, Mu¨ller cells and astrocytes seem interchangeable; elsewhere in the central nervous system the glia limitans at the pial surface is formed by astrocytes (Fig. 13-4 in Peters et al., 1976). A radial form of macroglia forms throughout the brain during development (Schmechel and Rakic, 1979, and see also the Chapter by Wolff and Chao), but the radial form survives only in the retina, where it is called a Mu¨ller cell. 3.2. The outer limiting membrane (OLM) The outer limiting membrane is the name given to a sharp alignment of the Mu¨ller cell processes near the outer surface of the retina. Along a distinct line, the outer tips of the Mu¨ller cell processes connect to each other with adherent junctions. This line of processes corresponds to the ventricular surface of other CNS regions that, in most brain regions, is formed by ependymal cells. These cells also adhere to each other, and form a ciliated epithelium, the cilia extending into the ventricle. Ependymal cells can be considered a specialized form of macroglia. In the wall of the third ventricle, for example, where the wall is thin, cells lining the ventricle have both ependymal properties (forming a ciliated epithelium to line the ventricle) and astrocytic properties (extending astrocyte-like processes into the neuropil) (Peters et al., 1976). Cells with this mixture of ependymal and astrocyte properties are called tanycytes; the mixture suggests the common lineage of astrocytes and ependyma. As the retina and optic nerve form from the optic stalk, however, the ventricle of the stalk is obliterated. The obliteration brings neural retina (the anterior wall of the cup) into contact with the RPE (the posterior wall). A ciliated epithelium is not required to secrete CSF and this region of retina – RPE contact undergoes a spectacular transition. Late in retinal development, each photoreceptor grows a cylindrical extension, which protrudes through the OLM. The proximal part of the extension is cytoplasmic (the inner segment); from it extends a cilium whose tip is massively expanded and folded to form the outer segment. The tips of the outer segments are embraced by processes of RPE cells (Anderson et al., 1978), but over a short but critical distance—from the apical process of RPE cells to the outer end-feet of the Mu¨ller cells—photoreceptor processes lie outside

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‘coverage’ of macroglial cells, or of cells, like those of the RPE, with glial properties. This short span—the subretinal space—is the only place in the CNS where neuronal processes extend beyond glial cover. Both the ILM and OLM can break down, destabilizing the structure of the retina. 4. Pathology of the ILM and OLM In the retina, Mu¨ller cells and astrocytes react to hypoxic stress in distinct ways, both destabilizing to the retina. Mu¨ller cells hypertrophy and proliferate causing glial scarring or gliosis (see also chapter by Bringmann et al.); astrocytes degenerate, leaving vessels free of a glia limitans, leaky and unstable. Protecting macroglia is as important for retinal function as the protection of neurones. 4.1. Gliosis Retinal gliosis is a damaging pathology, a blinding complication of retinal detachment. As analyzed by Fisher and colleagues in a feline model of retinal detachment (Fisher et al., 1991; Geller et al., 1995; Lewis and Fisher, 2000; Lewis et al., 2002), gliosis has (at least) two components, proliferation of the Mu¨ller cells and hypertrophy and growth of their processes at both the ILM and OLM. Hypertrophy of the ILM leads to the formation of a ‘pre-retinal membrane’ which may be vascular and opaque, and blurring to vision. Hypertrophy of the outer processes of Mu¨ller cells can lead to a glial scar forming at the outer surface of the retina, disrupting the normal and functionally critical relationship between photoreceptors and RPE cells. In humans, this gliosis is called proliferative vitreoretinopathy. The glial scar may contract, producing a whole-scale detachment of the retina, and complete blindness. A major element in these responses of the Mu¨ller cells to detachment is hypoxia, caused by the separation of photoreceptors from the choriocapillaris. When oxygen availability to the detached retina is increased (by increasing the proportion of oxygen in the air inspired) these gliotic changes are greatly reduced (Lewis et al., 1999). The grossness of the morphological changes in retinal gliosis highlights the ‘minimalism’ of the normal structure of the inner and outer limiting membranes. There is no thickening of the cells, no loss of transparency; just a narrow stratum of specialized adherent points between the membranes of adjacent glial processes (astrocytes and Mu¨ller cells at the ILM, Mu¨ller at the OLM). 4.2. Hypoxia-induced breakdown of astrocytic glia limitans The reaction of astrocytes to hypoxia is to degenerate and die, rather than to proliferate, as assessed in rodent models of hypoxia (Zhang and Stone, 1997). This removes the astrocyte components of the mosaic of macroglial processes, which make up the ILM, and may contribute to the growth of blood vessels from the retina into the vitreous humor (Section 6.2).

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5. Macroglia guide blood vessel formation 5.1. Astrocytes are required for the surface spread of the retinal vasculature The close relationship of astrocytes to vessels in the adult cerebral nervous system has been known for many decades. The developmental relationship between astrocytes and blood vessels was worked out in the retina (reviewed in Stone and Maslim (1997)). There were three distinct steps in the gaining of this understanding. 5.1.1. Astrocytes are found only in vascularized retinas When antibodies to cytoskeletal macroglia-specific proteins (such as vimentin and glial fibrillary acidic protein (GFAP)) became available and were applied to adult retina, it became evident that astrocytes are found in the retina only in species in which a retinal circulation forms (Stone and Dreher, 1987; Schnitzer, 1987). In the many mammalian species in which the retina is entirely avascular, its nutrients being derived entirely from the choroidal circulation, astrocytes are not found and in partially vascularized retinas, such as the rabbit retina, astrocytes are found only in the vascularized regions. Interestingly, avascular retinas are thin (, 200 mm from OLM to ILM (Chase, 1982)), while vascular retinas are consistently thicker (. 200 mm). 5.1.2. Retinal astrocytes are immigrants Second, when the development of retinal glia was traced, it became evident that the astrocytes are not generated in the retina, but are migrants, entering the retina from the optic nerve. An early developmental sign that a retinal circulation will form is the migration of astrocytes from the optic nerve head across the surface of the retina (cat (Ling and Stone, 1988), rat (Ling et al., 1989), monkey (Gariano et al., 1996), human (Provis et al., 1997)). The astrocytes spread from the margins of the optic disc radially, towards the edge of the retina in fairly straight lines. In the cat and human, the radial symmetry of the pattern of their spread is broken by an arcuate streaming of astrocytes superior and inferior to the future area or fovea centralis. In the cat, where a markedly specialized area centralis forms a few millimeters temporal (and superior) to the optic disc, astrocytes spread to the area centralis and, with a delay, spread over it. In the primate (monkey, human) astrocytes migrate to the edge of the highly specialized region fovea centralis, but do not enter it. Correspondingly, the retinal circulation does not form at the fovea centralis, and the retina there is thin (, 150 mm). 5.1.3. Astrocytes provide a developmental template for retinal vessels The function of astrocytes to form a physical template for developing retinal vessels became evident from four observations. First, the retinal circulation develops initially as a profuse capillary bed, from which larger vessels coalesce (Michaelson, 1954); it is not formed by the growth of large vessels from which small vessels sprout. Second, astrocytes show an intrinsically epithelial behavior termed ‘contact-spacing’ (Chan-Ling and Stone, 1991). Both on the retinal surface and in vitro (Tout et al., 1993b), they spread with a strong tendency to maintain contact, but then to space their somas. This behavior, in flat,

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process-less cells, produces a classical epithelium, a self-organizing sheet of cells. In stellate cells like astrocytes, the same contact-spacing behavior produces an astrocytic network (see Figs. 3 and 8 in Stone and Maslim (1997)). Third, astrocytes spread into the retina ahead of vessels (reviewed in Stone and Maslim (1997)). They are located in the right place to guide the formation of vessels. Fourth, astrocytes secrete the key factor that makes vessels form. The developing retina becomes hypoxic when photoreceptors start to function. This hypoxia induces the astrocytes to express the potent angiogenic factor vascular endothelial growth factor (VEGF) (Stone et al., 1995a) and vessels form along processes of astrocytes. The initial capillary bed then has the loop-size set by the astrocyte network.

5.2. Mu¨ller cells and the deeper vessels of the retinal circulation Once the inner layer of the retinal vasculature has formed to the template provided by astrocytes spreading at the inner surface of the retina, Mu¨ller cells provide structural guides for the growth of vessels radially outwards from the inner surface through the inner plexiform and nuclear layers. Like astrocytes, Mu¨ller cells express VEGF, induced by hypoxia of the middle layers of retina, before the radial vessels grow, and the growth of the deep vessels is followed by suppression of VEGF expression, presumably because the oxygen carried by these new vessels counters the hypoxia, which induced the VEGF expression. The ability of Mu¨ller cell to provide a template for the deep vessels of the retinal circulation, but not for the superficial vessels, emphasizes the role of macroglial morphology in the formation of retinal vessels. The need for two classes of macroglial is clinically important (Section 8.2) and deserves additional comment.

5.3. Angiogenesis in retina and brain compared The spread of astrocytes over the inner retinal surface is an unusual phenomenon, in the context of CNS development. The inner retinal surface corresponds to the pial surface of the rest of the brain and spinal cord, where a collagenous membrane, the pial mater, provides a substrate for the growth of branches of the carotid and vertebral arteries to spread over the brain, before penetrating along radial glial guides (see Fig. 3 in Stone and Maslim (1997), see also chapters by Mercier and Hatton and by Wolff and Chao). The pia does not form at the ‘pial’ surface of the retina (the ILM), presumably for optical reasons. Radial glial guides (Mu¨ller cells) exist to guide penetrating vessels. The inner end-feet of Mu¨ller cells lack the branched morphology needed to guide vessel formation across the retina surface (Section 5.1.3) and, because they span the thickness of the retina, Mu¨ller cells are wrongly placed to sense hypoxia at the inner surface. An astrocyte population restricted to the innermost layer of the retina is ideally placed to sense hypoxia at this surface and, in response, to form a template for the spread of vessels from the hyaloid artery (Fig. 1B, C).

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6. Macroglia stabilize adult retinal vasculature 6.1. Maintenance of the blood –retinal barrier (BRB) The permeability of capillaries is determined by their microenvironment. An important component of the microenvironment of CNS vessels is their glia limitans, formed in the brain by astrocytes and in the retina by both astrocytes (in the superficial vessels) and Mu¨ller cells (Hollander et al., 1991). There is experimental evidence for both astrocytes (Janzer and Raff, 1987) and Mu¨ller cells (Tout et al., 1993a) that macroglia induce barrier properties in the vessels they contact. Although signals that induce barrier properties remain under study, most authors conclude that macroglia contribute to that induction (Risau et al., 1998; Pardridge, 1999). It is striking, for example, that, when vessels grow abnormally out of the retina, away from glial cover (for example into the vitreous humor) their pre-retinal lengths lack barrier properties (reviewed in Aiello (1997)). 6.2. Vessel maintenance The expression of the angiogenic factor VEGF by astrocytes and Mu¨ller cells is important for vessel development (Section 5.1.3). In the adult retina, VEGF expression is important for the maintenance of vessels. Exogenously applied VEGF prevents the obliteration of retinal vessels caused by hyperoxia (Alon et al., 1995). Conversely, vessel thinning and retraction are consistent feature of retinas in which the photoreceptor population is depleted, e.g., in human retinitis pigmentosa (Heckenlively, 1988). The vessel thinning may be due to hyperoxia, since it is reduced by hypoxia (Penn et al., 2000). Photoreceptor depletion causes a rise in tissue oxygen levels in the retina (Stone et al., 1999; Yu et al., 2000), and hyperoxia downregulates VEGF expression by retinal macroglia (Stone et al., 1996). 6.3. Physical restraint Retinal neovascularization (the growth of new, abnormal vessels from the retina into the vitreous humor) is a feature of diseases in which the retina becomes hypoxic, such as retinopathy of prematurity (where hypoxia results from the oxygen-induced closure of developing retinal vessels), and diabetic retinopathy (diabetes-induced capillary closure). In animal models of hypoxic retinal disease there is marked degeneration of the astrocytes of the retina (Chan-Ling and Stone, 1992; Zhang and Stone, 1997). Hypoxia per se causes marked degeneration of astrocytes. When this degeneration is severe, patches of the walls of retinal vessels are left bare of a glia limitans (Zhang and Stone, 1997). These bare patches of vessel wall are then sites at which neovasculature forms, and grows into the vitreous humor. These observations suggest that macroglial cells, by adhering to each other at the ILM (Section 3.1) and around vessels, form an on-going restraint to retinal vessels. Although it would initially seem unlikely that vessels would be attracted to grow into the vitreous humor, where there are no cells to guide them, no metabolism and no specifically hypoxic regions, it is clear empirically that vessels do grow from the retinal

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circulation into the vitreous, when the retina is chronically hypoxic. The stimulus may be an accumulation of VEGF in the vitreous humor, known to occur in neovascularizing retinal diseases (Pe’er et al., 1996; Ferrara, 1999). 7. Macroglia stabilize the photoreceptor population Photoreceptors share the stability of all central nervous system neurones; they can last the lifetime of even long-lived species like Homo sapiens. Photoreceptors are, however, the most fragile of retinal neurones, their fragility evident in the diseases known as retinal degenerations. Retinal degenerations are specific to photoreceptors, although downstream changes such as vessel thinning (Section 6.2), pigmentary invasion of the retina and death of other retinal neurones occur at late stages. They occur in response to genetic factors (‘scores of mutations in dozens of genes’ (Dryja and Berson, 1995)) and environmental factors, including metabolic poisons (Graymore and Tansley, 1959; Noell, 1958), excess of normal metabolic substrates, such as oxygen (Noell, 1958; Yamada et al., 2001) and perinatal stress (Stone et al., 2001). It has become evident in recent years that the retina expresses factors (proteins, cytokines), which stabilize photoreceptors, enabling their survival in the face of stress, and that astrocytes and Mu¨ller cells are the cellular source of these factors. 7.1. Mu¨ller cell processes wrap every retinal neurone The somas of neurones throughout the central nervous system receive contact from glial processes (see for example Chapter VI in Peters et al. (1976)). In the retina, the glial wrapping of neuronal somas is particularly complete and consistent, and is provided by the processes of Mu¨ller cells (Cajal, 1893). Even where astrocytes appear to wrap the somas of ganglion cells closely, electron microscopy shows that a Mu¨ller cell process intervenes between the astrocyte process and the neuronal soma (Holla¨nder et al., 1991; Stone et al., 1995b). 7.2. Macroglia express factors that protect photoreceptors Stress induces the expression of protective factors by retinal cells, so that photoreceptors, which survive the stress, are resistant to a subsequent episode of stress. The effect is termed ‘conditioning’ or ‘pre-conditioning’. For example, a brief exposure to high light levels (capable at longer exposures of inducing total photoreceptor degeneration) makes surviving photoreceptors highly resistant to subsequent light challenge (Liu et al., 1998; Cao et al., 1997). Even daily experience of light well within normal parameters of time (exposure only during the ‘day’) and brightness (daylight or less) causes a significant increase in the resistance of photoreceptors to acute stress (Penn and Anderson, 1991; Stone et al., 1999). An edge-specific stress, in which hyperoxia is a factor (Mervin and Stone, 2002a,b), induces the high resistance of photoreceptors at the retinal edge to both genetic and environmental stress, in probably all mammalian retinas. Laser lesions to the photoreceptor layer increase the stability of surrounding

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photoreceptors (Humphrey et al., 1993), and damage to the optic nerve in the rat increases the stress-resistance of photoreceptors throughout the retina (Bush and Williams, 1991). Tests of factors expressed in stress-resistant regions of retinas and of proteins that protect photoreceptors when applied exogenously (Cao et al., 1997; Steinberg, 1994; Wahlin et al., 2000; LaVail et al., 1998) show the importance of (at least) three factors: basic fibroblast growth factor (FGF-2), CNTF (ciliary neurotrophic factor) and BDNF (brain derived neurotrophic factor). Two of these factors, FGF-2 and CNTF, are expressed specifically by astrocytes and Mu¨ller cells (Walsh et al., 2001). Further, although in stressed retina FGF-2 becomes prominent in the somas of photoreceptors (reviewed in Stone et al. (1999); Walsh et al. (2001)), in the unstressed retina, FGF-2 and CNTF are prominent only in macroglia (Walsh et al., 2001). The distribution of these two factors within macroglial cells is strikingly different, CNTF being prominent in cytoplasm and FGF-2 in nuclei; the significance of this difference remains unknown. However, the prominence of these factors in macroglia in the absence of stress suggests that these are the cells that produce the factors.

7.3. FGF-2/FGFR1 colocalization: sites of FGF-2 action The protective effect of FGF-2 on photoreceptors is believed to be mediated by FGFR1 (Desire et al., 2000) (reviewed by chapter by Bringmann et al.). In the unstressed retina, FGF-2 is prominent in the nuclei of Mu¨ller cells and astrocytes (Walsh et al., 2001), but it can be detected in the cytoplasm of ganglion cells, and in the processes of Mu¨ller cells extending across the outer nuclear layer. Evidence of where FGF-2 acts can be gained from the localization of the FGFR1 receptor. In the rat retina, FGFR1 is expressed in two sites, in the cytoplasm of photoreceptors and in their axon terminals, where it is closely associated with synaptic vesicles (Valter et al., 2003a). The presence of a high affinity receptor for FGF-2 in axon terminals had been predicted (Lewis et al., 1996). Colocalization of FGF-2 with FGFR1 is very limited in the unstressed retina. Stress, whether caused by light or edge-specific factors, causes upregulation of FGF-2 levels, and an increase in FGF-2/FGFR1 colocalization at both sites (Valter et al., 2003a). It is not yet established whether the FGF-2, which accumulates in photoreceptor somas following stress, is generated by the photoreceptors, or is transferred from Mu¨ller cells. One piece of evidence supports the latter possibility. When the time course of FGF-2 upregulation following the onset of bright light exposure is examined in detail it is evident (Walsh and Stone, 2001) that FGF-2 upregulation in Mu¨ller cells precedes its accumulation in photoreceptors by several hours. This issue deserves further analysis, however. In summary, the colocalization of FGF-2 and FGFR1 suggests ligand/receptor binding. The stress-induced increase in this colocalization at two sites in the photoreceptor suggests that these sites are functionally important. We have suggested (Valter et al., 2003a) that the synaptic vesicles in the axon terminal of photoreceptors are the site at which FGF-2 affects the b-wave of the ERG (Gargini et al., 1999), and that the photoreceptor cytoplasm

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Fig. 2. Colocalization of CNTF and CNTFRa in rat retina. In each of A–D, labeling for CNTF is green and for CNTFRa is red. The left panel shows CNTF labeling along the outer segment region of photoreceptors, and the asterisk shows the level of the OLM. The panel second from left shows CNTFRa labeling over the same region,

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is the site at which FGF-2 activates pathways which protect photoreceptors from stress. Detail of those pathways remains unknown, however. 7.4. CNTF, CNTFRa CNTF is a cytokine, one of a family which includes IL-6 and LIF, which share a common structure (4a-helices, , 20 kDa) and all signal through an intracellular pathway, the gp130/Jak/STAT pathway (Ip and Yancopoulos, 1996; Shelton, 1996) (see also chapter by Nakagawa and Schwartz). CNTF is prominent in the retina, in the cytoplasm of astrocytes and Mu¨ller cells (Walsh et al., 2001). The receptor for CNTF contains three major elements, known as LIFR, gp130 and CNTFRa (Hirano et al., 1997; Inoue et al., 1996). LIFR and gp130 contain the signaling elements; CNTFRa is the CNTF-specific element and is useful (since gp130 and LIFR are components of receptors for other CNTFlike cytokines) to identify the location of the receptor complex for CNTF. In the unstressed retina, CNTF was found throughout the cytoplasm of Mu¨ller cells, and CNTFRa was prominent specifically on the outer segments of photoreceptors (red in Fig. 1D – J). Using high-resolution confocal microscopy, CNTF þ granules could be found in the vicinity of the outer segments (left panel in Fig. 2A), but colocalization of CNTF and CNTFRa suggestive of ligand/receptor binding was not prominent (right panel in Fig. 2A). In stressed retina, CNTF protein expression was upregulated in Mu¨ller cells (Fig. 1K) (Chun et al., 2000; Walsh et al., 2001; Valter et al., 2003a) and CNTF/CNTFRa colocalization along outer segments became prominent (right panels in Fig. 2B –D). The stress-induced colocalizaton of CNTF and CNTFRa at the outer segment suggests that this is the site of the protective action of CNTF on photoreceptors. However, the mechanism by which CNTF reaches the outer segment, presumably from Mu¨ller cells, and the nature of the mechanisms activated by CNTF/CNTFRa binding at the outer segment remain unknown. 7.5. Mu¨ller cells provide metabolic protection for photoreceptors Photoreceptors are highly metabolically active, reviewed in Stone et al. (1999); particularly when dark-adapted their production of lactate (the end product of glycolysis) (Cohen and Noell, 1960) and their consumption of oxygen (Ames et al., 1992; Linsenmeier, 1986) are markedly higher than for any other neurones of the CNS. Despite these high requirements, the photoreceptor population is remarkably resistant to hypoxia and the panel third from left shows the CNTF- and CNTFRa-labeling superimposed. The right panel shows pixels that are yellow, because labeling is strong for both CNTF and CNTFRa, suggesting colocalization of ligand and receptor. (A) In the midperipheral region of a normal retina, the retina is unstressed. CNTF þ granules and CNTFRa þ granules are detectable in outer segments, but few colocalize (i.e., few yellow pixels in the right panel). (B). In a retina stressed by bright intense light (1000 L for 24 h) colocalization appears stronger on the outer segments of surviving photoreceptors (yellow pixels in right panel). (C) In photoreceptors near the edge of a normal retina, a region subject to oxidative stress (Mervin and Stone, 2002a,b), colocalization is stronger than in the midperipheral retina (yellow pixels in right panel). (D) In retina stressed by damage to the optic nerve 7 day previously, CNTF/CNTFRa colocalization also appears along outer segments (yellow pixels in right panel).

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and hypoglycaemia. In particular, the electroretinographic response of photoreceptors (the a-wave) is more durable than the response of inner retina (the b-wave) in the face of both hypoxia and hypoglycaemia (Winkler, 1983, 1995). Kang Derwent and Linsenmeier (2000) have confirmed the resistance of the a-wave to hypoxia. The mechanism of the photoreceptors’ resistance to the lack of oxygen or glucose was identified by the systematic study of retinal ATP production (Winkler, 1995). This demonstrated that aerobic and nonaerobic metabolic capacities of the retina are both high, allowing the retina to switch its pathway for ATP production, in hypoglycaemic conditions to predominantly oxidative production, and in hypoxic conditions to predominantly anaerobic production. The high aerobic capacity of photoreceptors resides in the concentration of mitochondria in their inner segments. The high glycolytic capacity associated with photoreceptors, however, may involve Mu¨ller cells. Photoreceptors show measurable glucose consumption and lactate production and early workers (Cohen and Noell, 1960) assumed that the lactate is produced by glycolysis in the perikaryal region of photoreceptors. However, there is evidence (Poitry-Yamate et al., 1995) that Mu¨ller cells produce lactate which is constantly transferred to photoreceptors for oxidative metabolism, suggesting that photoreceptors do not produce sufficient lactate to fuel their rapid oxidative phosphorylation, and rely for their full needs on production by Mu¨ller cells. The hypothesis that Mu¨ller cells underwrite the metabolic needs of photoreceptors by providing a take-as-necessary supply of pyruvate/lactate has proved attractive (Reichenbach et al., 1993). It gains support from evidence that glycogen and glycogen phosphorylase are prominent in Mu¨ller cells, but not in photoreceptors (Rungger-Bra¨ndle et al., 1996; Arroyo et al., 1997; Pfeiffer et al., 1994). A rise in extracellular [Kþ] causes Mu¨ller cells to hydrolyze glycogen to glucose (Reichenbach et al., 1993). It is possible then that ionic movements linked to photoreceptor signaling serve as a signal to mobilize glucose metabolism in Mu¨ller cells. Conversely, mitochondria are found in Mu¨ller cells but in vascularized retinas such as those of the human, monkey and rat, mitochondria are sparse in the Mu¨ller cell processes that wrap photoreceptors (reviewed in Rasmussen (1974)). This suggests that Mu¨ller cells do not compete with photoreceptors for oxygen diffusing from the choriocapillaris, but (arguably) limit their metabolism of glucose to glycolysis, and therefore release lactate. These hypotheses deserve further testing. 8. Implications for therapy The above considerations have several novel implications for the treatment of retinal disease. 8.1. Immediate oxygen for patients with retinal detachment The photoreceptor degeneration and glial proliferation that occur in the retina when it is detached are largely eliminated (at least in a feline model of detachment (Lewis et al., 1999; Mervin et al., 1999)) by supplemental oxygen. The implication is clear

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that patients with retinal detachment should do better if given supplemental oxygen between diagnosis and surgery. In the present context, the prevention of Mu¨ller cell proliferation and hypertrophy should ensure a more normal, stable and functional retina.

8.2. Neovascularizing diseases of the retina: the key importance of astrocyte survival At least in animal models, astrocytes are markedly sensitive to hypoxia, and their death can strip the glia limitans from superficial vessels and disrupt the ILM (Chan-Ling and Stone, 1992; Zhang and Stone, 1997). This loss of astrocytes is permissive for the sprouting of new vessels from the old, the new vessels forming at sites of ILM breakdown and growing through the defects into the vitreous humor. In hypoxic retinal disease the vitreous humor is primed with the potent angiogenic factor VEGF (Pe’er et al., 1995, 1996; Ferrara, 1999), probably leached into the vitreous from the retina, where hypoxia upregulates its expression. The implication is that intervention in such diseases could be usefully aimed ‘upstream’, at relieving retinal hypoxia, rather than downstream, for example to competitively inhibit VEGF receptors, or to block apoptotic mechanisms. Blocking the action of VEGF should prevent new vessel formation (Aiello, 1997; Penn et al., 2001), but would be less likely to prevent astrocyte death and the breakdown of the glia limitans. Blocking apoptotic mechanism should preserve the astrocyte population, but would not relieve the hypoxia-induced rise of VEGF in the retina and vitreous humor. The informed use of oxygen to prevent both astrocyte death and VEGF upregulation deserves testing in the treatment of such diseases (retinopathy of prematurity, venous occlusive disease, diabetic retinopathy).

8.3. Photoreceptor degenerations: the trade-off between neuronal protection and retinal sensitivity Knowledge of ability of retinal macroglial cells, especially Mu¨ller cells, to provide protective factors to photoreceptors should prove of value in the management of human photoreceptor degenerations (retinitis pigmentosa). The factors are produced by the retina, and one of their normal functions is to stabilize photoreceptors, for example, at the edge of normal retina (reviewed in Stone et al. (1999); Mervin and Stone (2002a)). The exogenous application of these factors, by injection into the vitreous humor (Faktorovich et al., 1992; Steinberg, 1994; Gargini et al., 1999) does slow degenerations, as does the upregulation of their retinal expression by viral vectors (Bok et al., 2002). However, the side effects are considerable, and include a loss of retinal function, measured as a reduction in the amplitude of the electroretinogram (Gargini et al., 1999; Bok et al., 2002). Nevertheless, the possibility of slowing degenerations by management of the expression of these factors remains an attractive potential therapy.

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9. Concluding remarks Retinal macroglia (astrocytes, Mu¨ller cells) play diverse roles in the homeostasis of the retina. This review has focused on their roles in stabilizing the vasculature of the retina, and in ensuring the long-term survival of neurones, particularly photoreceptors. The mechanisms involved are diverse. Some, such as the formation of the glia limitans of the retina and vessels, involve epithelial properties of macroglia, their specific adhesion to each other. Others are paracrine, the macroglial cells providing factors (proteins, cytokines), which have powerful effect on the behavior of endothelial cells or the stability of photoreceptors. Other mechanisms are metabolic and biochemical, the ability of Mu¨ller cells, for example, to contribute to the energy metabolism of photoreceptors. This diversity reflects the complexity of neurones and neural systems, such as the retina. Understanding that complexity opens avenues to therapy of still-intractable diseases of the retina.

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Function and dysfunction of enteric glia Tor C. Savidge,a,* Julie Cabarrocasb and Roland S. Liblaub,c a

Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA p Correspondence address: Tel.: þ1-617-7264169; fax: þ1-617-7264172 E-mail: [email protected](T.C.S.) b Institut National de la Sante´ et de la Recherche Me´dicale U546, Pitie´-Salpeˆtrie`re Hospital, Paris 75013, France c Laboratoire d’Immunologie, CHU Toulouse, France

Contents 1. 2. 3.

4. 5. 6.

Introduction Morphology and origin of enteric glia Physiological functions of enteric glia 3.1 Supply of trophic and nutritional factors 3.2 Potassium (Kþ) homeostasis 3.3 Glutamate and GABA 3.4 Nitric oxide, calcium and transmitters Pathological consequences of EGC dysfunction EGC and immunopathology of the gut Concluding remarks

Enteric glial cells (EGC) of the gastrointestinal tract constitute an extensive population of the enteric nervous system (ENS), and they show morphological and functional similarities to CNS-derived astrocytes. EGC provide trophic and cytoprotective functions towards enteric neurons, and are likely to be actively involved in regulation of neuronal activity. Transgenic models of EGC ablation have confirmed this neuroprotective role within the ENS, in addition to demonstrating a role for EGC in regulating intestinal immune responses. In a fashion akin to astrocytes, EGC are able to present antigens to T-lymphocytes, and may contribute to mucosal cytokine profiles that result in intestinal inflammation. EGC also perform other novel functions, such as directly regulating mucosal and vascular integrity, and as such may be an important target cell in inflammatory and permeability disorders of the gastrointestinal tract.

Advances in Molecular and Cell Biology, Vol. 31, pages 315–328 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction Gastrointestinal tissues are innervated by a highly complex and extensive component of the peripheral nervous system (PNS), known as the enteric nervous system (ENS) (Furness and Costa, 1987; Gershon and Rothman, 1991). Here enteric neurons control several aspects of gastrointestinal function, including motility, microcirculation of blood vessels, and mucosal secretion of fluid, ions and biologically active gut-derived factors. Although the ENS is regarded as an important component of the PNS, it differs substantially from its other family members. Notably, in addition to forming an integrated extension of the central nervous system (CNS), the ENS is also capable of performing reflex activities independently of the CNS (Gershon et al., 1994). Enteric neurons are clustered into two extensive, interconnected ganglionated plexi, comprising Meissner’s submucosal plexus and Auerbach’s myenteric plexus, respectively. Another unique feature of the ENS is that structural support for enteric neurons is provided exclusively by specialized enteric glial cells (EGC). This chapter will review the tentative functions of EGC, and will also consider the consequences of EGC dysfunction or ablation in the gastrointestinal tract. 2. Morphology and origin of enteric glia Multipotent neural crest-derived stem cells that colonize mouse embryonic gut at embryonic day (ED) 9.0 during development act as progenitor cells for both enteric neurons and EGC (Yntema and Hammond, 1954; Le Douarin and Teillet, 1973; Burns and Le Douarin, 1998). Cell-lineage specific markers have demonstrated that enteric neurons during development appear before differentiated EGC, which first appear at ED 16 (Rothman and Gershon, 1982; Rothman et al., 1986). Several molecular signals are able to distort cell lineage commitment to EGC, however, including neuropoietic cytokines, neurotrophic growth factors, glial-derived neurotrophic factor (GDNF), neurotrophin-3 and neuregulins (Gershon, 1998). Unlike the CNS and most other components of the PNS, neuronal plexi of the ENS consist exclusively of enteric neurons and EGC. The ENS lacks conventional Schwann cells, and contains no connective tissue or blood vessels that supply enteric neurons directly (Cook and Burnstock, 1976a,b; Gabella, 1972a,b; Thomas and Ochoa, 1984). Rather, structural and functional support for enteric neurons is provided by the EGC, which closely embrace neuronal cell bodies and processes. To achieve this neurosupportive function, EGC greatly outnumber enteric neurons and show similarities to CNSderived glia. Morphologically, EGC closely resemble astrocytes. They do not synthesize myelin (Cook and Burnstock, 1976b; Gabella, 1971; Gershon and Rothman, 1991), and they possess swollen end-feet processes that contact juxtaposed blood vessels and neighboring ganglia (Erde et al., 1985; Hanani and Reichenbach, 1994). EGC morphology is dependent on whether the cells are located within the ganglia, in which case they are star-shaped with short processes, or whether they are inter-ganglionic, in which case they have longer processes (Hanani and Reichenbach, 1994). Although EGC resemble astrocytes more closely than Schwann cells (these tend to possess a more uniform cell shape and a larger cell body), they perform a similar structural support role for neurons. However, rather than secreting a laminin- and collagen-based

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basal lamina to support neurons (Bannerman et al., 1986), EGC individually sheath several enteric neurons, thereby offering direct plasma membrane contact for structural and functional support (Gershon and Rothman, 1991). EGC also harbor other features in common with astrocytes that distinguish them from myelinating Schwann cells. For example, EGC and astrocytes abundantly express glycogen granules, L -arginine (Nagahama et al., 2001), type III intermediate filaments, glial fibrillary acidic protein (GFAP) and vimentin (Cook and Burnstock, 1976b; Gabella, 1987; Komuro et al., 1982; Jessen and Mirsky, 1980). Other distinguishing markers such as glutamine synthetase (Jessen and Mirsky, 1983) and calcium-binding protein S100 (Bishop et al., 1985; Ferri et al., 1982; Kobayashi et al., 1986; Scheuermann et al., 1989) are also evident. Although these distinguishing features may be used to delineate EGC from myelinating Schwann cells, less profound differences are found between EGC and nonmyelinating Schwann cells, which appear to be a more closely related cell type that differentiates into a distinct cell lineage relatively late during development (Dulac and Le Douarin, 1991). Markers that distinguish nonmyelinating Schwann cells from EGC in situ include expression of Schwann cell myelin protein (SMP) (Dulac et al., 1988) and rat neural antigen-1 (Ran-1) in nonmyelinating Schwann cells (Brockes et al., 1977; Fields et al., 1975), whereas Ran-2 is expressed on EGC (Bartlett et al., 1981) together with glutamine synthetase (Kato et al., 1990). However, gene expression is influenced somewhat by the local intestinal microenvironment, as EGC display Schwann cell-specific genes when cultured in vitro (Dulac and Le Douarin, 1991; Jessen and Mirsky, 1983; Bannerman et al., 1988). Specific lineage markers are therefore not available to identify EGC. This problem is complicated further by the substantial heterogeneity that is evident amongst EGC when studying these cells using morphology, immunohistochemstry or electrophysiology (Bernstein and Vidrich, 1994; Hanani and Reichenbach, 1994; Albrechtsen et al., 1984; Jessen et al., 1984; Broussard et al., 1993; Hanani et al., 2000). 3. Physiological functions of enteric glia Many similarities that the EGC and CNS-derived astrocytes share suggest that they may perform similar physiological roles. This is supported further by dye-coupling studies demonstrating that the EGC network is interconnected via gap-junctions in a similar way as astrocytes (Gutnick et al., 1981), allowing intercellular communication over extensive distances in the gastrointestinal tract (Hanani et al., 1989; Maudlej and Hanani, 1992). Traditionally held views, previously regarded glial cells as passive neurosupportive cells. More recent functional studies have demonstrated that glia, including EGC, also participate in the complex regulation of neuronal function and survival. 3.1. Supply of trophic and nutritional factors Glial-derived trophic factors are important for ENS development. Notably, GDNF is required for proliferation, differentiation and survival of enteric neurons, as is indicated by the lack of a functional ENS in GDNF null mice (Moore et al., 1996). Therefore, it is likely that EGC are involved in the development and survival of enteric neurons. Although other

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cells may provide alternative sources of GDNF in utero (Ba¨r et al., 1997; Hellmich et al., 1996), EGC-derived GDNF is the most likely source involved, as the characteristically large plasma membrane of EGC constitutes an effective permeability barrier that regulates substrate and metabolite exchange with enteric neurons (Maudlej and Hanani, 1992; Hanani and Reichenbach, 1994). Because of this ‘gut –brain’ barrier, EGC also control the immediate neuronal extracellular microenvironment, as blood vessels are absent from enteric neuronal plexi (Gershon and Bursztajn, 1978; Gabella, 1987). EGC therefore acts as the major cellular intermediary, providing for perineural nutrition and homeostasis. For example, EGC might supply glucose to neurons from stored glycogen granules in a fashion akin to what has been suggested for astrocytes (Cataldo and Broadwell, 1986; Koizumi, 1974). 3.2. Potassium (Kþ) homeostasis EGC also plays a pivotal role in buffering potentially excitotoxigenic mediators that are formed during neural activity. For example, perisynaptic potassium ions (Kþ) produced during neural activity need to be strictly controlled to facilitate subsequent synaptic signaling. In the retina, excess extracellular Kþ released during neuronal activity is removed via inward acting potassium channels, and eventually siphoned into the vitreous humor and the capillaries (Newman, 1986, 1993; Newman et al., 1984—see also chapters by Bringmann et al. and by Scemes and Spray). A similar mechanism may be operating in cerebral astrocytes, although Kþ homeostasis by brain cortex astrocytes mainly is dependent upon active transport mechanisms and efflux across the blood – brain barrier is very slow (see chapter by Walz). EGC express voltage-activated inward and outward acting potassium channels (Broussard et al., 1993; Orkand et al., 1966; Verkhratsky and Steinha¨user, 2000; Hanani et al., 2000), which may facilitate Kþ uptake in regions with elevated extracellular Kþ, followed by shedding of excess Kþ via the EGC syncytium into areas of low extracellular Kþ concentrations, and perhaps even into capillaries. 3.3. Glutamate and GABA Another feature of glial cells in general that relates to monitoring of neuronal activity and perineural homeostasis is the detoxification of neurotransmitters glutamate and g-aminobutyric acid (GABA), and of waste ammonia by glutamine synthetase (Cooper and Plum, 1987; Erecinska et al., 1986; Hertz, 1979; Kaneko et al., 1988). Within the ENS, the expression of glutamine synthetase is restricted to EGC (Kato et al., 1990). As such, EGC are not only likely to be involved in detoxifying glutamate and GABA, but they also provide a source of glutamine to neurons for glutamate and GABA synthesis (see chapter by Schousboe and Waagepetersen). 3.4. Nitric oxide, calcium and transmitters EGC are also the only cells to express L -arginine within the ENS, and may transfer this directly to enteric neurons (Nagahama et al., 2001). This represents an important substrate

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requirement for nitric oxide (NO) synthesis and, thereby is important for the neuronal intercellular signaling that occurs in the numerous NO synthase (NOS) expressing neurons that are located within the ENS (Costa et al., 1992; Ekblad et al., 1994; Llewellyn-Smith et al., 1992; Timmermans et al., 1994). In this way, EGC may indirectly regulate neuronal activity within the ENS. However, similar to the situation in the brain cortex (see chapter by Garcia and Baltrons), NO signaling is also important in EGC themselves (Sarosi et al., 1998). Regulation of ENS activity by EGC is also suggested by the presence of immunoreactive substance P and K (Bernstein and Vidrich, 1994), and by the induction of fos following vagal cholinergic stimulation of the ENS (Miampamba et al., 2001). EGC also respond physiologically, by elevating intracellular calcium concentrations, in response to various agonists that regulate ENS activity (Zhang et al., 1997), such as ATP (Sarosi et al., 1998) and endothelin family members (Zhang et al., 1997). Therefore, it would appear that EGC, as has been demonstrated for astrocytes (Araque et al., 2001), are active and responsive participants in the integration and modulation of ENS activity, as is summarized in Fig. 1.

4. Pathological consequences of EGC dysfunction Recent transgenic studies, specifically targeting EGC in mice has demonstrated that this cell population plays an important role not only in maintaining the integrity of the ENS, but also that of other tissues of the gastrointestinal tract. Conditional ablation of EGC utilizing two different transgenic approaches consistently resulted in the development of a fulminant intestinal inflammation and necrosis (Bush et al., 1998; Savidge et al., 1999; Cornet et al., 2001; Cabarrocas et al., 2003). Disease progression in these transgenic mice began with a breakdown of mucosal and/or vascular integrity, indicating that EGC also play a role in regulating the permeability of mucosal and vascular interfaces (Savidge et al., 2003; Bush et al., 1998; Cornet et al., 2001; Cabarrocas et al., 2003). The first of these transgenic models utilized the mouse GFAP promoter to express the herpes simplex virus thymidine kinase gene (HSVtk) (Bush et al., 1998). By treating these mice with a continuous infusion of the antiviral drug ganciclovir, proliferating EGC that express the HSVtk transgene may be specifically ablated in the gastrointestinal tract. Intracerebral astrocytes are not affected, because ganciclovir does not easily cross the blood – brain barrier. Specific EGC ablation is caused by the metabolism of ganciclovir by thymidine kinase, resulting in the production of toxic nucleotide intermediates that trigger cell death by interfering with DNA replication. HSVtk negative cells are unable to metabolize the drug, and therefore EGC may be specifically targeted with little or no toxic bystander effects on neighboring nontransgenic cells. Consequently, enteric neurons remain intact until secondary effects of intestinal inflammation and ischemic tissue damage ensue. However, even under these conditions gut motility and autonomic or sympathetic neuropeptide expression remains unaltered. Pathology in these GFAP – HSVtk transgenic mice is predominantly found in the distal small intestine and ganciclovir treatment is invariably fatal within 3 weeks from its initiation. Moribund animals characteristically display a patchy, but severe inflammation

320 T.C. Savidge et al. Fig. 1. Schematic illustration of EGC function. Enteric astrocytes form a barrier between the gut and the enteric neurons, enabling them to regulate neuronal supply of many essential substrates and factors. They release growth factors, such as GDNF, which are important for proliferation, differentiation and survival of enteric neurons. They might also supply neurons with glucose (or glucose metabolites) from glycogenolysis of contained glycogen stores. In contrast to enteric neurons, they express arginine, an essential precursor for synthesis of NO, and they may accordingly regulate synthesis of NO by the numerous NO synthetase expressing neurons in the ENS. They are the only cells in the ENS that express glutamine synthetase activity, necessary for detoxification of ammonia and continued glutamate supply to neurons. They express Kþ channels, indicating a role in Kþ homeostasis.

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and necrosis associated with transmural perforation and severe gastrointestinal bleeding. Initial signs of pathology are observed at the tips of villi and involve a loss of epithelial integrity. Vascular abnormalities are also commonplace and often include the formation of intravascular microthrombi in the lamina propria capillaries, resembling vascular summit lesions described in Crohn’s disease, a human inflammatory bowel disorder (Wakefield et al., 1991). The preferential ablation of EGC in the small intestine is probably a reflection of the high proliferation levels that are observed in this organ. We previously assumed that the high antigenic burden of the gastrointestinal tract also contributes to the intestinalspecific inflammation that is observed in this model (Bush et al., 1998; Savidge et al., 1999). However, sterile transgenic ectopic grafting studies into severe-combined immunodeficient mice, and studies using germ-free GFAP –HSVtk transgenic mice have now demonstrated that the gut flora is not essential in triggering intestinal inflammation and necrosis (Savidge et al., 2002). A different transgenic approach of ablating EGC yielded a similar finding (Cornet et al., 2001), indicating that the intestinal pathology observed in GFAP – HSVtk transgenic mice is due to a compromised ENS rather than being a specific feature of the suicide gene strategy as such. This model utilizes double-transgenic mice, in which EGC are targeted by cytotoxic T-lymphocytes. This was achieved by breeding mice that are transgenic for the influenza virus hemagglutinin (HA) receptor under control of the GFAP promoter (GFAP – HA), with another transgenic line (CL4-TCR) in which a HA-specific, Major Histocompatibility Complex (MHC) class I-restricted T-cell receptor (TCR) is expressed on 95% of the CD8þ T-cells. The resultant double-transgenic F1 offspring (GFAP –HA £ HA –TCR) develop a fulminant ileo-jejuno-colitis within the first week of life due to CD8þ T-cell infiltration of the gastrointestinal tract, and the subsequent autoimmune targeting of HA-expressing transgenic EGC. The submucosal plexus is initially targeted because of its close proximity to blood vessels that act as conduits for the autoreactive T-cells. The first evidence of pathology in these mice is a T-cell mediated apoptosis of EGC that progresses into a submucosal vasculitis. Fulminant intestinal inflammation subsequently results from a breakdown of the mucosal epithelial barrier and an induction of a type-1 cytokine response (Cornet et al., 2001). A noteworthy finding in this model is that disease progression mimics the early pathological manifestations of Crohn’s disease, which include T-cell infiltration of myenteric plexi, enteric neuronal pathology and vascular disturbances (D’Haens et al., 1998; Geboes and Collins, 1998; Wakefield et al., 1991). Interestingly, a compromised EGC network that responds poorly to inflammatory stimuli has also been demonstrated in Crohn’s disease patients (Cornet et al., 2001). Whether EGC in Crohn’s disease patients are also subject to a similar autoimmune targeting therefore merits further study. Autoimmune targeting of other cellular components of the ENS has already been described in patients with paraneoplastic and Chagas disease-associated intestinal pseudoobstruction, both of which show similarities to other T-cell-mediated paraneoplastic neurological disorders (Albert et al., 1998; Benyahia et al., 1999). It is of interest that ablation of EGC leads to a disruption of the mucosal barrier in both model systems, suggesting a novel role for EGC in maintaining the integrity of mucosal surfaces and in regulating their permeability. This hypothesis is consistent with the observation that EGC extend processes to the mucosal crypts and to the tips of the villi,

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ensheathing neuronal processes involved in mucosal physiological events. Whether EGC are involved in the regulation of epithelial permeability indirectly via their neuroprotective functions or directly via contact with epithelial cells is not known, although a recent study has demonstrated that EGC may directly promote intestinal epithelial cell integrity by altering the expression of specific tight-junction associated proteins occludin and ZO-1 (Savidge et al., 2003). In addition, in both models of selective depletion of EGC, early vascular abnormalities were also observed in the intestinal mucosa and submucosa, suggesting that EGC, through their contacts with local blood vessels (Hanani and Reichenbach, 1994), may also be important regulators of vascular function in the gastrointestinal tract. 5. EGC and immunopathology of the gut Transgenic models and studies of tissues from human inflammatory bowel disease have clearly demonstrated that ablation of EGC may be associated with intestinal immunopathology. However, it is not clear at this stage whether EGC directly regulate intestinal immune responses. Drawing a parallel to CNS-derived astrocytes, a plethora of literature now exists to demonstrate that these cells are potent regulators of local immune responses, and together with microglia they are often the first cells to respond to trauma, infection and inflammation (Ridet et al., 1997). Activation of astrocytes involves a characteristic proliferative and hyperplastic glial cell reaction known as ‘astrogliosis’ (see chapter by Kalman), that is designed to protect neurons, restrict tissue inflammation and maintain blood – brain barrier function (Eddleston and Mucke, 1993; Bush et al., 1999). A fine balance needs to be maintained as astrocytes are also required to promote inflammatory and T-cell mediated responses that protect the CNS against, for example, infection. It is not inconceivable that EGC may perform similar immunoregulatory and tissue protective roles in the gastrointestinal tract. EGC actively proliferate during experimental inflammatory bowel disease, showing similarities to astrogliosis (Bradley et al., 1997). In addition, EGC gliosis has been described in patients with inflammatory bowel disease (Cornet et al., 2001) and ENS inflammatory disease (Lhermitte et al., 1980). Several studies have demonstrated that EGC may directly regulate local intestinal immune responses. For example, although EGC do not express MHC class II molecules constitutively, the proinflammatory cytokines tumor necrosis factor (TNF)-a and interferon-g (IFN-g) readily upregulate its expression, as well as that of intercellular adhesion molecule-1 (Hollenbach et al., 2000). Expression of MHC class II molecules on EGC is upregulated during human inflammatory bowel disease (Geboes et al., 1992;

Fig. 2. Schematic illustration of EGC responses during gut inflammation. Enteric glia cells do not normally express MHC class II molecules constitutively, but proinflammatory cytokines upregulate its expression as well as that of ICAM-1. ECG also express substance P, which exerts potent proinflammatory effects on cells from the immune system, and they respond to inflammatory stimuli with the production of proinflammatory cytokines, such as IL-1, IL-6 and TNF-a. As a result both epithelial barrier and vascular barrier functions of the gut may become impaired.

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Koretz et al., 1984), and ultrastructural studies of Crohn’s disease lesions have demonstrated infiltrating T-cells establishing contact with MHC class II-expressing EGC (Geboes et al., 1992). Transgenic mice expressing HA viral epitopes from the GFAP promoter have clearly demonstrated that EGC can present a ‘neo-self’ antigen via MHC class I molecules to autoreactive CD8 þ T-cells (Cornet et al., 2001). These observations strongly suggest that EGC can act as potent antigen-presenting cells in vivo. EGC also secrete and respond to numerous immune mediators. For example, EGC express substance P, which exerts potent proinflammatory effects on macrophages, lymphocytes and mast cells. IL-1a, IL-1b, IL-6 and TNF-a are upregulated in the myenteric plexus of rats following gut inflammation induced by the parasite Trichinella spiralis (Khan and Collins, 1994). Early induction of IL-1 b may trigger the subsequent release of other cytokines, causing neuronal dysfunction in this model (Hurst and Collins, 1993; Main et al., 1993; Ru¨ehl and Collins, 1997). In vitro studies have demonstrated that EGC have functional cytokine receptors. Notably, IL-1b suppresses EGC proliferation, whereas IL-10 has a dose-dependent influence on cell division (Ru¨ehl et al., 2001b). EGC also produce IL-6 in response to IL-1b (Ru¨ehl et al., 2001a) and, as such, may play a role in regulating type-1 (inflammatory) versus type-2 (immunomodulatory) cytokine profiles during inflammatory bowel disease (Desreumaux et al., 1997). These findings indicate that EGC respond to inflammatory stimuli, notably via the production of proinflammatory mediators, and support the view that EGC are candidate regulators of intestinal immunopathology and tissue integrity (Fig. 2). 6. Concluding remarks This chapter has reviewed known and potentially novel functions of EGC. Morphological and functional similarities of EGC and CNS-derived astrocytes, confers an ability of EGC to impart protective and trophic regulation of enteric neurons, as well as control of neuronal activity. Recent transgenic studies have demonstrated that EGC ablation or dysfunction leads to intestinal vasculitis and inflammation. Hence, EGC are also important regulators of mucosal immune responses, and of mucosal and vascular permeability in the gastrointestinal tract. Acknowledgements This work was supported by the Crohn’s and Colitis Foundation, the National Institutes of Health (DK33506; DK40561), INSERM, the French MS society (ARSEP) and the French Research Ministry for grant support. References Albert, M.L., Darnell, J.C., Bender, A., Francisco, L.M., Bhardwaj, N., Darnell, R.B., 1998. Tumor-specific killer cells in paraneoplastic cerebellar degeneration. Nat, Med 4, 1321–1324. Albrechtsen, M., Von Gerstenberg, A.C., Bock, E., 1984. Mouse monoclonal antibodies reacting with human brain glial fibrillary acidic protein. J. Neurochem. 42, 86– 93.

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Schwann cell interactions with axons and CNS glial cells during optic nerve regeneration Mari Dezawa Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan Correspondence address: Tel.: þ 81-75-753-4343; fax: þ 81-75-751-7286. E-mail: [email protected]

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction The role of Schwann cells in PNS regeneration Neuronal and glial responses in injured CNS Mechanisms of optic nerve regeneration by Schwann cell transplantation Reconstruction of optic nerve circuit by Schwann cell transplantation Influence of Schwann cells on optic nerve glial cells Concluding remarks

One of the possible strategies for eliciting central nervous system regeneration is to adopt favorable properties through the transplantation of Schwann cells known to support axonal regeneration. This chapter describes the nature of the direct and dynamic communication that exists between the regenerating axon and the Schwann cells, which could play an important role in optic nerve regeneration. In addition, it describes changes and responses among the optic nerve glial cells, triggered by the transplantation of Schwann cells, which may also be associated with the mechanisms of regeneration.

1. Introduction Only little structural and functional regeneration of the central nervous system (CNS) occurs spontaneously following injury in adult mammals (Aguayo, 1985). In contrast, the ability of the mammalian peripheral nervous system (PNS) to regenerate axons after injury is well documented (Hall, 2001). Studies in the past decade have shown that the Schwann cell, one of the most important components of the peripheral glia that forms myelin, plays a key role during the process of regeneration. The proliferation and activation of Schwann Advances in Molecular and Cell Biology, Vol. 31, pages 329–345 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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cells leads to the production of various kinds of factors and other related molecules, so that the axons of the proximal nerve stump grow through the distal stump in close contact with these Schwann cells (Martini, 1994; Hall, 2001). The difference in abilities of the CNS and PNS to regenerate can largely be explained by neuronal and glial differences. There are some reports claiming that responses to the trophic signals differ between CNS and PNS neurons following injury. As to the glial cells, Schwann cells strongly promote growth of regenerating axons, whereas CNS glial cells actively inhibit re-growth. A number of factors are thought to contribute to this lack of recovery, including formation by astrocytes of a restraining glial scar, formation by oligodendrocytes of inhibitory myelin products, insufficient support of growth factors, and lack of permissive substrates for axonal growth (Dezawa et al., 1999; Schwartz et al., 1999; Lacroix and Tuszynski, 2000; Asher et al., 2001; Fournier and Strittmatter, 2001—see also chapter by Kalman). However, remarkable recent progress in the neurosciences has revealed that CNS neurons in principle have the ability to regenerate, a regeneration which under adequate circumstances can be triggered by proper artificial treatments (Iwashita et al., 1994; Quan et al., 1999; Fouad et al., 2001; Jones et al., 2001). Thus, one strategy for eliciting CNS regeneration is to adopt favorable properties, which can be achieved by providing neurotrophic factors and through the transplantation of cells known to support axonal regeneration. Above all, the Schwann cell is a strong candidate for transplantation, because CNS axons are known to regenerate, when the usual glial milieu is experimentally replaced by Schwann cells and/or peripheral nerve segments (So and Aguayo, 1985; Raisman, 1997). Indeed, several experiments, involving the spinal cord and some other areas of the CNS, have shown that either the injection of Schwann cells or transplantation of a polymer tube filled with cultured Schwann cells, can improve axonal growth across the injured site (Bunge, 1994; Harvey et al., 1995; Negishi et al., 2001; Oudega et al., 2001). This chapter describes the interaction between regenerating axons and Schwann cells during optic nerve regeneration, induced by transplantation of PNS and/or Schwann cells in the adult rat. The optic nerve is a readily accessible CNS tract that mostly contains glial cell bodies and processes as well as axons arising from retinal ganglion cells (RGCs). The relative anatomical simplicity has made it a valuable object for the investigation of axon – glia interactions during regeneration. Through selected examples evidence will be presented regarding the nature of the direct and dynamic communication that exists between the regenerating axon and the Schwann cells, which could play an important role in regeneration. In addition, changes and responses among the optic nerve glial cells, triggered by the transplantation of Schwann cells are described, since they may also be associated with the mechanisms of regeneration.

2. The role of Schwann cells in PNS regeneration In the PNS, injury produces a cascade of cellular and molecular events in the nerve stump distal to the injury site, which is known as Wallerian degeneration (based on the original observations by Waller, 1850; see also Torigoe et al. 1996; Hall, 2001). In the early stage, axons degenerate and the myelin sheaths break up into ovoids, which are subsequently phagocytosed by macrophages invading the distal nerve stump. The most

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characteristic feature of Wallerian degeneration is the proliferation and activation of Schwann cells within the distal nerve stump, which results in the formation of Schwann cell cordons. Proximal to the site of injury, damage to nerve axons immediately influences intracellular signaling pathways, which subsequently induces the expression of various kinds of genes related to the cell emergency system, including c-fos and c-jun (Soares et al., 2001). Considerable evidence suggests that neurotrophins are critical for both axonal growth and the survival of selected populations of neurons in the PNS (Goldberg and Barres, 2000; Lonze et al., 2002). Responding to neurotrophins, Ras/MAPK and PI3K/Akt pathways act as effectors of neurotrophin receptor signaling, and thus exercise survival and growth effects (Sofroniew et al., 2001). Moreover, the nuclear transcription factor, CREB, has been reported to serve as a key target in neurotrophin-stimulated cellular events, since null mutation in the Creb gene results in impaired axonal growth and an excess of apoptosis in PNS neurons (Lonze et al., 2002). Through these mechanisms, axons give rise to growth cones and enter into the Schwann cell tubes, which form the pathway for regenerating axons to the targets. Early in the process, one Schwann cell surrounds several regenerating axons, but eventually the cells segregate to form a 1:1 relationship between axon and Schwann cells, and the process of remyelination is completed (Hall, 2001). In the normal, undamaged PNS, Schwann cells have a reciprocal relationship with the axons they ensheathe. Axonal signals, whether acting by direct contact or by diffusible factors, regulate Schwann cell genes and control proliferation and differentiation. Conversely, Schwann cell signals regulate gene expression and intracellular signaling of axons (Bolin and Shooter, 1993). Such a tightly regulated relationship between axon and Schwann cell is disrupted by injury and the subsequent Wallerian degeneration. Schwann cells quickly down-regulate the expression of myelin protein genes and up-regulate the low affinity neurotrophin receptor p75, as well as neurotrophins, including NGF and brainderived neurotrophic factor (BDNF), GAP-43, and the neu-receptors, before starting to dedifferentiate and divide (Martinez, 1999; Hall, 2001). Neurotrophic factors are key regulatory proteins that modulate neuronal survival, axonal growth, synaptic plasticity and neurotransmission. Schwann cells clearly secrete a large variety of neurotrophic factors, including neurotrophins, transforming growth factor, basic fibroblast growth factor (bFGF), FGF-5, glial cell line-derived neurotrophic factor (GDNF), osteopontin, IL-6 and leukemia inhibitory factor (LIF). All of these are thought to make a contribution to a successful axonal regeneration (von Bartheld, 1998; Abe et al., 2001; Hall, 2001; Jander et al., 2002). A series of responses in the PNS regeneration is triggered by an intimate interaction between Schwann cells and macrophages. Following injury, Schwann cells directly attract macrophages. An autocrine cascade of IL-6 and LIF enhances Schwann cell secretion of monocyte chemoattractant protein-1, which directly attracts the infiltration of macrophages into the injured nerves (Tofaris et al., 2002). In turn, macrophages secrete cytokines and trophic factors, such as IL-1 and insulin-like growth factor (IGF), which cause a heightened activation of Schwann cells, followed by axonal growth (Toews et al., 1998). In addition, the rapid invasion of macrophages enhances phagocytotic activity to dispose of cell debris (Hall, 2001). Activated Schwann cells express a variety of cell adhesion molecules including neural cell adhesion molecules (NCAM), L1 and their close homologues CHL1, N-cadherin and

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integrins, represented by a1b1 and a6b1-integrin, that mediate interactions between Schwann cells and axons, including growth cones (Martini, 1994; Ide, 1996; Chernousov and Carey, 2000; Zhang et al., 2000). Besides these trophic factors and cell adhesion molecules, the Schwann cell supplies molecules of the extracellular matrix, such as fibronectin, laminin, J1/tenascin and merosin (laminin-2), to the injured axons, which then extend their processes (Chernousov and Carey, 2000). Among these extracellular molecules, laminin-alpha2 is known to play an important role in establishing remyelination, since its absence in mice led to reduced compactness and delay of myelination. Finally, reinnervated Schwann cells cease dividing, down-regulate expression of the molecules discussed above and revert to an axon-associated phenotype (Hall, 2001). Normally, Schwann cells express the gap junction proteins connexin32 (Cx32), connexin26 (Cx26) and connexin46 (Cx46), as well as lower levels of connexin43 (Cx43) (Ressot and Bruzzone, 2000). While Cx32 is concentrated at the incisures and paranodes, Cx26 and Cx43 are sparsely distributed in the plasma membrane of the cell body, and are not detected at the nodes of Ranvier. In response to nerve injury, Schwann cells in the degenerating region down-regulate the expression of Cx32. But dividing Schwann cells express Cx46 and other connexins, coupled through junctional channels, whereas nondividing cells are generally uncoupled. Moreover, the strength of junctional coupling among Schwann cells is modulated by a number of cytokines to which Schwann cells are exposed after injury; thus, tumor necrosis factor (TNF)-alpha and acidic-FGF decrease coupling between Schwann cells (Chandross et al., 1996; Reimers et al., 2000). In addition to gap junctions, activated Schwann cells are also linked via short focal tight junctions (Dezawa and Nagano, 1993). These observations suggest that enhanced junctional communication among Schwann cells could help to coordinate cellular responses to the injury. As a result of the integrated functions of these factors as well as of the activated Schwann cells, nerve regeneration can be induced.

3. Neuronal and glial responses in injured CNS Regeneration of the CNS, including optic nerve regeneration, in adult mammals is quite restricted. Once the CNS is damaged, the conditions for regeneration are very unfavorable. The reason why CNS neurons die or atrophy, but PNS neurons show vigorous regrowth after axotomy can be summarized as differences in both the neuronal response, and in the glial environment. The fundamental difference between CNS and PNS neuronal cell bodies has been the subject of intensive research. Recent findings suggest that CNS neurons die because of trophic deprivation caused by an axotomy-induced disconnection of the neuronal cell body from its target-derived trophic peptides, which normally convey a retrograde trophic signal to the cell body. Furthermore, they become less sensitive to the peptide trophic signals they do receive. Some types of central neurons depend on a physiological activation by electrical activity or elevation of intracellular cAMP (Meyer-Franke et al., 1998; Goldberg and Barres, 2000). On the other hand, adult PNS neurons are intrinsically

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responsive to neurotrophic factors and do not lose trophic responsiveness after axotomy (Goldberg and Barres, 2000). The glial environment is quite different between PNS and CNS. The PNS glial milieu consists mainly of Schwann cells, whereas astrocytes, oligodendrocytes, and microglia dominate in the CNS. A number of factors are involved in the lack of CNS regeneration, including myelin-mediated inhibition by oligodendrocytes, glial scarring, and insufficient trophic factor support (Dezawa et al., 1999; Schwartz et al., 1999; Lacroix and Tuszynski, 2000; Asher et al., 2001; Fournier and Strittmatter, 2001). Oligodendrocytes, which represent the myelinating glia in the CNS, carry on their surface axon growth-inhibiting molecules (Brittis and Flanagan, 2001; Fouad et al., 2001; Fournier and Strittmatter, 2001). It is in support of this conclusion, that the loss of regenerative capacity in the CNS during development correlates roughly with the onset of myelination (Fournier and Strittmatter, 2001). Specific components of the myelin produced by the oligodendrocytes, such as Nogo and myelin associated glycoprotein (MAG), have been shown to inhibit axonal growth, and antibodies against these proteins resulted in axonal regrowth in the CNS (Shen et al., 1998; Cai et al., 1999; Fournier and Strittmatter, 2001). The glial scar that forms at the site of injury is a biochemical and physical barrier to successful regeneration. It contains large numbers of reactive astrocytes, oligodendrocyte precursor cells, and CNS meningeal cells. A recent study suggests that injury-upregulated bone morphogenetic protein 7 (BMP7) synthesis within the CNS induces differentiation of astrocytes from neural progenitors, which may also contribute to the glial scar formation after CNS injury (Setoguchi et al., 2001). The expression of repulsive molecules such as semaphorin-3A, tenascin, NG2, neurocan, phosphacan, chondroitin and keratan sulfate proteoglycans are related to the repulsive nature of glial scars (Mckeon et al., 1991; McMillian et al., 1994; Pasterkamp et al., 1999). The reactive glial extracellular matrix is directly associated with the failure of axonal regeneration, whereas the myelinated white matter beyond the glial scar is rather permissive for regeneration (Davies et al., 1997). Nevertheless, Moon and Fawcett (2001) have shown that despite the reduction of scar formation by treatment with antibodies to transforming growth factors (TGFs), sufficient enhancement of spontaneous CNS regeneration was not obtained. It seems beyond doubt that the glial scars have a negative impact on CNS regeneration, although their precise contribution to the inhibitory nature of the CNS environment needs to be accurately ascertained. In contrast to the PNS, the injured adult CNS becomes very slowly and poorly invaded by macrophages. The importance of macrophage invasion is illustrated by the observation that macrophages stimulate regenerative responses in transected rat optic nerve axons (Schwartz et al., 1999; Leon et al., 2000). However, microglial activation is considered to be a double-edged response. The first stage of activation is towards a nonphagocytic state, where microglia become hypertrophic and produce molecules, which are cytotoxic to neuronal cells, such as TNF-a (Buttini et al., 1996). However, microglia also release cytokines beneficial to the promotion of regeneration; thus, TGFb could promote tissue repair by reducing astrocytic scar formation, and BDNF and GDNF, secreted by microglia, may also support regeneration (Batchelor et al., 1999).

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Fig. 1. Schematic summary of glial response to optic nerve injury (A; early phase, B; late phase). After injury, oligodendrocytes negatively regulate axonal regeneration by producing MAG, Nogo and inhibitory substances such as chondroitin and keratan sulfate proteoglycans. Few macrophages invade the optic nerve in the early phase (A). Later, glial scar, consisting of reactive astrocytes, oligodendrocyte precursors, meningeal cells and inhibitory extracellular matrix (chondroitin sulfate proteoglycans, NG2, neurocan, phosphacan) is formed and inhibits axonal regeneration (B).

For further discussion of the importance of reactive astrocytes as well as of macrophages and microglia in CNS regeneration, can be referred to the chapter by Kalman. A schematic summary of glial responses in the injured CNS is presented in Fig. 1. 4. Mechanisms of optic nerve regeneration by Schwann cell transplantation A feasible strategy for augmenting CNS regeneration is to promote axonal regeneration by adopting the favorable properties exhibited in injured peripheral nerves, specifically, the provision of trophic factors and the grafting of Schwann cells. A series of experiments have demonstrated that implantation of a peripheral nerve segment into the transected optic nerve promotes regeneration of nerve fibers by distances of several centimeters into the implanted tissue, and that the peripheral nerve graft can delay nerve cell death and prevent axonal degeneration (Villegas-Perez et al., 1988; Dezawa et al., 1997; Dezawa, 2002). It must be emphasized that the peripheral nerve segment transplanted into the optic

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nerve should be in the process of Wallerian degeneration, and therefore containing activated Schwann cells, which provide suitable substrates as described earlier. Optic nerve regeneration is sustained when the relationship between axons and activated Schwann cells is closely and intimately maintained, forming structural and molecular linkages (Dezawa and Nagano, 1993; Dezawa et al., 1997). In addition to producing neurotrophic factors and extracellular matrix molecules that support neuronal survival and axonal growth, Schwann cells express a wide range of cell adhesion molecules belonging to cadherins, integrins or the immunoglobulin superfamily, including NCAM (Fig. 2a). Extracellular molecules produced by Schwann cells are also known to stimulate neurite outgrowth through an interaction with integrins expressed on the axonal surface (Ide, 1996; Agius and Cochard, 1998). Recent studies demonstrated that neurite extension stimulated by cell adhesion molecules does not rely simply on adhesion, but instead requires that second messenger cascades in neurons are activated, thereby inducing further axonal elongation (Goldberg and Barres, 2000). Detailed observations of the mutual interactions between regenerating optic nerve axons and Schwann cells after peripheral nerve and/or Schwann cell transplantation have revealed that the cells involved are not simply connected via cell adhesion molecules, but seem to have established a closer structural and functional bond via tight and gap junctions (Fig. 2b,c). Usually, such connecting structures are formed between homologous cell

Fig. 2. Schematic summary of axon–Schwann cell interactions during optic nerve regeneration. a) and c) immunoelectron micrographs of NCAM and connexin43, respectively. b) freeze–fracture image of short tight junction between Schwann cell (S) and axon (A). (From Dezawa and Nagano, 1993, 1996).

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types, but the possibility exists of a temporary development of atypical tight and gap junctions between heterologous tissues of the nerve axon and Schwann cells during regeneration. Mature tight junctions in epithelial tissues show a belt-like meshwork in freeze – fracture images that provides a barrier to the free passage of molecules and ions across paracellular pathways (Anderson, 2001). This barrier is variable and physiologically regulated. Molecular components of the tight junction can be grouped into four categories: (i) peripherally associated scaffolding proteins like ZO-1; (ii) signaling proteins involved in barrier regulation and gene transcription; (iii) components targeting membrane vesicles; and (iv) transmembrane proteins such as occludin and claudin, creating the paracellular barrier (Anderson, 2001). However, tight junctions observed between regenerating axons and Schwann cells were short focal fusions of the outer leaflets of two adjoining plasma membranes (100 – 500 nm in length), and were mostly isolated (Dezawa and Nagano, 1993, 1996; Dezawa et al., 1997). Such structures seem to serve as sites of adhesion, providing mechanical links for regenerating axons to facilitate stable interactions with Schwann cells (Fig. 2b), rather than sealing structures (Dezawa and Adachi-Usami, 2000). There have been several reports of connexin 32-mediated gap junctions in nervous tissues, such as brain and peripheral nerves, but all were between homologous cell types, for example, Schwann cell processes of the Schmidt-Lanterman incisures and paranodal portions of the nodes of Ranvier, and between adjoining Schwann cells in regenerating peripheral nerves (Dermietzel and Spray, 1993; Chandross, 1998). Gap junctions have also been found between the immature and undifferentiated cells in early developing embryos (Tetzlaff, 1982; Warner, 1992). The gap junction between heterologous cell types of regenerating axon and Schwann cells was mostly a small plaque that consisted of several intramembrane particles (Fig. 2c), associated with either connexin 32 or connexin 43 immunoreactivity (Dezawa et al., 1998; Dezawa and Adachi-Usami, 2000). Intercellular coupling between the regenerating axon and its adjacent Schwann cell was confirmed by intracellular injection of biocytin, a membrane-impermeable dye of low molecular weight (373 Da). The axon and the slender process of an adjacent Schwann cell become labeled with biocytin, but the cytoplasm of other Schwann cells nearby does not (Dezawa et al., 1998; Dezawa and Adachi-Usami, 2000). This result implies that during nerve regeneration a direct path for gap junction-mediated intercommunication exists between axon and Schwann cells, through which nutrients and cytoplasmic factors can pass back and forth. Cell-adhesion molecules and neurotrophic factors, in most cases, act via membrane receptors. In contrast, the existence of gap junctions suggests that these structures might provide a ‘hot line’ for the direct trafficking of small but significant quantities of essential intracellular factors between axons and Schwann cells in the process of regeneration. In conclusion, Schwann cells contribute to nerve regeneration by (i) production of trophic factors and cell-adhesion molecules; and (ii) provision of junctional structures to stabilize cell contact and facilitate traffic of substances between the regenerating axon and the Schwann cell. A schematic summary of axon – Schwann cell interactions during optic nerve regeneration is shown in Fig. 2.

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5. Reconstruction of optic nerve circuit by Schwann cell transplantation Some kinds of cells, such as gene-transfected astrocytes, amniotic cells, olfactory ensheathing cells, ependymal cells, differentiated ES cells, and neuronal stem cells, can induce an elongation of CNS nerve fibers (Bankiewicz et al., 1994; Castillo et al., 1994; Lundberg et al., 1997; Ramon-Cueto et al., 1998; Ide et al., 2001; Kitada et al., 2001; Sawamoto et al., 2001). However, it has to be remembered that many CNS axons are myelinated by oligodendrocytes. The optic nerve tract is one of the typical examples. Myelinating cells, either of Schwann cell or oligodendrocyte origin, mediate the spacing of sodium channel clusters at the nodes of Ranvier to enable saltatory conduction, which is a prerequisite for normal neuronal activity and function. Moreover, myelinating cells are known to control the number of neurofilaments, and they can elevate the phosphorylation state of the neurofilaments in the axon, thereby leading to ‘large-calibre axons’ (Martini, 2001). Conversely, absence of myelin results in lower amounts of neurofilaments, reduced phosphorylation levels and smaller axon diameters (Martini, 2001). Therefore, even if the CNS can elongate its axons, remyelination of regenerated axons is indispensable for the re-establishment of CNS function. Although Schwann cells originally myelinate peripheral axons, they can also remyelinate CNS axons when transplanted. Consequently, they are ‘cells with a purpose’ and represent one of the best candidates for implantation to support regeneration of the CNS. Considering the evidence that intimate and direct interactions are maintained between regenerating optic nerve axons and Schwann cells, regeneration success can be expected to depend upon the extent of opportunities for axons to encounter Schwann cells. To obtain a fully functional recovery of transected optic nerve fibers, artificial grafts were designed, consisting of Schwann cells at high-density (Negishi et al., 2001; Dezawa, 2002). Cultured Schwann cells were purified from dorsal root ganglia of newborn rats (Wistar strain), and approximately 106 cells/mL were suspended in an extracellular matrix containing NGF (100 ng/mL) and bFGF (100 ng/mL), so that Schwann cell density in the tube was approximately 100 times higher than in the normal PNS. NGF and bFGF might activate and prolong the survival of Schwann cells, and also act on damaged RGCs (Yip and So, 2000). In rats, almost all optic nerve axons project to the contralateral side of the superior colliculus. Normally, optic nerve fibers run beneath the skull, but in the experimental design, optic nerve fibers of the left were dissected just behind the eyeball, and bridged to the right superior colliculus with an artificial graft lying on the upper surface of the head. Soon after the transplantation, BDNF (100 ng/mL), ciliary neurotrophic factor (CNTF) (50 ng/mL) and forskolin (FSK) (5 mmol) were injected into the vitreous body. Such supplies of neurotrophins together with FSK have been reported to be efficient in the survival of RGCs (Meyer-Franke et al., 1998; Cho et al., 1999; Goldberg and Barres, 2000). The optic nerve fibers regenerated successfully within the artificial graft for a few centimeters to the CNS target of the superior colliculus. Intravitreous injection of cholera toxin B subunit labels RGC axons anterogradely, and detection of the cholera toxin B by immunohistochemistry enabled us to distinguish regenerating RGC axons from other neuronal axons. There was abundant penetration of cholera toxin B-positive RGC axons within the superior colliculus, and most of the axons

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were positive for synaptophysin, indicating that regenerated optic nerve fibers participated in reformation of synapses. Moreover, injection of the tracer diI into the superior colliculus showed retrograde labeling of approximately 18% of the total RGC axons, indicating that almost one fifth of the severed axons had regenerated into the superior colliculus. Axons within the artificial graft tube were mostly ensheathed by Schwann cells, and some of the neurites labeled with cholera toxin B were myelinated by Schwann cells (Negishi et al., 2001; Dezawa, 2002). Recovery of the visual function in regenerated optic nerve fibers was evaluated by a visual prepulse task, and some animals did show signs of light perception (Dezawa, 2002). In the past, it was assumed that optic nerve fibers had no capacity to regenerate neurites under any circumstances. However, it has now been shown that optic nerve fibers can regrow neurites, up to several centimeters long, under appropriate circumstances like an artificial environment of Schwann cells. Furthermore, visual function may recover, once the regenerating optic nerve fibers reach the superior colliculus and are in a position to make functional connections.

6. Influence of Schwann cells on optic nerve glial cells The transitional zone between the transected optic nerve stump and transplanted peripheral nerve and/or Schwann cell grafts is the site where optic nerve elements first encounter a PNS environment. This site allows us to investigate how optic nerve glial cells are affected by Schwann cells during regeneration. Astrocytes are known to participate in glial scar formation, but they do not always inhibit axonal growth. When the transitional zone between the optic nerve stump and the peripheral nerve graft was observed by electron microscopy, most of the axons were in contact with either astrocytes or Schwann cells. Consequently, naked axons were rarely observed. The astrocytes possessed their own basement membranes and contained numerous glial filaments, similar to the cells which delineate the intact optic nerve at the glia limitans (Dezawa et al., 1999). Penetration of host astrocytic processes into the Schwann cell column and their subsequent incorporation into the transplant has also been observed in the thalamus (Brook et al., 2001). In some instances, direct attachments of both astrocytes (from the CNS) and Schwann cells (from the transplant) were seen, with regenerating axons held in-between (Dezawa et al., 1999). Since, regenerating axons were invariably covered by either astrocytes or Schwann cells in the transitional zone, both cell types may be indispensable as glial pathways for the successful regeneration of optic nerve. Recent studies also suggest that the inhibitory character of mature astrocytes may be reversed in some situations: purified cortical astrocytes, seeded into the gap in a transected sciatic nerve, inhibited regeneration, whereas a mixture of astrocytes and Schwann cells promoted the axonal regrowth of peripheral nerves (Baehr and Bunge, 1990; Gue´nard et al., 1994). Taken together, these data indicate that astrocytes in the presence of Schwann cells may function to support optic nerve axons, rather than interfering with the regenerating axons, and that the inhibitory properties of astrocytes can be partially changed in vivo, when they are combined with Schwann cells (Fig. 3). Oligodendrocytes, which normally inhibit axonal elongation, showed a decrease in the

Schwann Cell Interactions with Axons and CNS Glial Cells Fig. 3. Schematic summary of glial response to Schwann cell transplantation (A; early phase, B; late phase). In the presence of Schwann cells, macrophages migrate into the optic nerve (A). Astrocytes penetrate into the Schwann cell column, followed by axonal elongation sustained either by astrocytes or by Schwann cells (B). 339

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intensity of O4, a marker of mature oligodendrocytes after Schwann cell transplantation (Dezawa et al., 1999). This suggests that the distribution of oligodendrocytes and their characteristic properties may also be influenced by Schwann cells. There are positional similarities between Schwann cells (both myelinating and unmyelinating) and astrocytes, present in the glia limitans of the CNS since both of these cell types by nature attach themselves between the neuronal elements and the extracellular matrix of the basement membranes (Fig. 4). Proliferation of connective tissue is induced in the damaged zone, so that optic nerve axons emerging from the transected retinal end encounter this connective tissue environment during the initial stage of regeneration. Schwann cells and astrocytes may work as footholds to provide glial guidance in this situation. By contacting both regenerating axons and extracellular matrix, Schwann cells as well as astrocytes are able to cover, guide, and protect elongating axons within the connective tissue jungle of the transitional zone. Factors derived from invading macrophages are thought to regulate glial proliferation and to induce a transformation of Schwann cells and astrocytes that permits axonal growth (Logan et al., 1994; Hall, 2001—see also chapter by Mercier and Hatton). After Schwann cell transplantation, an increase in density of ED1-positive macrophages within the optic nerve was observed, possibly because cytokines and factors such as LIF and IL-6, secreted from the Schwann cell, diffused to the adjacent area, facilitating the invasion of macrophages into the CNS (Dezawa et al., 1999) (Fig. 3). This result suggests that the Schwann cell environment positively influences the invasion and activation of macrophages, which may also participate in the successful regeneration of the optic nerve. In conclusion, optic nerve regeneration induced by peripheral nerve transplantation does not simply represent axonal elongation into the graft, but it is accompanied by

Fig. 4. Schematic diagram showing similarity between Schwann cell (S) (myelinated; A, unmyelinated; B) and astrocyte (as) (C). Both are covered by the basement membrane and are also attached to the axons (A).

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changes and responses among glial cell populations, encompassing astrocytes, oligodendrocytes and macrophages. In particular, Schwann cells may modulate the response of neighboring astrocytes, rendering them suitable for supporting axonal regrowth. 7. Concluding remarks Despite the fact that Schwann cells as well as astrocytes and oligodendrocytes are constituent members of the nerve tissue, they carry out very different functions during regeneration. This may reflect that there are certain qualitative differences between them. During development, CNS glial cells derive from the neural tube, whereas Schwann cells derive from stem cells of the neural crest (Jessen and Mirsky, 1999). Thus, firstly, central macroglia (astrocytes and oligodendrocytes) and Schwann cells are originally derived from different precursors. Secondly, astrocytes and oligodendrocytes contrast sharply with Schwann cells by neither reverting to an immature phenotype nor altering their characteristics, when the CNS is injured. Instead they maintain their highly differentiated structure as in normal situations. On the contrary, although Schwann cells in the mature PNS are fully differentiated and associated with myelin sheaths, they rapidly change their structure and characteristics after injury, to become undifferentiated, return to the cell cycle and proliferate. Unlike astrocytes and oligodendrocytes, Schwann cells are more flexible in nature and can rapidly adapt to emergencies, such as nerve injury. Along with the well-known functions of the Schwann cell to produce trophic factors and cell adhesion molecules, Schwann cells contribute to nerve regeneration by providing tight junctions to stabilize cell contacts with injured nerve cells and by establishing gap junctions to facilitate traffic of substances between the cells. The detailed functional mechanisms of the junctions should be elucidated, but it is presumed that this structure might act as a foothold in the region where tissue remodeling takes place. If the overall goal of CNS reconstruction is initially to secure the survival and maintenance of nerve-cell bodies, then to establish the right conditions for elongation of nerve fibers and eventually to achieve reconstruction of neural connections and functional recovery, then our final goals are still distant. Cell transplantation to repair CNS injury is a vibrant area of research, where the goal is to alleviate functional deficits. Recent studies indicate that implantation of Schwann cells (as well as of some other types of cells) can induce neurite elongation in the CNS. However, considering that almost all fibers of the intact CNS, such as optic nerve fibers, are myelinated, complete functional recovery may not be achieved without proper remyelination. Fortunately, Schwann cells do not only induce axons of the optic nerve to elongate, but they are also able to remyelinate the elongated RGC axons. Accordingly, implantation of Schwann cells can be regarded as a promising approach for the purpose of nerve regeneration. Acknowledgements I wish to acknowledge the excellent support of Dr Masahiko Takano, Department of Ophthalmology, Kitasato University School of Medicine, for the optic nerve

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transplantation, and Drs Hitoshi Sasaki, Hajime Sawai and Yutaka Fukuda, Department of Physiology, Osaka University Graduate School of Medicine, for the visual prepulse task experiment. I would like to express my cordial thanks to Prof. Dr. V. B. Meyer-Rochow (International University Bremen, Germany) for his valuable reviewing of the manuscript. I also wish to thank Professor Emeritus Toshio Nagano, Department of Anatomy, Chiba University School of Medicine, for his support and encouragement.

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Control of microglial activity by protective autoimmunity Michal Schwartz Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel Correspondence address: Tel.: þ 972-8-9342467; fax: þ 972-8-9346018. E-mail: [email protected]

Contents 1. 2. 3. 4. 5. 6. 7. 8.

The central nervous system as an immune-privileged site Acute degenerative conditions: destruction by the enemy within Conflicting reports related to the role of inflammation in CNS repair A stress signal from the damaged CNS evokes an ‘anti-self’ immune response T-cell-dependent protection against glutamate toxicity Possible connection between T-cell-based protection and microglia at the lesion site Synthesizing the findings Therapeutic vaccination for neurodegenerative conditions

Microglia have been viewed as the quiescent though potentially active arm of the innate immune response in the central nervous system (CNS). After injury they become activated, and migrate to the lesion site. The participation of activated CNS-resident microglia in the inflammatory response that follows traumatic injuries or chronic neurodegenerative diseases in the CNS, like that of activated blood-borne macrophages and infiltrating lymphocytes, has generally been viewed as detrimental. Accordingly, attempts have been made to treat these conditions by anti-inflammatory therapy. Based on our recent studies, however, we suggest that microglia act as stand-by cells in the service of both the immune and the nervous systems. Yet, their abiliy to fully benefit damaged tissue rather than to be inactive or even destructive reflect their activation. They exercise their neural function by buffering harmful self-compounds and clearing debris from the damaged site and their immune function by providing immune-related requirements for recovery. Proper regulation of the inflammatory (autoimmune) response to injury arrests degeneration and promotes regrowth, whereas inappropriate regulation leads to ongoing degeneration. We further suggest that regulation is achieved by the operation of a T-cellmediated response directed against abundant self-antigens residing in the damaged site. Advances in Molecular and Cell Biology, Vol. 31, pages 347–365 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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Since this regulatory mechanism was found to be protective against harmful endogenous factors released as a result of the injury (such as glutamate toxicity, a major factor in neurodegenerative disorders), boosting of an autoimmune response while avoiding autoimmune disease induction might constitute the basis for development of a therapeutic vaccination against neurodegenerative or other brain-related diseases.

1. The central nervous system as an immune-privileged site The unique interaction between the central nervous system (CNS) and the immune system has long been regarded as a relationship characterized by immune privilege, in which local immune responses within the CNS are restricted (Streilein, 1993, 1995). Unlike most peripheral tissues, the CNS functions through a network of postmitotic cells (neurons) that are incapable of regeneration and hence cannot be replaced when aging or impaired. Being simultaneously essential for survival and unable to recover fully from injury, the CNS is in special need of protection from pathogens and insults. However, the CNS is also vulnerable to damage that might be caused by the very means that the immune system uses to defend peripheral tissues from pathogens. Consequently, immune privilege in the CNS has been viewed as an evolutionary adaptation developed to protect the intricate neuronal networks of the CNS from incursion by the immune system (Lotan and Schwartz, 1994). An early definition of CNS immune privilege was based on the assumption that the immune system ignores the CNS. This assumption was supported by the observed tendency of the CNS not to reject allografts (tissue grafts from the same species but from a different major histocompatibility complex (MHC) haplotype) (Medawar, 1948). Immune privilege was thought to be maintained by the isolation of antigens within the CNS and the inability of immune cells to enter the CNS under normal physiological conditions. Any leukocyte accumulation was viewed as evidence of pathology. Several observations have challenged this definition. Firstly, CNS antigens can escape and induce immune responses in the host (Cserr et al., 1992; Cserr and Knopf, 1992). Second, activated T-cells have been found to enter the CNS in the absence of discernible neuropathology (Flugel et al., 2001; Hickey et al., 1991; Shrikant and Benveniste, 1996). Third, leukocyte recruitment into the CNS appears to successfully resolve some CNS viral infections, such as Sindbis virus encephalitis, without the development of any apparent long-term bystander effects (Griffin et al., 1997). Fourth, based on the relatively prolonged survival of xenografts (tissue grafts from different species) in immunosuppressed individuals, it was suggested that the immune system participates in CNS xenograft failures (Czech et al., 1997). Taken together, these findings and others indicate that the CNS is accessible to immune cells, and that local immune responses within the CNS are regulated by a number of resident mechanisms. It seems likely that some of these mechanisms help to limit immune responses, with vital consequences for the functioning of the healthy CNS. According to this view and the data summarized below, it seems that the healthy CNS is hostile to immune activity (Flugel et al., 2001; Moalem et al., 1999a,b), and that it is destroyed by it,

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whereas immune activity is an essential requirement for the protection and maintenance of the damaged CNS. 2. Acute degenerative conditions: destruction by the enemy within Acute mechanical or biochemical injury to the mammalian CNS often results in an irreversible functional deficit (Kalb, 1995; Schwab and Bartholdi, 1996) for several reasons, including the poor ability of injured axons to regrow, and a destructive series of injury-induced events that result in the spread of damage to neurons that escaped the direct injury (Faden, 1993; Hauben and Schwartz, 2003; Povlishock and Jenkins, 1995; Yoles and Schwartz, 1998). This spread of damage is known as secondary degeneration. Attempts to promote CNS recovery have focused on two goals: (i) stimulation of regrowth (Caroni and Schwab, 1988; Cheng et al., 1996; Chong et al., 1999; Davies et al., 1997; Li et al., 1997; Miwa et al., 1997; Neumann and Woolf, 1999; Rapalino et al., 1998; Reier et al., 1992; Wang et al., 1998), and (ii) neuroprotection, or the arrest of self-perpetuating degeneration (Basso et al., 1996; Bavetta et al., 1999; Beattie et al., 1997; Behrmann et al., 1994; Bethea et al., 1998; Blight, 1989; Constantini and Young, 1994; Crowe et al., 1997; Gruner et al., 1996; Moalem et al., 1999a,b; Sanner et al., 1994; Schwartz et al., 1999a,b; Yong et al., 1998). The recognition that a significant tissue loss after acute injury results from a process of delayed degeneration and not only from the primary insult has led to an emphasis on neuroprotection in the approach to therapy. The spread of damage is mediated by numerous factors, some of which have been characterized. Interestingly, however, the factors that operate in different insults and across species were found to be similar, suggesting that the progression of damage might reflect a loss of control over mechanisms that regulate self-components. Although these self-components are normally essential, they may contribute to the damage spread under pathological conditions because of a toxic increase in their concentrations. The increased presence of infiltrating or activated immune cells that accompanies degeneration often led scientists and clinicians to conclude that these cells contribute to the pathology. As discussed below, it seems that this conclusion is an oversimplification. 3. Conflicting reports related to the role of inflammation in CNS repair Studies of recovery after CNS injury have traditionally been based on the assumption that the CNS is a unique tissue whose postinjury behavior is governed by rules different from those underlying the recovery of other types of injured tissue, including the peripheral nervous system (PNS). Studies during the early 1980s showed, however, that although transected CNS axons fail to regenerate in their own degenerative environment, they can grow into transplanted peripheral nerve bridges (David and Aguayo, 1981). This key discovery indicated that at least some CNS axons are intrinsically capable of regeneration, and that the hostility to growth comes from their environment. From this starting point some studies began to focus on comparisons, both within and among species, between regenerative and nonregenerative nervous systems. This line of research

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established the concept that CNS regeneration failure may be attributable, at least in part, to an inability of the cellular components surrounding the injured axons to create a balanced environment capable of permitting and supporting growth. Among the factors found to be hostile to regrowth in adult CNS nerves are myelin-associated proteins (Cai et al., 2002; McKerracher et al., 1994; Qiu et al., 2002; Schnell and Schwab, 1990; Schwab and Caroni, 1988; Shibata et al., 1998) and extracellular matrix proteins such as chondroitin sulfate proteoglycans (CSPG) (Rudge and Silver, 1990; Zuo et al., 1998) (see also chapter by Kalman). Accordingly, for regeneration to occur, these inhibitors must be masked, neutralized or eliminated. These and other discoveries prompted a series of studies whose findings were translated into strategies for the treatment of transected CNS axons, with the goal of rendering the axons capable of regeneration. As an example, the transplantation of intercostal nerve segments (peripheral nerves) into completely transected spinal cords of adult rats was shown to lead to partial regeneration, with restoration of hindlimb function (Cheng et al., 1996). Monoclonal antibodies directed specifically against the myelin-associated inhibitor IN-1 were introduced into partially transected rat spinal cords, with consequent promotion of regeneration (Schnell and Schwab, 1990). More recently, inhibitors of signal transduction associated with myelin-related growth inhibitors were found to neutralize the inhibition of CNS regrowth (McKerracher, 2002; Qiu et al., 2002). Likewise, regrowth was promoted by treatment with proteases that degrade CSPG (Bradbury et al., 2002; Rudge and Silver, 1990). Other approaches aimed at promoting recovery of CNS axons have been based on modification of the environment by local transplantation of embryonic tissue (Grady et al., 1985), Schwann cells (Xu et al., 1997) (see also chapter by Dezawa), or cells that provide a source of trophic factors to increase the survival rate of the cell bodies (Coumans et al., 2001; Tuszynski and Gage, 1995). Although immune cells are known to play a key role in tissue repair, their activity in the CNS, as outlined above, has mostly been viewed as detrimental. A comparative study of the local inflammatory response in injured PNS and CNS axons revealed that in the injured PNS, where regeneration takes place spontaneously, both degeneration and regeneration appear to be brought about and controlled with the prominent participation of macrophages. Macrophages are also a source of cytokines and growth factors that actively participate, both directly and indirectly, in regrowth. As an example, the regeneration of optic nerves in lower vertebrates, as well as of peripheral nerves in mammals, was found to correlate with up-regulation of macrophage-derived apolipoproteins, which participate in the recycling of lipids needed for membrane rebuilding (Harel et al., 1989; Heumann et al., 1987; Ignatius et al., 1987) (see also chapter by Itoh and Yokoyama). Similarly, the synthesis of nerve growth factor seems to be regulated by stimulated macrophages. Interleukin (IL)-1 and tumor necrosis factor (TNF)-a, both secreted by macrophages, probably induce nerve growth factor transcription in Schwann cells (Lindholm et al., 1987, 1988). Literature reports suggest that microglia in the intact CNS are in a down-regulated form, and therefore that their activation is likely to be restricted relative to that of macrophages which invade or reside in other tissues (Hailer et al., 1997; Neumann et al., 1996). In early experiments aimed at overcoming the apparently inherent inadequacy (in terms of numbers and the timing and intensity of activity) of the innate response in the injured

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CNS, our research group found that implantation of homologous macrophages in completely transected spinal cord resulted in a partial recovery of motor function (manifested by locomotor activity scored in an open field) and electrophysiological activity (assessed by motor-evoked potential responses) (Rapalino et al., 1998). Behavioral manifestations of recovery were also reflected in the electrophysiological recovery of motor-evoked potential responses in the implanted rats. The extent of recovery, in terms of the numbers of recovered muscles and bilaterality, varied among the rats (Rapalino et al., 1998). The effect on recovery was found to be a function of the macrophage dose. Other studies have also provided evidence that microglia and macrophages can, under certain conditions, promote regeneration (Franzen et al., 1998; Prewitt et al., 1997). On the other hand, there is evidence suggesting that the damaged spinal cord benefits from macrophage depletion (Popovich et al., 1999). These apparently contradictory findings might be attributable to differences in the animal strains in which the experiments were conducted and the experimental paradigm, as will be discussed below. Thus, for example, whereas experimental implantation of macrophages was found to promote axonal regrowth in Sprague –Dawley (SPD) rats, the depletion of macrophages was conducted in Lewis rats. The former represent the more common type of strain characterized by relative resistance to injurious conditions (Kipnis et al., 2001), whereas the latter are relatively susceptible to the consequences of injury (Hauben and Schwartz, 2003; Schwartz and Kipnis, 2002). Moreover, close examination of the processes of neuronal regrowth and neuronal protection suggests that within the same process the requirements for recovery might vary at different postinjury stages. As an example, the anti-inflammatory cytokine IL-10 (Bethea et al., 1999), which has a strong positive effect on recovery when applied immediately after spinal cord injury, loses its effect after 24 h. Observations of effective neuroprotective treatment, for example, by steroids (Bracken, 1991; Hess and Sellon, 1997), by hypothermia (Chatzipanteli et al., 2000), or by antichemokines (Ghirnikar et al., 2000) have been interpreted to mean that inflammation is bad for recovery. Along the same lines, anti-inflammatory treatment after traumatic spinal cord injury was found to be neuroprotective only if applied within the first few hours after the insult (Bracken and Holford, 1993; Young et al., 1994). In interpreting these findings, it should be borne in mind that apparent discrepancies might derive from the fact that the mere presence of activated microglia/macrophages does not necessarily provide any indication as to their effect (Hauben and Schwartz, 2003; Schwartz and Hauben, 2002). Activity is determined by phenotype, which is profoundly affected in turn by the nature of the tissue and the presence or absence of other immune cells. Thus, the phenotype of the activated microglia can be both cytotoxic and protective (Shaked, I., Butovsky, O., Mizrahi, T., Gersner, R., Xiao, X., Soteropoulos, P., Tolias, P., Hart, R. P., and Schwartz, M. unpublished observations).

4. A stress signal from the damaged CNS evokes an ‘anti-self’ immune response The adaptive immune response has been generally perceived as an immune activity evoked to enable the organism to cope with stressful conditions caused by pathogens. Thus, immunologists have viewed the functions of the adaptive immune response as

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helping the body to neutralize pathogens, prevent pathogen invasion of the tissue, or counteract the damage caused by pathogens that manage to invade. The damage caused by trauma, however, does not involve pathogens, and was therefore not viewed by immunologists as posing the type of danger to the tissue that necessitates an adaptive immune response. It was suggested many years ago (Burnet, 1971, 1959) that an adaptive immune response would be evoked unless the pathogen was recognized as self. Opinions differ as to the mechanisms by which self becomes invisible to the immune system (for example by clonal deletion, anergy or tolerance) (Bretscher and Cohn, 1970; Cohen, 1988; Jameson et al., 1995; Janeway, 1992; Jerne, 1984). Some workers have proposed that autoimmunity, once established, might be harmless or even useful, but none has described a situation in which an anti-self response is regarded as a source of help for the body. Even according to the ‘danger signal’ model the response to self is viewed as a by-product of a response to a pathogen, a side effect that soon decays in the absence of a second signal to maintain it (Matzinger, 1994). This model basically argues against discrimination between self and nonself in characterizing the signals triggering a beneficial immune response. Our results suggest that autoimmunity is a purposeful physiological mechanism that operates after any CNS insult, with the object of reducing the spread of damage initiated by the insult-induced release of harmful self-compounds. In our earlier studies, we observed that optic nerve injury in rats could benefit from passive transfer of autoimmune T-cells directed against myelin proteins (Moalem et al., 1999a,b). The benefit was manifested by morphological and functional criteria. We further showed that passive transfer of T-cells directed against myelin antigens could benefit the damaged spinal cord (Hauben et al., 2000a,b). The beneficial effect, manifested by locomotor activity and demonstrated by morphological and imaging techniques (Butovsky et al., 2001; Nevo et al., 2001), was obtained even if the T-cell injection was delayed (Hauben et al., 2000a,b). Similar benefit was obtained when passive transfer of T-cells was replaced by active immunization using myelin-derived peptides (Hauben et al., 2001a,b). The same effect was obtained with different myelin peptides, such as myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP) (Fisher et al., 2001), and Nogo (Hauben et al., 2001a,b). These and other observations led us to postulate that in order for the T-cells to be beneficial they should locally encounter their specific antigen, presented to them by antigen-presenting cells. This hypothesis was further substantiated by the finding in rats and mice that myelin proteins fail to protect retinal ganglion cells (RGCs) from direct insult to the retina, a myelin-free site (Mizrahi et al., 2002; Schori et al., 2001a,b). On the basis of our results, we suggest that the anti-self immune activity in response to trauma in the CNS (Popovich et al., 1996) is a purposeful physiological event (Yoles et al., 2001). If trauma can indeed act as a stress signal that activates a helpful immune response, a number of questions arise: Does this occur in all tissues? And if not, why not? Does the trauma-related stress signal vary from tissue to tissue? Since the role of the immune system incorporates tissue protection, defense and maintenance, does this signal always operate for the organism’s benefit? How it is related to autoimmune disease? In an attempt to determine whether the beneficial effect of autoimmune T-cells represents a physiological phenomenon, we subjected rats to two consecutive insults at

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distinct CNS sites (spinal cord followed by optic nerve). Survival of RGCs after the second injury was significantly better than that obtained when the optic nerve was injured without prior injury to the spinal cord (Yoles et al., 2001). We further showed that the benefit is attributable to an injury-induced T-cell response. Other studies from our laboratory demonstrated that recovery from optic nerve or spinal cord injury is significantly reduced if the animals lack mature T-cells (Hauben et al., 2002; Kipnis et al., 2001; Yoles et al., 2001). Interestingly, however, not all strains were equally capable of harnessing a T-celldependent protective response (Kipnis et al., 2001). Moreover, strains that were genetically capable of more favorable regulation of their response, and did not develop an autoimmune disease upon active immunization with myelin proteins, were found to be more capable of withstanding the consequences of axonal injury (Kipnis et al., 2001). Our more recent studies led us to suggest that both the neuroprotective autoimmune T-cells and the autoimmune T-cells that cause an autoimmune disease are Th1 cells (Kipnis and Schwartz, 2002). Moreover, while the phenotype and the antigenic specificity of the T-cells are identical in both cases, protection can be achieved by both cryptic (i.e., nonpathogenic) and pathogenic epitopes within the same antigen (Fisher et al., 2001; Moalem et al., 1999a,b), whereas disease is caused by pathogenic epitopes only. Accordingly, we suggested that in degenerative conditions the default mechanism is protective autoimmunity, and that an autoimmune disease results only when the autoimmunity is malfunctioning (Fisher et al., 2001; Hauben et al., 2001a,b; Schwartz and Kipnis, 2002a,b). The demonstration of autoimmunity as a physiological mechanism of repair raised some questions about the balance between the need for autoimmunity and the risk of autoimmune disease. Our most recent data suggest that naturally occurring CD4þCD25þ regulatory T-cells, which have been viewed as the cells responsible for avoiding autoimmunity via suppression of peripheral autoimmunity, are actually the cells that can preserve the balance by allowing the autoimmune T-cells to exist in a state of alert in the healthy individual, ready to be activated when needed for protection, while not causing an autoimmune disease (Kipnis et al., 2002a,b; Schwartz and Kipnis, 2002a,b).

5. T-cell-dependent protection against glutamate toxicity In further experiments, we attempted to determine whether a T-cell response is spontaneously manifested after an insult caused directly by exposure to glutamate toxicity, and if so, whether it effectively protects the site against induced stress (Schori et al., 2001a, b). The glutamate toxicity induced in mice by intra-ocular injection of glutamate into the vitreous body was found to be significantly greater in mice deprived of T-cells than in wild-type mice (Schori et al., 2001a,b). As with axonal injury, different strains differed in their ability to withstand the consequences of the toxicity (Kipnis et al., 2001; Schori et al., 2001a,b). Thus, T-cell deprivation had almost no effect on neuronal survival in strains with poor resistance to glutamate toxicity, whereas it caused substantial loss in resistant strains. These studies (Kipnis et al., 2001; Schori et al., 2001a,b) provided the first indication that local coping mechanisms against a glutamate insult receive support from a systemic immune response. This unexpected observation that an immune response to a self-antigen can reinforce the overburdened local CNS coping mechanisms led us to extend the

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traditional view of immune function as protective against foreign microorganisms to include protection against the enemy within (Nevo et al., 2003; Schori et al., 2001a,b; Schori et al., 2002; Schwartz, 2001; Schwartz and Kipnis, 2002a,b). Response to treatment with receptor antagonists showed that not only the extent of toxicity is strain-dependent, but also its underlying mechanism (Schori et al., 2002). Administration of the AMPA/KA-receptor antagonist significantly reduced glutamateinduced death of RGCs after intravitreal glutamate injection in all mouse strains examined, whereas the NMDA-receptor antagonist MK801 reduced RGC death only in mice that were devoid of mature T-cells (nude mice) or were incapable of manifesting a protective T-cell response (such as C57Bl/6J mice, found to be highly susceptible to glutamate toxicity) (Kipnis et al., 2001; Schori et al., 2001a,b; Schori et al., 2002). In the relatively resistant Balb/c mice, treatment with MK801 not only did not improve the outcome, but even exacerbated the toxic effect, i.e., fewer RGCs survived (Schori et al., 2002). This observation is in line with reports suggesting that in Wistar rats undergoing progressive neurodegeneration or subjected to traumatic brain injury, neuronal loss is increased by treatment with NMDA receptor antagonists but reduced with AMPA/KA antagonists (Ikonomidou et al., 2000). It is possible that glutamate interacts with the neuronal NMDA receptor to activate microglia, which consequently express MHC class II (MHC-II) molecules (Neumann et al., 1996). The activated microglia then interact directly with glutamate via their nonNMDA receptors (Nakajima et al., 2001; Noda et al., 1999; Noda et al., 2000). In addition, our findings might suggest that protective T-cells engage in cross-talk with MHC-II-expressing microglia, with or without their direct exposure to toxic concentrations of glutamate, thereby enhancing their ability to buffer the potentially harmful glutamate-induced conditions (Shaked, I., Butovsky, O., Mizrahi, T., Gersner, R., Xiao, X., Soteropoulos, P., Tolias, P., Hart, R. P., and Schwartz, M. unpublished observations). The discovery that glutamate toxicity is reduced by a systemic adaptive immune response prompted our group to search for a way to boost this response as a means of reducing toxicity. Based on our earlier experience with axonal injury of the optic nerve (Fisher et al., 2001), we first chose proteins and peptides associated with myelin as our antigens. This choice proved to be unsuitable for a site such as the eye, which contains no myelin (Mizrahi et al., 2002; Schori et al., 2001a,b). In retrospect, we realized that these antigens could not be effective, because T-cells become activated only after homing to the site of damage and encountering their relevant antigens there. We subsequently discovered that T-cell-dependent resistance to toxicity could indeed be boosted by vaccination with antigens that are abundantly expressed in the eye (Mizrahi et al., 2002), such as peptides derived from the inter-photoreceptor retinoid-binding protein (IRBP) (Adamus and Chan, 2002; Avichezer et al., 2000). It therefore seems that a peptide which boosts beneficial autoimmunity at the site of stress is also potentially capable of inducing an autoimmune disease at the same site (Fauser et al., 2001). We found that protection from glutamate toxicity in the eye is not restricted to peptides derived from one protein, as similar protective effects in the rat visual system were obtained with two uveitogenic peptides derived from S-antigen, another retinal protein. Moreover, synthetic compounds designed to evoke an immune response without causing disease also enhanced RGC survival, indicating that nonpathogenic peptides derived from pathogenic ones can be used to

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protect RGCs from insult-induced death without risk of autoimmune disease development (Mizrahi et al., 2002). The same principle was found to apply in the case of damage to myelinated axons, i.e., the protective myelin antigens, such as myelin basic protein (MBP) or MOG, were also instrumental in causing myelin-related autoimmune disease (Fisher et al., 2001; Moalem et al., 1999a,b). In each of those cases, cryptic nonpathogenic peptides derived from the same immunodominant protein induced protective autoimmunity without showing signs of autoimmune disease (Fisher et al., 2001; Mizrahi et al., 2002; Moalem et al., 1999a,b). These and other observations further support our proposed concept of protective autoimmunity (Moalem et al., 1999a,b; Schwartz et al., 1999a,b) as the body’s rigorously controlled mechanism of maintenance and repair. Most recently, neonatal immunization with a homogenate of retinal proteins was found to deprive mice of their normal ability to respond to abundant eye proteins as adults. The deprivation was accompanied by lowered resistance to glutamate toxicity in the eye (Avidan, H., Kipnis, J., Caspi, R., and Schwartz, M., unpublished observations). This finding further substantiated our hypothesis that the physiological protective response is directed against abundant proteins residing at the site of stress. Neuroprotection and autoimmune disease are invoked by T-cells of the identical phenotype (Th1) (Kipnis et al., 2002a,b) and antigen specificity (immunodominant proteins) (Schwartz and Kipnis, 2002a,b). As discussed above, this does not preclude—at least in strains resistant to the consequences of a CNS insult—the evocation of a spontaneous and risk-free T-cell-dependent protective response against cryptic (but not against pathogenic) epitopes of a particular protein (Schwartz and Kipnis, 2002a,b). It should be noted that such protection may not stop neuronal loss altogether (Fisher et al., 2001; Hauben et al., 2000a,b; Mizrahi et al., 2002; Nevo et al., 2003; Schwartz and Kipnis, 2002a,b), but that as long as the system does not collapse in the chaos of unchecked degeneration, the benefit outweighs the risk. In our view, a threatened tissue endangers some cells for the purpose of saving others. We suggest that the operative antigen evokes an immune response which, in the event of malfunction, induces disease, but not necessarily in the cells under direct threat [the RGCs in uveitis or the myelinated CNS neurons in experimental autoimmune encephalomyelitis (EAE)]. Nevertheless, in the absence of appropriate regulation, the intensive autoimmune response might eventually lead to neuronal loss as well. Accumulating information indicates that autoimmune diseases of the CNS in humans are often accompanied by neuronal loss and an increase in extracellular glutamate (Bjartmar et al., 2001; Bjartmar and Trapp, 2001; De Stefano et al., 2001; Schwartz and Kipnis, 2002; Steinman, 2001).

6. Possible connection between T-cell-based protection and microglia at the lesion site In the normal healthy CNS, there is no need for immune activity, and immune activity might even be harmful. Any local buffering of glutamate required is therefore done primarily by astrocytes. Under degenerative conditions, however, astrocytes at the lesion site may be in short supply (Blaugrund et al., 1993). Astrocytes delineating the lesion site not only show poor ability to migrate to the lesion site and repopulate it, but also might not

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be fully active, as their principal function in this situation is probably scar formation (Faber-Elman et al., 1996). Microglia, on the other hand, apparently have the potential for migration, and in addition they express both chemokines and chemokine receptors (Aloisi, 2001). When activated T-cells interact with microglia, they endow them with the ability to present antigen and to buffer glutamate (Shaked, I., Butovsky, O., Mizrahi, T., Gersner, R., Xiao, X., Soteropoulos, P., Tolias, P., Hart, R. P., and Schwartz, M. unpublished observations). It thus seems that microglia serve a dual function, acting both as antigenpresenting cells (i.e., immune cells) and as glutamate-buffering cells (i.e., neural cells). In view of their ability to function in both capacities, microglia might be regarded as stand-by cells at the service of the two systems when needed. The two functions are complementary, as glutamate removal from the extracellular microenvironment is required not only for the neurons, but also for T-lymphocyte activation (Angelini et al., 2002). Recent studies in our laboratory showed that interferon (IFN)-g, a characteristic product of the Th1 cells that is both protective and destructive, can simulate the effect of activated T-cells or microglia (Shaked, I., Butovsky, O., Mizrahi, T, Gersner, R., Xiao, X., Soteropoulos, P., Tolias, P., Hart, R. P., and Schwartz, M. unpublished observations). Thus, once activated, the Th1 cells—acting at least partially via secretion of IFN-g—boost the ability of microglia to remove glutamate. These findings substantiate the contention that the innate response, in coping with self-inflicted damage, is assisted by a T-cell-mediated autoimmune response, just as it is assisted by a T-cell-mediated immune response to cope with invading microorganisms (Nevo et al., 2001; Schwartz and Kipnis, 2002). The results presented above argue against the notion that pro-inflammatory microglia have a detrimental effect in neurodegenerative conditions, as MHC-II-expressing microglia are the ones that buffer glutamate. This conclusion is also in line with recent data suggesting a link between proinflammatory microglia and tissue repair (Gasque et al., 2002). In patients with neurodegenerative diseases, microglia have often been implicated in the ongoing process of degeneration. Our results suggest that activated microglia comprise many phenotypes, not all of them detrimental. Glutamate uptake was found, unexpectedly, to be increased in rats with an experimental autoimmune disease (Ohgoh et al., 2002). On the other hand, since our results show that the same mechanism is responsible for both protection and destruction, this finding might have been expected. It thus seems that nothing beyond a modest constitutive level of immune activity is needed by the healthy brain, and that anything beyond that might even have a destructive effect in individuals susceptible to autoimmune disease. In contrast, in the diseased or damaged brain a heightened level of activity is both necessary and beneficial—as long as it is well regulated. It thus seems that the dialog between activated T-cells and microglia enables the latter to acquire a protective rather than a destructive phenotype. This appears to be achieved by the T-cell-induced activation of a microglial glutamate clearance mechanism, which in the normal resting CNS is quiescent (and not necessary) and depends upon T-cell-induced activation for mobilization of its protective action against an environment which has turned hostile. The results further suggest that the heightened ability of microglia to clear glutamate is in line with the enhanced expression of a battery of genes known to participate in protection from oxidative stress and other threatening situations and thus confers better ability to withstand such hostile conditions.

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The beneficial outcome of T-cell-mediated protection against the consequences of insults could also be a function of supply of neurotrophic factors. Data from our laboratory suggest that treatment with autoimmune T-cells leads to a transient increase in expression of such neurotrophic factors as NGF, BDNF, and NT-3. Since neurotrophins (NT)s are known to play an important role in the posttraumatic maintenance, survival, regeneration and neuroprotection of neuronal tissue (see chapter by Nakagawa and Schwartz), it seems likely that the beneficial effect of autoimmune T-cells is mediated, at least in part, by NTs (Kerschensteiner et al., 1999; Olsson et al., 2003). The NTs may influence neuronal survival directly through binding to their receptors on neurons, or indirectly by modulating the local immune response, or both. The cellular source of NTs could be microglia, astrocytes or infiltrating T-cells (Barouch and Schwartz, 2002; Moalem et al., 2000).

7. Synthesizing the findings The T-cell-mediated response directed to self-antigens residing at the site of CNS damage is spontaneously evoked. T-cells isolated from the lymphocytes of spinally injured Lewis rats, a strain susceptible to EAE, are capable of causing neurological deficits and histopathological changes similar to those seen in EAE, when injected into naı¨ve syngeneic rats (Popovich et al., 1996, 2001). Our findings have shown that T-cells isolated from spinally injured rats of a strain known to be resistant to autoimmune CNS diseases, when transferred into newly injured Lewis rats, are capable of conferring neuroprotection without EAE-like changes. We therefore suggest that an insult to the CNS, caused either by exogenous pathogens or by pathogen-free trauma, signals the immune system for assistance in protecting the tissue against the spread of damage. Experiments using different T-cell lines suggest that the neuroprotection induced by weakly encephalitogenic and by strongly encephalitogenic autoreactive T-cells is similar (Moalem et al., 1999a,b), supporting the notion that autoimmune T-cells may be stimulated to facilitate protection of the damaged tissue against further damage, without inducing an autoimmune disease. The fact that the protection evoked by the spontaneous T-cell response to a CNS insult is too weak to cause significant recovery might be attributable, at least in part, to the activity of naturally occurring CD4þCD25þ regulatory T-cells, whose presence might reflect an evolutionary compromise between two conflicting needs, namely for the presence of patrolling autoimmune T-cells on alert for action in healthy individuals, and avoidance of the risk of autoimmune disease posed by these autoimmune T-cells (Kipnis et al., 2002a,b; Schwartz and Kipnis, 2002a,b). This view of beneficial autoimmunity in the context of CNS trauma might explain the presence of autoimmune T-cells frequently observed in healthy individuals. Our theory that inflammation, as the body’s healing process, is mediated by microglia with the assistance of activated autoimmune T-cells, appears to contradict the conclusions of other studies on neurodegenerative diseases (Hauben and Schwartz, 2003; Schwartz and Kipnis, 2002a,b). Given that the observations are the same, the difference between our interpretation and those of others might be attributable to a number of possible reasons: (i) the fact that inflammation in general and postinjury inflammation in particular is not a single event, but a series of events that together produce a multifactorial and

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multiparameter dialog between the tissue and the immune systems; (ii) differences in the insult model selected for study (transection, contusion, injection of yeast, etc.); (iii) strain differences (susceptible or resistant to autoimmune disease); (iv) differences in the duration of the assessment period (short or steady-state) and in the parameter assessed (morphological or behavioral); (v) the fact that the beneficial effect of inflammation does not come free of charge (in terms of additional neuronal loss); and (vi) the need for an optimal balance in terms of the time of onset, duration, phenotype and intensity of inflammation in order to achieve a beneficial response. It is clear from our results that if the immune cells are not active enough, or if their numbers are insufficient, the damage continues to progress in the presence of the activated innate immune cells, leading observers to assume that these were the cells causing the ongoing spread of damage (Eikelenboom et al., 2002; Lue and Walker, 2002; Magnus et al., 2002; McGeer and McGeer, 2002; Polazzi and Contesabile, 2002; Srebro and Dziobek, 2001; Taylor et al., 2002). Our data suggest, however, that the presence of activated immune cells at the lesion site does not necessarily mean that these cells are contributing to the ongoing damage. It seems more likely that they were recruited to help but are unequal to the task. We therefore envisage the following scenario: Microglia, which can be either destructive or protective, are recruited to the lesion site. If they are properly activated but there are too few of them to be effective, the degeneration continues. This is what happens in the case of neurodegenerative disorders. If, on the other hand, the activation is not appropriate, or if the immune system is malfunctioning, the autoimmune T-cells might contribute to the ongoing degeneration. In the former case, boosting of the autoimmune response without risk of autoimmune disease will be beneficial. In our experiments with rats and mice, we have successfully adopted this approach as a therapeutic measure, using a weak self-antigen to make sure of activating only T-cells with low affinity and avoiding pathogenic T-cell clones.

8. Therapeutic vaccination for neurodegenerative conditions In our view, the role of immune cells in the injured CNS does not differ from their role in any tissue under stress from exogenous (nonself) pathogens. A balance is required so that the cost of the immune activity does not exceed the protective benefit (Kipnis and Schwartz, 2002). In the absence of proper regulation, activated microglia might either be insufficiently effective or cause deleterious, chronic hyperinflammation. All individuals, even those not inherently equipped with resistance to autoimmune diseases, can benefit postinjury from vaccination with myelin peptides (Hauben et al., 2001a,b; Kipnis et al., 2002a,b). In susceptible strains, this beneficial effect might (depending on the choice of antigen used for the vaccination and the adjuvant) come at the price of a transient autoimmune disease (Hauben et al., 2000a,b), accompanied by some loss of neurons (Mizrahi et al., 2002). In humans, because of major histoccompatibility complex diversity, cryptic (nonpathogenic) epitopes cannot be predicted (Kipnis and Schwartz, 2002). Therefore, to design a safe therapeutic strategy for individuals who are susceptible to developing an autoimmune disease in the CNS, it will be necessary to use weak, selfreactive antigens, such as altered-peptide ligands (Hauben et al., 2001a,b). Our group has

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discovered that the weak self-reacting antigen Cop-1 (glatiramer acetate) can provide effective neuroprotection in several models of CNS damage (Kipnis et al., 2000; Schori et al., 2001a,b). Cop-1, a drug approved by the Food and Drug Administration (FDA) for use in multiple sclerosis, has been proposed as an antigen that interacts weakly with a wide range of self-reacting T-cells (Hafler, 2002; Kipnis and Schwartz, 2002). It is interesting to note that after traumatic injury to myelinated axons, many different myelin-related antigens, including Nogo, can lead to the same beneficial outcome (Hauben et al., 2001a,b). Rescue of damaged nerves by therapeutic T-cell-mediated vaccination is accompanied by sprouting (O. Butovsky, unpublished data). Research in the past few years has demonstrated obstacles to sprouting and regeneration in the injured CNS caused by myelin-associated inhibitors of axonal regrowth, scar formation, and cavitation (Huang et al., 1999; Merkler et al., 2001; Qiu et al., 2002; Schnell and Schwab, 1990). Antibodies that neutralize myelin inhibitors facilitate sprouting and regrowth (Schnell and Schwab, 1990). It was demonstrated that pre-trauma immunization with myelin-associated proteins results in regeneration, which was attributed to antibodies (Huang et al., 1999). Moreover, passive transfer of Nogo-A-specific antibodies facilitates recovery by a mechanism that leads to efficient myelin clearance (Merkler et al., 2001). It thus seems that recovery can be facilitated not only by autoimmune T-cells but also by antibodies to self-antigens. However, the mechanisms that underlie the beneficial effects of these immune factors are still a matter of debate (Schwab, 2002; Schwartz and Hauben, 2002). Immune vaccination is one of several ways to augment the local innate response. Another way is by local application of autologous activated macrophages (Rapalino et al., 1998). Both apparently have the effect of helping the local environment to cope with the injurious conditions, allowing better survival of the remaining tissue and cell bodies and the possibility of sprouting and regeneration. Because both degeneration and repair of the damaged CNS are highly complex processes that comprise multiple operations, it is unlikely that a single intervention with a single compound (cytokine, neurotrophic factor or drug) will be sufficient to achieve repair. The approach of harnessing the immune system is more likely to supply the multiplicity of factors and processes in the right amounts and at the right time. We suggest that strategies based on T-cell-mediated therapeutic vaccination with a weak self-reactive antigen might be the optimal choice. For clinical application the choice of antigen and adjuvant should be based on considerations of safety, such as the risk of autoimmune disease and plasticity in adult CNS (Raineteau et al., 2001). This therapeutic strategy should be viewed not simply as another pharmacological therapy, but as an approach that stimulates the body to use a mechanism that has developed through evolution (its own immune system) rather than experimental manipulation (Schwartz and Kipnis, 2001).

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A role for lactate released from astrocytes in energy production during neural activity? Eugene L. Roberts Jr.a,b,* and Ching-Ping Chihb b

a Department of Neurology, University of Miami School of Medicine, Miami, FL 33136, USA Geriatric Research, Education, and Clinical Center, Miami VA Medical Center, Miami FL 33125, USA p Correspondence address: Department of Neurology, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, USA E-mail: [email protected]

Contents 1. 2.

3. 4.

Introduction Major assertions of the ANLSH 2.1. Assertions 2.2. Do active neurons prefer lactate to glucose? 2.3. Convincing evidence showing that active neurons prefer lactate to glucose is still lacking 2.4. Does activity-induced glucose uptake occur predominantly in astrocytes? 2.5. Are neurons lactate consumers, and astrocytes lactate producers? Role of lactate as an energy substrate in the brain Concluding remarks

Glucose has long been viewed as the main energy source for brain cells, while lactate served as a secondary energy source. Recently, the astrocyte –neuron lactate shuttle hypothesis (ANLSH) has proposed that, when neurons are activated, they preferentially consume lactate generated by astrocytes to meet their energetic needs. In this chapter, we examine the theoretical basis and current evidence for the ANLSH. From this examination, we find little support for the ANLSH and much credibility for the conventional view of glucose utilization. 1. Introduction Glucose has long been viewed as the primary energy source for brain cells. Lactate, a glucose metabolite, has been thought of as a secondary energy source utilized by brain cells when its levels are high (Nemoto et al., 1974) or when glucose levels are low Advances in Molecular and Cell Biology, Vol. 31, pages 391–407 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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(Thurston et al., 1983). Recently however, a new model of brain glucose utilization termed the astrocyte –neuron lactate shuttle hypothesis (ANLSH) (see Pellerin et al., 1998a; Sibson et al., 1998; Magistretti and Pellerin, 1999; Magistretti et al., 1999; Rothman et al., 1999; Magistretti, 2000) has drastically redefined the role of lactate in brain energy metabolism. According to the ANLSH, lactate released from astrocytes is the primary energy source for active neurons. Neuronal activity is thought to stimulate astrocytic production of lactate indirectly by releasing glutamate, which is taken up by astrocytes via high affinity Naþ-dependent transporters. Increases in glutamate and Naþ in glia activate glutamine synthetase and Naþ – Kþ ATPase, respectively. The latter stimulates astrocytic ATP consumption, leading to activation of anaerobic glycolysis (conversion of glucose to lactate) in astrocytes. The resulting lactate is envisaged to be transported out of astrocytes and into neurons, where it is oxidized to fuel the activity-related energy needs of neurons. The conventional (Fig. 1) and ANLSH (Fig. 2) models of glucose metabolism in brain tissue differ most noticeably in where and how glucose is utilized. In the conventional model, neural activation stimulates Naþ – Kþ ATPase activity, causing increased ATP utilization. ATP consumption activates glycolysis (the conversion of glucose to pyruvate) and increases glucose oxidation in both neurons and astrocytes. In contrast, the ANLSH compartmentalizes glucose and lactate consumption resulting from neural activity. According to the ANLSH, neural activation stimulates glucose consumption in astrocytes, but not in neurons, via anaerobic glycolysis. Instead of metabolizing glucose during neural activity, neurons consume lactate released from astrocytes. The ANLSH has received widespread attention since it represents a significant departure from the classical view of glucose utilization in the brain. If true, the ANLSH would require a major change in how we think about brain energy metabolism. In this chapter, we examine the theoretical background and critically review the experimental evidence regarding the ANLSH to see whether such a change in thought is warranted. 2. Major assertions of the ANLSH 2.1. Assertions Studies supporting the ANLSH assert that (i) active neurons preferentially use lactate instead of glucose (Magistretti and Pellerin, 1996, 1999; Tsacopoulos and Magistretti, 1996; Magistretti, 1999; Magistretti et al., 1999), (ii) activity-induced glucose utilization occurs predominantly, if not exclusively, in astrocytes (Sibson et al., 1998; Magistretti, 1999), and (iii) lactate is produced by astrocytes and shuttled to neurons during activity (Magistretti et al., 1999; Magistretti, 2000). In Section 2.2, we discuss the theoretical background and experimental evidence regarding these assertions. 2.2. Do active neurons prefer lactate to glucose? The brain has similar concentrations of glucose and lactate (about 1 mM; Kuhr and Korf, 1988; Prichard et al., 1991; Harada et al., 1992; Silver and Erecinska, 1994; Fray et al., 1996; Hu and Wilson, 1997; Lowry et al., 1998). Several questions deserve

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Fig. 1. Conventional view of glucose metabolism in astrocytes and neurons when neural activity increases. Increased Naþ, Kþ-ATPase activity boosts ATP consumption in both cell types, because decreasing ATP levels leads to rapid activation of the glycolytic enzymes hexokinase (HK) and phosphofructokinase (PFK). Thus, increased neural activity leads to increased consumption of glucose in both neurons and astrocytes via either aerobic or anaerobic pathways. Glucose has the following advantages over lactate as an energy substrate during neural activity (see numbers on figure): (1) HK and PFK are very sensitive to declines in ATP levels and activate rapidly when energy demand increases. Also, conversion of glucose to pyruvate is more favorable energetically than conversion of lactate to pyruvate (see Section 2.2.3). (2) Glucose is readily available to both neurons and astrocytes. In particular, glucose concentrations in brain cells exceed greatly the Km for HK (see Section 2.2.1). Also, GLUT3 transports glucose seven times faster than GLUT1. (3) When glucose is metabolized during neural activity, both NADH shuttles (see Section 2.2.4) and lactate production are available to maintain the cytoplasmic redox balance. These advantages are in contrast to the limitations of the ANLSH pointed out in Fig. 2. Abbreviations: GLUT1 and GLUT3: glucose transporters 1 and 3, respectively; GAD-3-P: glyceraldehyde3-phosphate; 1,3-BPG: 1,3-bisphosphoglycerate; LDH-1 and LDH-5: lactate dehydrogenases 1 and 5, respectively; M: mitochondrion.

consideration when comparing these substrates as energy sources for active neurons: (1) Are neurons equipped to use these substrates? (2) Which metabolic pathway (glucose or lactate utilization) responds faster to increases in energy demand? (3) Which metabolic pathway is energetically more favorable? (4) Which pathway makes it easiest to maintain the cytoplasmic redox balance? and (5) Is glycolysis under some conditions advantageous for local synthesis of ATP in neurons? In the following analysis, we discuss whether lactate can be the principal energy substrate of active brain cells, as proposed by the ANLSH.

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Fig. 2. Neuronal and astrocytic glucose metabolism during neural activity according to the astrocyte–neuron lactate shuttle hypothesis (ANLSH). The ANLSH requires that astrocytes do not consume glucose oxidatively during neural activity, but instead metabolize all glucose to lactate. The ANLSH also requires that active neurons do not metabolize glucose but instead metabolize lactate produced by astrocytes. In the figure, dashed lines and open arrow heads show metabolic pathways not available to neurons (the glycolytic pathway) and to astrocytes (the oxidative phosphorylation pathway), according to the ANLSH. Limitations in the ANLSH’s view of energy metabolism pointed out in the figure are: (1) In contrast to HK and PFK, LDH is not regulated by changes in ATP levels, so LDH responds slowly to increased cellular energy demands. Also, conversion of lactate to pyruvate may not be favored thermodynamically during neural activity (see Section 2.2.3). (2) Increases in extracellular lactate levels lag well behind the start of neural activity. This tends to limit lactate utilization as a choice for meeting the energy demands of neural activity since increases in lactate oxidation depend heavily upon an increase in available lactate (see Section 2.2.2). (3) If lactate is the only available substrate for neurons, then the cytoplasmic redox balance in neurons will depend entirely upon NADH shuttles (see Section 2.2.4). Also, LDH must compete with glyceraldehyde-3-phosphate-dehydrogenase (the enzyme converting GAD-3-P to 1,3-BPG) for NADþ. This competition can limit lactate utilization. These limitations in the ANLSH model contrast with the advantages of the conventional model (Fig. 1).

2.2.1. Neurons and astrocytes are well equipped to utilize both glucose and lactate Both neurons and astrocytes have high activities of glycolytic enzymes (Cimino et al., 1998; Lai et al., 1999). High specific activities of glycolytic enzymes are found within synaptosomal membranes and nerve endings (Wilson, 1972; Knull, 1978; Kao-Jen and Wilson, 1980; Lim et al., 1983). This high glycolytic capacity in neurons and astrocytes is coupled to high capacity glucose transporters (Vannucci et al., 1997). The dominant glucose transporter in neurons (GLUT3) transports glucose seven times faster than the dominant glucose transporter in astrocytes (GLUT1) (Vannucci et al., 1997). Much evidence exists for

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both neurons and astrocytes increasing their glycolytic rates in response to increased energy demands (e.g., Walz and Mukerji, 1988; Peng et al., 1994; Sokoloff, 1999). A high concentration of blood glucose and efficient glucose transporters in the brain help maintain brain glucose levels between 0.35 and 2.6 mM (Silver and Erecinska, 1994; Fray et al., 1996; Hu and Wilson, 1997; Lowry et al., 1998). Glucose is evenly distributed between the intra- and extracellular compartments of the brain (Pfeuffer et al., 2000). Increased blood flow accompanying brain stimulation (Fellows and Boutelle, 1993) may increase glucose delivery to the brain. Even with this possible increased delivery of glucose, brain glucose levels have been found to either increase (Fellows and Boutelle, 1993) or decrease (up to 20%; see data of Fray et al., 1996; Hu and Wilson, 1997) transiently during neural stimulation. However, glucose levels were well above the Km for hexokinase (0.04 mM; Lowry and Passonneau, 1964) in these studies. Thus, although neuronal processes are not directly coupled to the circulation as astrocytic processes are, the high glucose concentrations in the brain and the presence of GLUT3 suggest that neuronal glucose supplies are adequate. Lactate dehydrogenase (LDH) and lactate transporters are also present in both neurons and astrocytes (Bittar et al., 1996; Pellerin et al., 1998b). In vitro studies have shown that neurons and astrocytes can oxidize lactate at similar rates (Peng et al., 1994). 2.2.2. Activation of glycolysis occurs rapidly during increased energy demand During neural activity, oxygen utilization goes up rapidly (Malonek and Grinvald, 1996; Malonek et al., 1997), so rapid increases in the supply of pyruvate to mitochondria are necessary to maintain oxidative metabolism. Both glucose and lactate can supply pyruvate to mitochondria. However, the glycolytic pathway, which converts glucose to pyruvate, can be activated much faster than lactate dehydrogenation, which converts lactate to pyruvate. The activities of key glycolytic enzymes are tightly regulated by energy demand (Clarke et al., 1989). Thus, when energy demand is low, glycolysis is inhibited due to down-regulation of the key glycolytic enzymes hexokinase and phosphofructokinase by high ATP levels (Clarke et al., 1989). For example, brain hexokinase is 97% inhibited under resting conditions (Clarke et al., 1989). This basal inhibition gives cells the potential to respond quickly to increased energy demands. This is particularly important for neurons because they shift energy demands drastically between their active and inactive states (Ames, 2000). During neural activity, increased Naþ – Kþ ATPase activity in neurons should augment their ATP utilization. The resulting changes in adenine nucleotide and phosphate levels then activate several key regulatory enzymes in glycolysis and oxidative phosphorylation. Thus, rapid increases in the rates of glucose utilization can occur without increases in glucose concentrations. For example, in cultured cerebellar neurons, neuronal glucose utilization increased two-fold without an increase in glucose concentration during heightened energy demand (Peng et al., 1994). In contrast to glycolytic enzymes, the activity of LDH is not coupled to energy demand, and it is high enough that the reaction will be close to its thermodynamic equilibrium. Thus, for lactate dehydrogenation to increase, either lactate levels must go up, or pyruvate levels must decrease. An increase in lactate levels may be the only viable choice since

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pyruvate levels go up during increased neural activity (Goldberg et al., 1966; Ferrendelli and McDougal, 1971). For example, in cultured cerebellar neurons exposed to heightened energy demand and constant levels of either glucose, pyruvate, or lactate, the rates of both glucose and pyruvate oxidation increased while the rate of lactate oxidation was not significantly altered (Peng et al., 1994). Thus, for lactate to be a major substrate for neuronal oxidative metabolism during neural activity, its extracellular concentrations must increase, and do so quickly. However, instead of going up rapidly with neural activity, increases in extracellular lactate in situ lag behind neural activity (Fellows et al., 1993; Fray et al., 1996; Hu and Wilson, 1997). For example, in rats undergoing 5 min of continuous tail pinch stimulation, lactate showed no measurable increases for the first 2.5 min of stimulation, and peaked (55 –80% greater than control) 5 min after cessation of stimulation (Fellows et al., 1993). Sometimes extracellular lactate does not increase until after neural activity has ended. For example, in the electrically stimulated rat brain, lactate levels began increasing 10– 12 s after termination of a 5 s stimulation, and peaked (40 –100% above control) after another 50– 60 s (see Hu and Wilson, 1997). These increases in extracellular lactate appear far too slow to meet the energy demands of neural activity. Some investigators have also argued that lactate may become a significant energy source for neurons during prolonged activation when lactate levels increase (Hu and Wilson, 1997). However, increases in extracellular lactate concentrations during prolonged neural activity are relatively modest (35 – 135%; Fellows et al., 1993; Fray et al., 1996; Hu and Wilson, 1997), and may not create conditions thermodynamically favorable for driving the LDH-catalyzed reaction in neurons toward lactate oxidation. Also, such a utilization of lactate would come at the expense of glucose utilization since both lactate dehydrogenation and glycolysis need NADþ. This means lactate must compete with glucose for utilization. Early during neural activity, as glycolytic rates increase, pyruvate (Goldberg et al., 1966; Ferrendelli and McDougal, 1971) and Hþ (Hochachka and Mommsen, 1983; Chesler and Kaila, 1992) levels go up, and the cytosolic NADþ/NADH ratio decreases (Clarke et al., 1989). These changes favor the production of lactate via the LDH-catalyzed reaction rather than its utilization. Even if conditions support lactate oxidation in neurons during activity, LDH must compete with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for NADþ (Fig. 2). Both LDH and GAPDH are present in high concentrations in brain cells (McIlwain and Bachelard, 1985). Heightened glyceraldehyde-3-P levels resulting from increased glycolytic activity boost the activity of GAPDH (Williamson, 1965). Also, increases in pyruvate levels caused by greater glycolytic activity may limit lactate oxidation in neurons since LDH-1 is highly sensitive to pyruvate product inhibition (Stambaugh and Post, 1966, also see Section 2.3.2). Thus, high glycolytic rates may preclude lactate use in neurons. 2.2.3. Glucose oxidation is energetically more favorable than lactate oxidation Glucose conversion to pyruvate is more advantageous energetically than conversion of lactate to pyruvate. The reason for this is that the HK- and PFK-catalyzed reactions of glycolysis are very favorable thermodynamically and are essentially irreversible under intracellular conditions (Lehninger et al., 1993; Chih et al., 2001a). In contrast,

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thermodynamic conditions are highly unfavorable for the conversion of lactate to pyruvate during neural activity (also see Section 2.2.2). In addition, conversion of glucose to pyruvate yields two net ATPs, while conversion of lactate to pyruvate yields no ATP. These factors favor glucose utilization during increased ATP consumption. Thus, neurons gain an energetic advantage by oxidizing glucose instead of lactate once increased energy demand has activated key glycolytic enzymes. 2.2.4. Maintaining the cytoplasmic redox balance may be more difficult with lactate as the principal energy substrate Both glycolysis and lactate dehydrogenation decrease the cytoplasmic redox ratio (NADþ/NADH). This means that NADþ must be regenerated faster to accommodate higher rates of glycolysis or lactate dehydrogenation during increased energy demand. When glucose is used as a substrate, the cytoplasmic redox balance can be maintained by NADH shuttles (oxidative metabolism) and lactate production (anaerobic glycolysis). The fact that lactate is produced during neural activity suggests that activation of NADH shuttles in the brain may lag behind increases in energy demand. Glycerol-3-P dehydrogenase (glycerol-3 phosphate shuttle) and malate dehydrogenase (malate –aspartate shuttle) (Cheeseman and Clark, 1988; McKenna et al., 1993) are involved in reoxidizing NADH generated from glycolysis via GAPDH. Each NADH shuttle system depends on the availability of carrier metabolites (Berry et al., 1992). The carrier for the glycerol-3-P shuttle, dihydroxyacetone-3-P, is also a glycolytic intermediate. When the glycolytic rate increases, dihydroxyacetone-3-P levels also go up (Williamson, 1965), so the rate of NADþ regeneration can increase (Berry et al., 1992) to maintain a high glycolytic rate. When lactate is used as substrate, the cytoplasmic redox balance can rely only on NADH shuttles. Also, without increases in glycolysis, dihydroxyacetone-3-P levels will not go up with increased energy demand, eliminating a driving force for the regeneration of NADþ via one shuttle mechanism. Moreover, the accessibility of LDH to the glycerol3-P shuttle may differ from that of GAPDH. For example, in hepatocytes, NADH coming from lactate dehydrogenation failed to access the glycerol-3-P shuttle, possibly because the enzymes for glycolysis are segregated from those for gluconeogenesis (Berry et al., 1992). Thus, the NADH shuttle may be a limiting factor for lactate oxidation. Indeed, Peng and Hertz (Peng et al., 1994) found that, in cultured cerebellar granule neurons exposed to either glucose, pyruvate, or lactate and to high Kþ concentrations, the rates of glucose and pyruvate oxidation doubled, while the rate of lactate oxidation showed no statistically significant change. The fact that pyruvate oxidation, but not lactate oxidation, can increase with elevated energy demand shows that lactate oxidation is limited at the conversion of lactate to pyruvate, possibly due to a limited supply of NADþ. Thus, the ability of neurons to increase lactate oxidation during heightened energy demand is limited even in the absence of glucose. 2.2.5. Glycolysis may generate ATP locally in both neurons and astrocytes for ion pumping and other processes Anaerobic glycolysis may be an essential source of energy for localized areas of brain cells (Wu et al., 1997). This point is particularly true for synapses and for the most

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distal and finest astrocytic processes (see chapters by Derouiche and by Hertz, Peng et al.). For example, mitochondria occur with low frequency in axonal varicosities, and are indeed absent in 50% of synaptic boutons in the CA3 to CA1 projection in the hippocampus (Shepherd and Harris, 1998). Also, mitochondria are rarely observed in dendritic spine heads in the cerebral cortex (Wu et al., 1997). However, glycolytic enzymes are bound to postsynaptic densities (PSDs; Wu et al., 1997), which are proteinaceous structures attached to the surface of dendritic spine heads containing neurotransmitter receptors, ion channels, and protein kinases. These protein kinases consume ATP generated in PSDs via glycolysis (Wu et al., 1997). The presence of glycolytic enzymes, LDH (Wu et al., 1997), and the monocarboxylate transporter (MCT) (Bergersen et al., 2001) in PSDs, and the rarity of mitochondria in dendritic spine heads in the cerebral cortex (Wu et al., 1997), suggest that at least some glucose may be used anaerobically and that the lactate produced via glycolysis in PSDs may be released via the MCT. This segregation of glycolytic enzymes from mitochondria may provide a reasonable explanation for the increase in lactate production during heightened neural activity even when there is no shortage of oxygen. Also, energy generated from glycolysis has been linked to ion transport via Naþ –Kþ ATPase. Studies of erythrocytes (Proverbio and Hoffman, 1977), smooth muscle (Paul et al., 1979), and cardiac muscle (Weiss and Hiltbrand, 1985) have provided compelling evidence that glycolytically generated ATP is linked to Naþ –Kþ ATPase. Although no direct evidence for a similar link has been identified yet in the brain, both glycolytic enzymes and Naþ –Kþ ATPase are bound at high specific activity within synaptosomal membranes (Knull, 1978; Lim et al., 1983). Also, several studies have shown that ion transport is less robust when glucose is replaced either by lactate or by pyruvate (Lipton and Robacker, 1983; Raffin et al., 1992; Roberts, 1993; Silver et al., 1997). The association of glycolytic enzymes with Naþ –Kþ ATPase in synaptic membranes (Knull, 1978; Lim et al., 1983) suggests that glucose, rather than lactate, is used as the substrate to provide energy for the Naþ – Kþ pump in neurons.

2.3. Convincing evidence showing that active neurons prefer lactate to glucose is still lacking Several studies have been considered as strong evidence for the concept that active neurons use lactate instead of glucose. In the following sections, we discuss the uncertainties of these studies. 2.3.1. Substrate utilization in the isolated retinal preparation Poitry-Yamate et al. (1995) reported that Mu¨ller cells fed 14C-labeled glucose released 70% less radioactive 14C-lactate to the bathing medium when photoreceptors were included in the cell suspension. They concluded that lactate released from retinal glia (Mu¨ller cells) was used preferentially by photoreceptors even in the presence of glucose. However, because the mixed neuronal –glial samples contained only about half the number of glial cells per mg of protein (Poitry-Yamate et al., 1995), the radioactivity of

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bath lactate in the mixed photoreceptor-Mu¨ller cell cultures had to be half that of Mu¨ller cells alone (see Chih et al., 2001a). This dilution of radioactivity, and not an increased lactate utilization by photoreceptors, would account for most of the decrease in radiolabeled lactate/mg protein in the bath. Thus, results from this study do not support the assertion that neurons prefer to metabolize lactate from astrocytes instead of ambient glucose. 2.3.2. Distributions of LDH-1 and LDH-5 The dominant presence of the LDH isoform LDH-1 in neurons has also been cited as support for the ANLSH. ANLSH proponents suggest that LDH-1 drives the LDH-catalyzed reaction toward pyruvate production (Bittar et al., 1996). However, the direction of a reaction is determined by the relative concentrations of its substrates and products, and is not influenced by the enzyme catalyzing the reaction (Lehninger et al., 1993). Compared with LDH-5, LDH-1 has a lower Km for pyruvate or lactate, a lower Vmax ; and is more easily inhibited by its substrates and products (Cahn et al., 1962; Stambaugh and Post, 1966; Everse and Kaplan, 1973; Nitisewojo and Hultin, 1976). The fact that LDH-1 is more easily inhibited by its substrates and products may have important functional consequences (Cahn et al., 1962). For example, LDH-1 has a higher sensitivity to pyruvate substrate inhibition and lactate product inhibition, which may help direct pyruvate generated from glycolysis toward the TCA cycle instead of toward lactate production (Cahn et al., 1962). This would allow full oxidation of glucose in cells where LDH-1 is the dominant isoform, such as neurons and cardiac muscle cells (Cahn et al., 1962). Also, LDH-1 has a high sensitivity to pyruvate product inhibition ðKi ¼ 0:18 mMÞ (Stambaugh and Post, 1966). Since physiological pyruvate levels are between 0.1 and 0.2 mM (McIlwain and Bachelard, 1985), any slight increase in pyruvate levels during heightened glycolysis may help inhibit LDH-1 and prevent lactate oxidation. Thus, the dominant presence of LDH-1 does not help lactate compete with glucose as an energy substrate in neurons. 2.3.3. Substrate utilization in isolated chicken embryo ganglia A study of substrate utilization in chicken embryo ganglia (Larrabee, 1995) has also been cited as evidence that brain tissue preferentially uses lactate instead of glucose (Tsacopoulos and Magistretti, 1996). Briefly, this study showed that isolated chicken embryo ganglia produced more CO2 from lactate than from glucose when both substrates were present. However, as stated earlier, the rate of glycolysis is tightly regulated by energy status and is largely inhibited under resting conditions. In contrast, lactate utilization can be increased by increasing the concentration gradient for lactate. In Larrabee’s (1995) study, lactate concentrations were about five times greater than physiological lactate levels. Since glycolysis is down-regulated in resting brain tissue, lactate may become the dominant substrate. An analogous situation occurs in resting red muscle (Pearce and Connett, 1980), where lactate oxidation can account for 70% of energy production when ambient lactate levels are 8 mM, which is high enough to suppress both glucose and glycerol oxidation. However, this result does not mean that lactate is the preferred substrate for red muscle cells during activity. It means only that lactate levels are

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high enough to inhibit utilization of other substrates when cellular energy demand is low. Since the ANLSH concerns energy utilization by active neurons, the study by Larrabee (1995) does not apply to the ANLSH. It is also important to note that in this study, the cell type(s) using lactate was (were) not identified. As will be discussed below, astrocytes are equally as able to use lactate as neurons (Peng et al., 1994). 2.3.4. In vivo measurements of lactate levels during neural activity In another study thought to support the ANLSH (Hu and Wilson, 1997), transient changes in extracellular lactate and glucose levels were measured in the rat hippocampus in response to repetitive trains of electrical stimulation. After each stimulus train, glucose and lactate declined transiently, and the data were interpreted as showing that lactate declined more in later stimulus trains after mean lactate levels had gone up (Hu and Wilson, 1997). However, results from only one experiment were reported, and no statistical analysis of the data was provided. Also, each repetitive stimulation was given when lactate was sharply declining and glucose was gradually increasing. This complicated interpretation of the actual changes in glucose and lactate caused by stimulation. Finally, this kind of experiment provides no details regarding what cell types were using glucose or lactate. 2.3.5. Utilization of lactate under stress Hippocampal slices subjected to hypoxic (Schurr et al., 1997) or excitotoxic (Schurr et al., 1999) stress fail to recover synaptic transmission after these stress conditions, even in the presence of sufficient glucose, if the monocarboxylate transport inhibitor 4-CIN (alpha-cyano-4-hydroxycinnamate) is present (Schurr et al., 1999). These studies have been viewed as strong support for the ANLSH (e.g., Magistretti et al., 1999), because they suggest that neurons need lactate during recovery from stress, and that glucose alone is insufficient for such recovery. However, 4-CIN blocks pyruvate and lactate entry into isolated mitochondria (Halestrap and Denton, 1974; Cox et al., 1985) besides blocking lactate transport. Also, 4-CIN compromises normal oxidative metabolism and blocks neural activity (e.g., Amos and Richards, 1996; Chih et al., 2001a). These effects of 4-CIN on mitochondrial function, rather than inhibition of lactate transport, may explain why hippocampal slices do not recover from hypoxia or excitotoxic stress in these experiments. Moreover, these studies apply to pathological stress, and not to stress resulting from normal physiological activity. 2.4. Does activity-induced glucose uptake occur predominantly in astrocytes? A study by Shulman and coworkers is thought by proponents of the ANLSH to provide the most conclusive evidence that activity-induced glucose utilization occurs mainly in astrocytes (Sibson et al., 1998). Briefly, Sibson et al. (1998) found a 1:1 ratio between total oxidative glucose consumption and astrocytic glutamate cycling in the rat brain. They concluded that activity-induced glucose utilization is driven by astrocytic glutamate cycling, and that their results were consistent with the ANLSH.

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However, the 1:1 ratio obtained from this study can vary significantly depending on the assumptions used to calculate this ratio. For example, if the rate of glutamine synthesis via the anaplerotic pathway during normal activity is 30% of total glutamine synthesis, as others have observed (Ku¨nnecke et al., 1993; Lapidot and Haber, 2000), instead of being unchanged, as assumed by Sibson et al., then the stoichiometry between oxidative glucose consumption and glutamate cycling would be roughly 1.5:1 rather than 1:1 (estimated from data in Sibson et al., 1998). It is consistent with this conclusion that Gruetter and coworkers have found a much higher rate of glucose oxidation than of glutamate cycling (see chapter by Gruetter). Also, because the mode of astrocytic glucose consumption was not identified, it is impossible to determine the percentage of glucose being used by astrocytes or neurons (Chih et al., 2001b) even if the 1:1 stoichiometry holds true. If as little as 6% of glucose is metabolized aerobically in astrocytes, then 50% of glucose could have been metabolized by neurons and still satisfy the 1:1 stoichiometry. In this context it is interesting that Rothman and coworkers recently have determined by aid of the astrocyte-specific precursor, acetate, that oxidative metabolism in astrocytes accounts for 15% of oxidative metabolism in the brain in vivo (Lebon et al., 2002). The ANLSH assumes that activity-induced glucose consumption in astrocytes is entirely anaerobic. However, many studies (Swanson, 1992; Eriksson et al., 1995; Hertz et al., 1998) have shown that oxidative metabolism can support astrocytic glutamate uptake. Thus, the reported 1:1 ratio between oxidative glucose consumption and astrocytic glutamate cycling does not necessarily support the ANLSH. Activity-induced 2-DG uptake occurs primarily in the neuropil (Kadekaro et al., 1985), a region enriched in axon terminals, dendrites, and synapses. Since the membranes of synaptic terminals contain a high density of the glucose transporter GLUT3 (McCall et al., 1994; Leino et al., 1997), little apparent reason exists to speculate that neurons do not take up glucose as the ANLSH suggests. Critical experiments determining the actual locus of glucose uptake during activity in situ are in their infancy, but evidence has recently been obtained that both cell types may contribute about equally to glucose utilization (Wittendorp-Rechenmann et al., 2001).

2.5. Are neurons lactate consumers, and astrocytes lactate producers? A key point of the ANLSH is that, during neural activity, lactate is produced by astrocytes and then shuttled to and used by neurons. Although astrocytes produce lactate (Pellerin and Magistretti, 1994; Demestre et al., 1997), no evidence exists showing that neurons use lactate released from astrocytes during neural activity. In fact, in vitro studies have shown that neurons also produce lactate when energy demands increase (Walz and Mukerji, 1988; Peng et al., 1994). The fact that neuronal cultures produce less lactate than cultures of astrocytes has been used as indirect evidence that neurons are primarily lactate consumers, and that astrocytes are mainly lactate producers (Schousboe et al., 1997). However, less neuronal lactate production may simply mean that neurons oxidize glucose more fully (also see Section 2.2.2). As a result, neuronal cultures may not need to consume as much glucose as

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astrocytic cultures (Lopes-Cardozo et al., 1986). It should be stressed that less lactate production by neurons at rest does not mean neurons have a smaller glycolytic capacity than astrocytes, because when oxidative metabolism is blocked, neurons produce as much lactate as astrocytes (Walz and Mukerji, 1988; Peng and Hertz, 2002). The fact that LDH-1, the dominant LDH isoform in neurons, has a lower Km for lactate than LDH-5 has also been used as evidence that neurons may be better suited to use lactate than astrocytes (Bittar et al., 1996), which have both LDH-5 and LDH-1. However, the lower Vmax of LDH-1 may offset the effect of a lower Km : For example, LDH-1 catalyzes a lower rate of lactate production than LDH-5 despite its lower Km for pyruvate (Pesce et al., 1967). Also, in vitro studies have shown that astrocytes oxidize lactate at approximately the same rate as neurons (Peng et al., 1994). Thus, the distribution of LDH apparently does not play a significant role in affecting the rate of lactate utilization in neurons and astrocytes. The heterogeneous distribution of MCT subtypes between neurons and glia has also been suggested as being consistent with the ANLSH (Broer et al., 1997; Pellerin et al., 1998b). However, the kinetic characteristics of the MCT isoforms currently provide little support for the ANLSH. Indeed, the initial rates of lactate uptake by cultured astrocytes and neurons are similar for lactate concentrations of 1– 5 mM (Dienel and Hertz, 2001), suggesting that differences in MCT isoforms do not play a significant role in lactate transport under normal conditions. Also, at physiological lactate concentrations, metabolically driven lactate uptake in both neurons and astrocytes corresponds to at most 25% of the resting rate of glucose oxidation in the brain (Dienel and Hertz, 2001). Thus, lactate probably does not replace glucose as the principal metabolic substrate in neurons.

3. Role of lactate as an energy substrate in the brain The above discussion does not rule out the possibility that lactate is used as a substrate in the brain. Because of the relatively slow transport of lactate across the blood – brain barrier (McIlwain and Bachelard, 1985), most of the lactate produced during neural activity probably has to be utilized by brain cells. Also, lactate can become an important energy substrate when blood lactate levels increase above normal (Ames, 2000). Besides, many in vitro studies have shown that brain tissue can use lactate to maintain neural function (Schurr et al., 1988). Thus, lactate can clearly serve as an alternative energy substrate in the brain. The major difference between the conventional hypothesis and the ANLSH concerns how lactate is produced and used in the brain during neural activity. The conventional hypothesis contends that glucose is the major substrate for both neurons and astrocytes during normal neural activity. Lactate production occurs during neural activity when increases in the glycolytic flux surpass increases in the rate of oxidative phosphorylation, when oxygen is temporarily in short supply, or in areas where mitochondria are rare, such as dendritic spine heads (Wu et al., 1997). Lactate produced by active neurons and astrocytes is then used by inactive brain cells and by those cells with little glycolytic capacity, such as oligodendrocytes (Dringen et al., 1993a). Lactate may also be used by astrocytes for glycogen synthesis (Dringen et al., 1993b). The ANLSH

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contends that lactate is produced by astrocytes and used by neurons during neural activity. So far, there is no clear evidence that shuttling of lactate between astrocytes and neurons actually occurs in situ during neural activity. Also, as discussed above, the kinetic properties of enzymes and transporters involved in the metabolism of glucose and lactate do not support the ANLSH. Indeed, the lack of mechanisms for activating LDH in response to increases in energy demand, and the slow increases in lactate levels during neural activity, suggest that lactate is not intended for use as the major energy substrate during neural activity. Instead, lactate is used by inactive brain cells to keep brain lactate levels low.

4. Concluding remarks Both neurons and astrocytes metabolize glucose and lactate. The ANLSH compartmentalizes the use of glucose and lactate during neural activity by identifying neurons as exclusive lactate consumers and astrocytes as exclusive glucose consumers and lactate producers. However, the ANLSH model of substrate utilization during neural activity is not well supported by current evidence. From a theoretical point of view, glucose is a better substrate for both neurons and astrocytes during neural activity: First, glycolytic enzymes are activated rapidly in response to increased energy demands while LDH is not regulated by energy demand. Second, increases in lactate levels, which drive lactate oxidation, lag well behind neural activity. Third, the conversion of glucose to pyruvate is thermodynamically more favorable than the conversion of lactate to pyruvate. There is no apparent reason why neural activity should only activate astrocytic glycolysis but not neuronal glycolysis, as suggested by the ANLSH, since neurons have a high glycolytic capacity and glucose is readily available to neurons. Experimental evidence also does not support the major assertions of the ANLSH (1) that active neurons prefer lactate to glucose, (2) that activity-induced glucose uptake occurs predominantly in astrocytes, and (3) that astrocytes are the lactate producers and neurons are the lactate consumers. In particular, convincing evidence for shuttling of lactate between astrocytes and neurons during neural activity is still lacking. Thus, both theoretical considerations and experimental evidence provide little support for the ANLSH and much credibility for the conventional view of glucose utilization. Experiments are needed to determine where glucose uptake takes place during neural activity so that the issues raised by the ANLSH may be resolved.

Acknowledgements We would like to thank Dr Peter Lipton (Department of Physiology, University of Wisconsin School of Medicine) for his invaluable advice and ideas regarding the topics covered in this chapter. This work was supported by an award from the American Heart Association, Florida/Puerto Rico Affiliate.

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Principles of the measurement of neuro-glial metabolism using in vivo 13C NMR spectroscopy Rolf Gruetter Departments of Radiology and Neuroscience, University of Minnesota, Minneapolis, MN, USA Correspondence address: Center for MR Research, 2021 6th Street SE, Minneapolis, MN 55455, USA. Tel.: þ 1-612-625-6582; fax: þ1-612-626-2004. E-mail: [email protected](R.G.)

Contents 1. 2.

3.

4.

5.

Introduction Key elements of 13C tracer methodology measured by in vivo 13C NMR spectroscopy 2.1. ‘Tracer methods’ 2.2. Dynamic isotopomer analysis 2.3. 13C NMR methodological aspects Glial metabolism I: brain glycogen 3.1. Brain glycogen, an endogenous store of fuel 3.2. Human brain glycogen metabolism in vivo Glial metabolism II: the glutamate –glutamine cycle 4.1. Glutamate turnover: neuronal oxygen metabolism and the malate – aspartate shuttle 4.2. Glutamine turnover: the hallmark of glial metabolism 4.3. Anaplerosis and the astroglial TCA cycle Concluding remarks

This chapter reviews some recent achievements and insights obtained by 13C NMR in the brain in rats and humans in vivo. The studies discussed include (i) the demonstration that brain glycogen is an important store of glucose equivalents in the brain, providing significant fuel during hypoglycemia; (ii) the demonstration of slow brain glycogen metabolism in non-activated awake brain; (iii) the presence of significant anaplerosis (pyruvate carboxylase activity) in brain in vivo; (iv) the measurement of the energy metabolism of neurons and glia and the metabolic trafficking of glutamate between these two major metabolic compartments; (v) the measurement of a major regulatory metabolic element of oxidative metabolism in the brain, the malate– aspartate shuttle; and (vi) the finding that brain glycogen metabolism is deranged following hypoglycemic episodes, Advances in Molecular and Cell Biology, Vol. 31, pages 409–433 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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suggesting an involvement in the hypoglycemia unawareness syndrome clinically observed in diabetes. 1. Introduction The propagation of electrical impulses between brain cells is accomplished by chemical transmission, achieved by releasing signaling molecules (neurotransmitters) from the presynaptic bouton that interact with receptors on the postsynaptic neuron and thus mediate the transmission of electrical signals from one neuron to the next. It is becoming increasingly clear that normal brain function not only involves the function of neurons on both sides of the synaptic cleft. In addition to the pre- and the postsynaptic neuron, the astroglial compartment has recently gained increased attention by virtue of its importance in maintaining the functionality of the synapse (Schousboe et al., 1993a; Hansson and Ronnback, 1994; Vernadakis, 1996; Magistretti and Pellerin, 1999). Among the many neurotransmitter systems, that of glutamate is probably most abundantly distributed in the central nervous system. The accepted mechanism for the action of glutamate (Shank and Aprison, 1979; Westergaard et al., 1995; Daikhin and Yudkoff, 2000; Lieth et al., 2001), is a prime example for the importance of the interplay of electrical events and metabolism in the action of this important neurotransmitter, as illustrated in the scheme of Fig. 1. Glutamate uptake from the synaptic cleft is characterized by concurrent electrical events due to electrogenic glutamate transporters which are critical in maintaining a low extracellular glutamate concentration in order to avoid excitotoxicity and to maintain

Fig. 1. Scheme depicting the metabolism of neurotransmitter glutamate, which forms the basis for the concept of the glutamate–glutamine cycle, with the rate VNT. Uptake of glutamate in astrocytes from the synaptic cleft (not shown as a separate step) is followed by conversion into electrophysiologically inactive glutamine.

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postsynaptic excitability. Most of the synaptic glutamate is cleared into the astrocytes surrounding the synaptic cleft (Yudkoff et al., 1993; Bergles et al., 1997; Zigmond, 1999; see also the chapter by Schousboe and Waagepetersen). Following uptake into the glial cell, glutamate is converted by glutamine synthetase into the electrophysiologically inactive glutamine, which is transported back to the neuron and converted to glutamate, thereby maintaining the nerve terminal concentration of neurotransmitter glutamate, in a ‘glutamate – glutamine cycle’ (Yudkoff et al., 1988; Kanamori and Ross, 1995; Westergaard et al., 1995; Brand et al., 1997; Rothman et al., 1999; Daikhin and Yudkoff, 2000). It is therefore quite clear that glutamatergic transmission involves metabolism (through the glutamate– glutamine cycle), as well as energy metabolism of the astroglial compartment due to the requirements for the restoration of the ion balance and for glutamine synthesis (Magistretti and Pellerin, 1996; Attwell and Laughlin, 2001). Indeed, it has been reported that the uptake of Glu into the astrocytes was associated with increased glucose metabolism in the astrocyte (Magistretti et al., 1993; Eriksson et al., 1995), thereby linking stimulated energy metabolism between the astroglial and neuronal compartments during neurotransmission. Because of the mostly neuronal localization of glutamate and the exclusively astroglial localization of glutamine synthesis, the measurement of label transfer from glutamate to glutamine, uniquely possible using 13 C NMR spectroscopy (Gruetter, 1993; Gruetter et al., 1994; Hassel et al., 1997; Rothman et al., 1999; Chhina et al., 2001; Bluml et al., 2002; Gruetter, 2002), in principle could serve as an indicator of the rate of the glutamine –glutamate cycle, thought to reflect the rate of glutamatergic action (Yudkoff et al., 1988; Gruetter et al., 1998a; Rothman et al., 1999). While conceptually very simple in its formulation, the interpretation of the labeling of glutamate and glutamine needs to take into account many additional reactions, whose activity cannot be neglected in vivo, such as the rate of glial oxidative metabolism and pyruvate carboxylase, and, potentially, glycogen metabolism. This chapter deals with the requirements on the modeling that are necessary to understand in order to use in vivo NMR spectroscopy for the assessment of glutamatergic metabolism in the intact brain in vivo, but it does not deal with the technical requirements to implement a successful 13C NMR spectroscopy program and NMR methodology.

2. Key elements of spectroscopy

13

C tracer methodology measured by in vivo

13

C NMR

NMR spectroscopy is a non-destructive method that allows the measurement of signals from several compounds and distinct positions within the molecule. The information content of detecting 13C label by NMR is illustrated in Fig. 2. While detection of the 1H NMR signal of hydrogen nuclei adjacent to 13C nuclei is clearly most sensitive (Fig. 2B), the detection of label by directly measuring the signal of 13C provides more information (Fig. 2A). A full review of the methodology involved is beyond the scope of this paper, however, it is important to recognize that many methodological difficulties need to be overcome in measuring 13C label in vivo and this field is far from being fully developed as illustrated by some examples are provided at the end of this section.

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Fig. 2. In vivo NMR detection of 13C label. (A) illustrates a spectral region depicting the detection of 13C label for the C3 and C4 region of amino acids in a 400 ml volume of rat brain at 9.4 Tesla. Reproduced from (Choi et al., 2000). (B) shows the spectral region of the 1H spectrum covering the 13C label in all compounds but glucose in a 120 ml volume. Reproduced from Pfeuffer et al. (1998). (NAA - N-acetyl-aspartate; Asp - aspartate; Glu glutamate; Gln - glutamine; Lac - lactate; Ala - alanine; Crtot - phosphocreatine + creatine).

2.1. ‘Tracer methods’ The administration of a tracer, whether stable or radioactive, and the ability to follow its metabolism non-invasively opens unique opportunities to study the brain in action. When the highly sensitive radiotracers, label in different metabolic pools cannot be distinguished, unless the measurement of the incorporated radioactivity is performed for each compound separately which is only possible using postmortem analysis or if one uses

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non-metabolizable analogs, such as deoxyglucose, which is trapped following phosphorylation by hexokinase (Sokoloff et al., 1977). The fundamental mathematical principle that underlies the modeling of tracer turnover curves is in principle the same, regardless of the type of tracer used: In all cases, the rate of label appearance in the product pool P is given by the sum of metabolic fluxes from any substrate multiplied by the probability that this particular substrate was labeled, 13 S=S; resulting in Eq. (1) which is from (Gruetter, 2002): X ðinÞ 13 Si ðtÞ X ðoutÞ 13 PðtÞ d13 P ðtÞ ¼ Vi 2 Vj dt Si P i j

ð1Þ

For example, the Sokoloff method measures the tissue radioactivity 45 min after administering a measured bolus of labeled glucose, when the radioactivity from nonphosphorylated sources is negligible and when dephosphorylation is not significant. A further extension of the Sokoloff method is the measurement of tissue radioactivity as a function of time, to which a suitable model of the tracer compartments is fitted. The elegance of the Sokoloff method is its operation in the true tracer mode, i.e., when the kinetics of the product buildup do neither affect the tracer kinetics nor the biochemical reaction, leading in principle to a simplified mathematical problem, as indicated by the schematic representation of label incorporation (Fig. 3). Label incorporation into a product from a precursor, such as into glutamate from glucose, is based on the same fundamental mathematical principles as, e.g., the Sokoloff method, yet several quite profound differences must be discussed. In contrast to the radiotracer method, the signal is detected in a naturally occurring compound, which, because of the inherently lower sensitivity of NMR, must be highly concentrated and enriched in order to be measurable, and inherently includes an upper limit of the measurable label incorporation in tissue. These aspects typically lead to ‘tracer’ curves, i.e., label incorporation curves, similar to what is shown in the middle in Fig. 3, with the label in a specific compound (such as the highly concentrated glutamate) reaching a steady-state value after some time. 2.2. Dynamic isotopomer analysis The true power of modeling label incorporation into tissue pools, as measured by NMR, however, is harnessed by taking into account the ability of NMR to measure the rate of label incorporation not only into different molecules, but also into different positions in a given molecule (Cerdan et al., 1990; Badar-Goffer et al., 1992; Mason et al., 1992; Schousboe et al., 1993b; Shank et al., 1993; Gruetter et al., 1998a), such as the C2, C3 and C4 of glutamate (Fig. 3). The measurement of label incorporation into multiple positions in a molecule in effect is very similar to the measurement of the label distribution in a molecule, traditionally dubbed isotopomer analysis (Malloy et al., 1990; Jeffrey et al., 1991). It has been shown that the time-resolved measurement of label incorporation into the glutamate C3 and C4 is equivalent to the dynamic measurement of the simultaneous, but separate measurement of [4-13C] glutamate and [3,4-13C2] glutamate from the 13 C – 13C singlet and doublet signals (isotopomers) at the C4 position (Jeffrey et al., 1999).

414 R. Gruetter Fig. 3. Measurement of labeling kinetics using radiotracer methods in comparison to in vivo NMR methods. (Top) Radiotracer methods such as the Sokoloff method use a non-metabolizable analog such as deoxyglucose (DG) to measure the accumulation in the metabolic products (Cptissue), mostly due to deoxy-glucose-6-P, leading to simplified first-order tracer kinetics (Sokoloff et al., 1977). (Middle) The traditional method of measuring metabolic fluxes is based on dynamic NMR spectroscopy, mostly focusing on the measurement of label incorporation from a precursor, such as glucose (Glc) into the C4 of a molecule, such as glutamate (13Glu4) (Malloy et al., 1987; Fitzpatrick et al., 1990). (bottom) The full power of NMR spectroscopy is exploited by dynamic isotopomer analysis, which in its generality measures the label incorporation into multiple positions of the same molecule, leading to measurement of multiple time courses of label incorporation into the C2, C3 and C4 of glutamate.

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It is therefore proposed to name the time-resolved measurement of label incorporation into multiple positions of a given molecule ‘dynamic isotopomer analysis’ (bottom in Fig. 3). When fitting a model of compartmentalized cerebral metabolism to such a model, it is important to recognize that the cost function will be evaluated for all fitted time courses simultaneously. In strictly mathematical terms, this is an extension of the one-dimensional case applicable to the measurement of label incorporation into the glutamate C4 for instance. In this context it may be confusing that some models seem to be governed by many more differential equations than parameters that are fitted or number of time courses that are measured. However, in practice, even with so-called simpler models involving only a few explicit differential equations, many more differential equations are in fact involved. In those cases they do not enter the model explicitly because it has been assumed that the small pool size compared to the metabolic rate leads to a negligible effect on the measured metabolic rate. In other words, in the case of a series of chemical reactions involving at least three pools of metabolites, when assuming that the second pool is small compared to the third pool and compared to metabolic flux, the labeling rate of the third pool is not likely to be substantially affected whether that of the second pool was explicitly calculated or not. For metabolic branching points, such as 2-oxoglutarate, however, it is necessary to explicitly include the calculation of the rate of labeling of 2-oxoglutarate, even though the pool size of 2-oxoglutarate itself is unlikely to affect the label turnover curve. Hence, the argument that the number of differential equations is too large needs to take into account whether this implies more reactions with a significant impact on the labeling curves or just a more explicit mathematical formulation of reality. Lastly, an important difference between NMR measurements using isotopes and radiotracer methods lies in the fact that the degree of isotopic labeling in the NMR studies typically is very high, such that small fluctuations of the isotopic enrichment of the precursor pool are not likely to affect the outcome of the analysis. In any of these methodologies it is clear that the rate of label incorporated as a function of time can in principle be related to the metabolic rate and thus allows measuring absolute metabolic fluxes. Another interesting approach consists in measuring the relative amount of label in different molecules or even different positions between different molecules when metabolic steady-state has been achieved (Gruetter et al., 1998a; Gruetter et al., 2001; Bluml et al., 2002; Lebon et al., 2002), and some relative fluxes can be derived using equations such as the following: P V ðinÞ 13 Sn ¼ X n ðoutÞ P Sn Vj

13

ð2Þ

j

Eq. (2) is derived from Eq. (1) assuming steady-state and only one substrate Sn leading to label incorporation into the product P: Such steady-state analysis can lead to a simplified analysis and has also been used for the measurement of differential labeling in the C4 following acetate labeling (Lebon et al., 2002). In the case of acetate labeling, the scrambling of label into many molecules needs to be taken into account. For example, in

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the case of the glutamate –glutamine cycle, not only are there four metabolic pools to be considered (glutamate and glutamine in the neuronal and glial compartments) that can be labeled, but also the magnitude of the fluxes between the mitochondrial Krebs cycle intermediate and the cytosolic glutamate, Vx ; is expected to have an effect on the calculated relative metabolic rates. That the derived labeling can depend on this exchange can be appreciated from the following ‘Gedankenexperiment’: Consider a case where the glutamate does not have a significant exchange with 2-oxoglutarate in the neuron (small Vx ). In this case the relative labeling of neuronal glutamate (13Glu(n)/Glu(n)) will be identical to that of glial glutamine. However, as the exchange rate increases, so does the contribution of unlabeled carbon from the neuronal Krebs cycle to neuronal glutamate, leading to increasingly different labeling in glutamate relative to glutamine, which may very well affect the interpretation of the relative quantitative magnitude of the glutamate– glutamine cycle. Therefore, for the calculation of relative rates of the glutamate– glutamine cycle, at least 6 pools into which label is accumulated, need to be considered even for the case of labeling from acetate or [2-13C] glucose.

2.3.

13

C NMR methodological aspects

Unfortunately, the technical development of 13C NMR spectroscopy in vivo has been limited to a handful sites worldwide (Gruetter et al., 1998a; Bluml, 1999; Rothman et al., 1999; Chen et al., 2001; Chhina et al., 2001; Henry et al., 2002) and largely requires further development. The technical underpinnings of 13C NMR in vivo is not the focus of this chapter, but shall be briefly illustrated by reviewing a few key advances in this field. In 1992, two important advances in MR methodology were introduced to in vivo 13C NMR spectroscopy: First, the use of automated shimming (i.e., in vivo optimization of the main static magnetic field, B0 ; such that it becomes largely independent of the spatial coordinates) dramatically improved sensitivity by narrowing linewidths by almost an order of magnitude compared to what was reported at the time (Gruetter and Boesch, 1992; Gruetter, 1993). Second, the introduction of three-dimensional localization allowed for well-defined volumes to be measured (Gruetter et al., 1992a,b). Two improvements were immediately realizable, namely (i) the complete elimination of the intense scalp lipid signals from the extra-cerebral tissue, which overlap with numerous signals from amino acids, and (ii) the collection of signals from a well-defined volume, which together with excellent shimming improved the spectral resolution. The increases in sensitivity were demonstrated with the rather surprising observation that natural abundance signals from brain metabolites can be observed in vivo, such as those from myo-inositol (Gruetter et al., 1992b). These methodological advances lead to the landmark discovery that labeling of glutamine can be detected in the in vivo brain (Gruetter, 1993; Gruetter et al., 1994), which is now recognized as a window to study cerebral metabolic compartmentation (Sonnewald et al., 1994; Bachelard, 1998; Cruz and Cerdan, 1999; Magistretti et al., 1999; Rothman et al., 1999) and provides a unique window on the brain. Localization has also proven critical in the ability of NMR to detect and measure the signals of brain glycogen, because the

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several-fold higher concentration of muscle glycogen requires dedicated efforts to eliminate any non-cerebral source of glycogen signal (Choi et al., 1999; Choi et al., 2000; Oz et al., 2003). 3. Glial metabolism I: brain glycogen The brain relies on a continuous supply of glucose from the blood for maintaining normal brain function, yet the brain maintains a significant level of brain glycogen making it the largest endogenous carbohydrate reserve in the brain. The concentration of brain glycogen has been estimated at a few mmol/g (Choi and Gruetter, 2003) and references therein, however, several recent studies have suggested that due to the rapid postmortem glycogenolysis (Lowry et al., 1964; Swanson et al., 1989; Choi et al., 1999), as well as glycogen breakdown during the assay itself, these brain glycogen concentrations may have been underestimated (Cruz and Dienel, 2002; Kong et al., 2002). The problems with the biochemical determination of brain glycogen clearly point to the need of a non-invasive method for its measurement. In vivo NMR spectroscopy has unique capabilities in that regard, as shall be illustrated below. 3.1. Brain glycogen, an endogenous store of fuel Brain glycogen metabolism and concentrations obviously can be measured by NMR when using suitable methodology (Choi et al., 1999, 2000). Pulse-chase experiments demonstrated that glycogen breakdown in the a-chloralose anesthetized rat was very slow with a turnover rate on the order of 0.5 mmol/g/h during glucose infusions (Choi et al., 1999). The study further precluded label turnover as the only mechanism by which label was transferred to the brain glycogen pool and it was concluded that net brain glycogen synthesis must have occurred (Choi et al., 1999). In a follow-up study, the effect of insulin was measured by measuring the effect of hyperinsulinemia on label incorporation at a controlled plasma glucose level fixed close to normoglycemia (Choi et al., 2003). The results showed that in vivo at mild hyperglycemia, plasma insulin has a profound effect on brain glycogen metabolism and leads to a net accumulation. These findings were in agreement with data from cell cultures showing an effect of high insulin concentrations on culture glycogen concentrations (Nelson et al., 1968; Dringen and Hamprecht, 1992; Sorg and Magistretti, 1992; Swanson and Choi, 1993). The study extends previous studies suggesting an effect of insulin on brain glycogen content at supraphysiologic hyperglycemia (Daniel et al., 1977). It is quite plausible that brain glycogen may serve as a reservoir of glucosyl units that are mobilized whenever demand for glucose is in excess of supply and such a neuroprotective role for brain glycogen can be implied from, e.g., the mechanism of glutamate neurotransmission (Fig. 4), where glycogen is able to provide energy during hypoglycemia to maintain a low extracellular glutamate concentration. Such a neuroprotective role for brain glycogen has been suggested on the basis of preloaded astrocytes in co-culture with neurons (Swanson and Choi, 1993) and for axonal survival during glucodeprivation (Wender et al., 2000). A recent study demonstrated that degradation of brain glycogen

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Fig. 4. Potential mechanism of a neuroprotective role of brain glycogen during hypoglycemia. In this scheme, an impaired supply of glucose to the brain (as indicated by the shaded bars) leads to an energy deficit, that ultimately can result in glutamate excitotoxicity, as the mechanism of the glutamate neurotransmission involves glial energy metabolism. However, glycogenolysis can produce the extra fuel to maintain glial function and thus a low extracellular glutamate concentration. Some of the glycogen is metabolized oxidatively to CO2 and some of it can be exported to the neuron for oxidative metabolism (Magistretti and Pellerin, 1996).

initiated by hypoglycemia started when the brain glucose concentration approached zero, which is the point at which glucose transport became rate-limiting for metabolism (Choi et al., 2003). Interestingly, at this point cerebral blood flow (CBF) was increased abruptly (Fig. 5A), indicating an attempt by the brain to increase supply, by decreasing the arteriovenous gradient for glucose (Choi et al., 2001). The textbook literature implied that brain glycogen must be a limited storage form for glucose due to its low content and, thus, the role of brain glycogen as a glucose reservoir has been generally dismissed in the literature. Nonetheless, during hypoglycemia, glycogen need only account for part of the deficit in glucose supply and hence can survive longer periods of sustained hypoglycemia. Indeed, a preliminary estimate indicated conservatively that brain glycogenolysis accounted for a majority of the deficit in glucose supply, supporting the quantitative importance of brain glycogen in hypoglycemia (Choi et al., 2003). Measurements of brain glycogen during hypoglycemia indicated that brain glycogen degradation occurred at a rate during hypoglycemia that resulted in brain glycogen concentrations to be substantial even after 2 h of moderate hypoglycemia (Fig. 5B). It is therefore likely that the brain tries to defend itself against moderate hypoglycemia by using brain glycogen and by increasing CBF and that these defenses are triggered by the point at which glucose transport becomes rate-limiting for metabolism, or by the point at which the brain glucose concentrations become ratelimiting for metabolism. Because brain glycogen is an insulin-sensitive glucose reservoir it is interesting to explore whether brain glycogen metabolism is deranged following hypoglycemia. Indeed,

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Fig. 5. Effect of hypoglycemia on cerebral blood flow, brain glucose concentrations and brain glycogen metabolism as measured by NMR. (A) Measurements of brain glucose concentration (left scale) as a function of plasma glucose concentration. The solid line is the fit of the reversible Michaelis–Menten model to the eu- and hyperglycemic brain glucose (open circles). The open squares indicate brain glucose measurements below 4.5 mM plasma glucose. When brain glucose approaches zero (dotted vertical line), the measurement of cerebral blood flow (solid triangles, right scale) indicate CBF values above the 95% confidence interval (shaded area) and this is also the point where brain glycogenolysis started (arrow in A and vertical dotted line in B). (B) Measurement of the effect of insulin-induced hypoglycemia at 4 h on brain glycogen metabolism and glucose concentrations. When the brain glucose concentrations (open squares) approached zero, brain glycogenolysis started (dotted vertical line) at a rate that sustained brain glycogen concentrations (solid circles) for at least 2 h. Restoration of brain glucose concentrations at t ¼ 7 h typically resulted in a brain glycogen rebound (supercompensation). Modified from (Choi et al., 2001) and (Choi et al., 2003).

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data suggest that brain glycogen concentrations following hypoglycemia increase substantially above normal (Fig. 5B) leading to increased neuroprotection. It is therefore reasonable to conclude that brain glycogen metabolism may play a role in the development of defective recognition of hypoglycemia (hypoglycemia unawareness) by the brain as proposed recently (Choi et al., 2003).

3.2. Human brain glycogen metabolism in vivo The studies mentioned in Section 3.1 have been performed in anesthetized animals and, consequently, the legitimate question arises as to whether the slow brain glycogen turnover observed also translates to the awake brain. This is of interest, since several studies suggesting an involvement of brain glycogen in brain activation have done these measurements in the conscious animal (Swanson, 1992; Dienel et al., 2002). Our results thus far suggest that brain glycogen is only utilized when supply is insufficient to cover demands in metabolism, possibly only when brain glucose concentrations become so low that they significantly limit the rate of glucose phosphorylation. Some of the reported increases in brain glucose metabolism observed during focal activation imply increased usage of carbohydrates other than blood glucose because the reported increases (Hyder et al., 1997) exceed the transport capacity of the blood-brain barrier by several-fold (Choi et al., 2001). One such source of glucose equivalents is brain glycogen, which is present in sizable amounts in brain (Sagar et al., 1987; Choi et al., 1999; Cruz and Dienel, 2002; Kong et al., 2002). It is possible that parts of the glycogen molecule may undergo rather rapid metabolism. However, in line with our results in the a-chloralose anesthetized rat brain (Choi et al., 1999), we found that metabolism of bulk brain glycogen was also very slow in the awake rat brain, with a turnover time on the order of that of NAA (Choi and Gruetter, 2003) and a total brain glycogen concentration of , 3 mmol/g wet weight in line with the literature. However, the important question arises how these measurements relate to human brain glycogen metabolism, which has never been measured in vivo. The question remained as to whether in the conscious human, brain glycogen metabolism is also slow. We have adapted our previously developed methods to the measurement of brain glycogen in humans and measured the rate of label incorporation into brain glycogen during administration of [1-13C] glucose in humans (Oz et al., 2003). The results indicated a much slower rate of label incorporation in the human than in the rat (Fig. 6) with a turnover rate of approximately 0.15 mmol/g/h. Such a slow rate of turnover certainly does not suggest an involvement of brain glycogen metabolism in the background activity of the conscious human brain. Nonetheless, it does not preclude the activation of the reservoir in conditions of extreme metabolic demand. Instead, it favors the overall influence of the sleep –wake cycle on brain glycogen metabolism as reported (Kong et al., 2002), and supported by altered gene expression (Petit et al., 2002): For example, it is quite conceivable that small bursts of brain activity will lead to transient mismatches in glucose supply and demand causing, e.g., small decreases in brain glycogen that can accrue over time during the day. Such a slow rate of turnover of glycogen in humans suggests that turnover of the glucosyl units in brain glycogen may require days and that altered brain glycogen concentrations

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Fig. 6. Measurement of the label incorporation into human brain glycogen C1. The solid squares represent the measurement of 13C label in glycogen C1 following administration of 80 g of 1-13C glucose in three humans begun at t ¼ 0 min. The solid line is the result of a linear regression of the measurements in the first 5 h of the study and indicates a rate of label incorporation consistent with a very slow glycogen turnover rate on the order of 0.1–0.2 mmol/g/h. From Oz et al. (2003).

(such as a super-compensation following a hypoglycemic episode) may require time on the order of a week to be restored to normal. This time scale of brain glycogen metabolism is consistent with the time scale it takes to revert the syndrome of hypoglycemia unawareness and is consistent with the proposed involvement of brain glycogen in the pathogenesis of impaired recognition of hypoglycemia.

4. Glial metabolism II: the glutamate –glutamine cycle Because of the ever increasing importance of functional MRI, a mechanism of which is the activation-dependent change in the venous concentration of deoxyhemoglobin, the question whether there is tight coupling between glucose and oxygen consumption in the brain has become of paramount importance. The landmark study by Fox and Raichle in the late 1980s suggested that there are indeed large increases in glucose metabolism and CBF that exceed the changes in oxygen metabolism (Fox et al., 1988). In principle, NMR provides the unique capability to measure cerebral concentration changes of brain glucose and lactate, both of which are key components in addressing this question, and increases in

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lactate and glucose concentration have been reported (Prichard et al., 1991; Sappey-Marinier et al., 1992; Frahm et al., 1996). In addition, from the incorporation of label from a suitable precursor such as glucose, into glutamate, the cerebral oxygen consumption could be computed. A majority of studies in the brain have focused on measuring glutamate turnover (Rothman et al., 1992; Hyder et al., 1997; Lukkarinen et al., 1997; Pan et al., 2000), which was motivated by the fact that glutamate turnover is linked to the metabolism of the Krebs cycle (Mason et al., 1992; Chatham et al., 1995; Mason et al., 1995; Yu et al., 1997; Gruetter et al., 1998a, 2001; Cruz and Cerdan, 1999), and that the glutamate C4 resonance, which is labeled in the first turn of the Krebs cycle, presents a readily detectable NMR signal due to the high concentration of glutamate. Using this methodology, one such study compared the rate of label incorporation and found a significant difference between the activated and the resting visual cortex, indicating that the cerebral oxygen consumption increased at most by 30%, which is approximately half of the blood flow increase measured using this stimulation paradigm (Chen et al., 2001). This study supported the idea that oxygen consumption increases are less than the associated blood flow increases, leading to a net decrease in deoxy-hemoglobin content during focal activation, which forms the basis of blood-oxygen-level-dependent functional MRI (Ogawa et al., 1998). Perhaps the major advantage of in vivo NMR is not to provide neuroscientists with an alternative alternative method to measure CMRO2 and CMRglc (although this may be very useful as indicated above), but to shed light on metabolic processes not accessible by any other method, one of which (glycogen) was addressed above and some of which will be discussed below.

4.1. Glutamate turnover: neuronal oxygen metabolism and the malate– aspartate shuttle The measurement of cerebral oxygen consumption from turnover of glutamate (as referred to in the previous paragraph) assumes a direct stoichiometric relationship between that measurement and the rate of oxygen consumption. Unfortunately, this relationship is not directly inferred, as the brain is intricately compartmentalized, which shall be discussed further below. In addition, most of the glutamate signal that is observed is in the cytosol, whereas the labeling occurs in the mitochondrion and hence label has to be transported across the charged inner mitochondrial membrane (Fig. 7), which has been shown to be the rate limiting step in many tissues, such as the heart (Chatham et al., 1995; Yu et al., 1997; Sherry et al., 1998) and the liver (Garcia-Martin et al., 2002). Initially it was thought that the exchange between 2-oxoglutarate and glutamate, Vx ; is very fast in the brain in vivo. However, many studies in heart, liver and muscle have indicated the opposite in these tissues (Chatham et al., 1995; Yu et al., 1997; Sherry et al., 1998; Garcia-Martin et al., 2002). More recent evidence now suggests that in the brain Vx is on the order of the flux through pyruvate dehydrogenase, VPDH (Gruetter et al., 2001; Choi et al., 2002), which may vary in pathologic conditions (Henry et al., 2002). The observation that Vx was comparable to the flux through pyruvate dehydrogenase implied that the malate –aspartate shuttle may be a major mechanism mediating the

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Fig. 7. Measurement of oxidative glucose consumption from the flow of label from glucose to glutamate. The label in glucose (or any other precursor, such as pyruvate or acetyl-CoA for that matter) is metabolized in the mitochondrion and then transferred to glutamate, most of which is in the cytosol. The flow of label into glutamate thus is in principle a combined effect of Krebs cycle flux (VPDH) and label exchange across the mitochondrial membrane ðVx Þ:

exchange of label across the mitochondrial membrane (LaNoue and Tischler, 1974; Yu et al., 1997; Gruetter, 2002). The assumption that Vx is very fast will affect the modeling results depending on the pool sizes that participate in this exchange (Gruetter et al., 2001; Gruetter, 2002). Furthermore, it is important to recognize that because of the mostly neuronal localization of glutamate, the measurement of glutamate turnover alone, as done in several previous studies (Rothman et al., 1999; Pan et al., 2000; Sibson et al., 2001), mainly measures neuronal metabolism. The astroglial compartment does contain significant oxidative capacity for metabolism, as pointed out recently (Gruetter et al., 2001), despite some previous assumptions to the contrary (Sibson et al., 1998). This shall be discussed in Section 4.2. 4.2. Glutamine turnover: the hallmark of glial metabolism It is well known that brain metabolism is characterized by at least two major compartments with a large neuronal and a small glial glutamate pool associated with the Krebs cycle. As pointed out previously, these two pools are metabolically linked by the glutamate– glutamine cycle. The compartmentation of brain metabolism is based on that of several enzymes. In addition to those of glycogen (see above), glutamine synthetase (Martinez-Hernandez et al., 1976) and pyruvate carboxylase (Shank et al., 1985) are almost exclusively in the glial compartment, as summarized in more detail elsewhere

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(Bachelard, 1998; Cruz and Cerdan, 1999; Gruetter et al., 2001; Gruetter, 2002). Compartmentation furthermore extends to metabolites, such as glutamate (neuronal) and glutamine (glial) (Ottersen et al., 1992; Shupliakov et al., 1997), as well as to mitochondria and other systems (Schousboe et al., 1993b). It is of interest to note that the exclusively glial localization of glutamine synthetase implies that the observation of glutamine synthesis in vivo, first achieved in human brain (Gruetter, 1993; Gruetter et al., 1994), is a direct manifestation of glial metabolism, whereas the observation of label incorporation into glutamate implies a mainly neuronal event. Clearly, glutamine synthesis can be measured non-invasively by NMR, as shown in Fig. 2, and therefore demonstrates the ability of NMR to study cerebral compartmentation non-invasively in intact brain. The mechanism of inactivation of glutamate by uptake into the astroglial compartment implies a much more active role for astrocytes than is conventionally assumed, due to the imperative involvement of glial energy metabolism (Eriksson et al., 1995; Magistretti and Pellerin, 1996; Silver and Erecinska, 1997). The neuron-astrocyte triade thus has to be considered the functional unit intimately involved in achieving chemical transmission (Magistretti et al., 1993, 1999; Tsacopoulos and Magistretti, 1996). The link between astrocytes and neurons is generally accepted from a metabolic as well as from a neurophysiological standpoint (Bergles et al., 1997), yet differences exist as to the precise coupling and the specific energetics involved. The simplest scheme for measuring glutamate neurotransmission in vivo is shown in Fig. 8. This model assumes very rapid exchange Vx and negligible glial Krebs cycle rate, as well as negligible anaplerosis. Based on this simple and elegant scheme, it was proposed that the rate of glutamate/glutamine inter-conversion (the glutamate– glutamine app cycle), identified in the scheme in Fig. 8 by VNT ; is equal to the glucose consumption rate (Sibson et al., 1998; Rothman, 2001; Shulman et al., 2001a,b; Shen and Rothman, 2002; Rothman et al., 2003). This elegant, but perhaps oversimplified model assumed that the two ATP produced by glycolysis were almost completely consumed by glutamine synthesis and restoration of the ion balance through the Na/K ATPase with a negligible oxidative metabolism in the glial compartment. Under these circumstances it was postulated that glucose metabolism must be directly linked to glutamate neurotransmission with a 1:1 stoichiometry. The proposal put forth by Shulman and coworkers (Sibson et al., 1998; Rothman et al., 1999), that the glial ATP production needed to maintain neuronal glutamate is solely provided by glycolysis pathway is intriguing as it emphasizes the coupling between neurons and glia at the level of energy metabolism. However, only a few percent of pyruvate molecules need to be diverted to the Krebs cycle to generate as many ATP as are formed in the absence of oxidative metabolism of glucose in the astrocytes. Furthermore, a recent study (Choi et al., 2002) measured brain glucose and glycogen metabolism in deep pentobarbital anesthesia under conditions similar to what was used (Sibson et al., 1998) and what had been shown to result in isoelectric coma (Contreras et al., 1999). In that study it was shown that the brain glucose concentration changed only slightly despite a drastic reduction in electrical activity and that a substantial gradient in brain glucose concentration relative to that in plasma persisted, as illustrated in Fig. 9.

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Fig. 8. Proposed link between glucose metabolism and glutamate neurotransmission. Adapted according to Sibson et al. (1998). The proposed stoichiometric coupling between glucose metabolism and glutamate neurotransmission is based on the assumption that all of the glucose is metabolized in the glia and that the two ATP produced are consumed by the need to restore ion potential following glutamate uptake and by glutamine synthetase. Oxidative metabolism of glucose and anaplerosis (pyruvate carboxylation) are neglected in this simple, yet elegant model.

In addition, that study indicated that when using the simplified scheme shown in Fig. 8 (Choi et al., 2002), similar metabolic rates as those reported by (Sibson et al., 1998) were obtained, however, the rate of label incorporation into glutamate C4 and C3 was inconsistent with that observed (dashed line in Fig. 10). Thus, the study reiterated the importance of minimizing the number of assumptions made in the modeling, which was also emphasized by two other independent studies (Gruetter et al., 2001; Henry et al., 2002). One additional assumption of the study by Sibson et al. (1998) was that the magnitude of glutamine synthesis not related to neurotransmitter cycling was constant over a large range of electrical activity. A surprising observation of our study was that under deep pentobarbital anesthesia, astrocyte metabolism was as significant as was neuronal metabolism with approximately equal magnitude per volume brain tissue. This observation is consistent with results from previous studies in culture, suggesting an effect of barbiturates on neuronal metabolism that is different in magnitude from that on astrocytes (Yu et al., 1983; Hertz et al., 1986; Swanson and Seid, 1998; Qu et al., 2000). Oxygen consumption has been reported to increase in cultured astrocytes when exposed to extracellular glutamate (Eriksson et al., 1995) and large increases in oxygen consumption have been reported in brain during functional activity (Hyder et al., 1996, 1997), which support the idea that oxygen metabolism in astrocytes is stimulated during focal

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Fig. 9. Effect of pentobarbital anesthesia on brain glucose content in the rat brain. Shown is a comparison of the brain glucose concentration between light a-chloralose anesthesia and deep pentobarbital anesthesia. Modeling of brain glucose transport according to previous studies (Gruetter et al., 1998b; Choi et al., 2001; Seaquist et al., 2001) indicated a decreased rate of glucose metabolism (CMRglc) relative to the apparent maximal rate of glucose transport (Tmax). Even under deep pentobarbital anesthesia, brain glucose concentrations were significantly lower than expected if glucose metabolism was abolished (as indicated by the dashed line).

Fig. 10. Effect of the exchange rate between 2-oxoglutarate and glutamate, Vx ; on the relative labeling of glutamate C3 and C4 during deep pentobarbital anesthesia. When assuming Vx ¼ 0:57 mmol/g/min and fitting to the measured label incorporated into the C4 of glutamate only, an oxidative glucose consumption rate similar to that reported by Sibson et al. (1998) was obtained (VPDH ¼ 0.15 mmol/g/min), however, the label incorporation into the C3 of glutamate relative to that into the C4 (solid squares) was not very well reproduced (dashed line). In contrast using the scheme in (Gruetter et al., 1998; Gruetter et al., 2001; Gruetter, 2002), lead to a much better approximation (solid line), indicating that Vx is on the order of VPDH also in deep pentobarbital anesthesia, and thus brain activity dependent.

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activation. Glucose metabolism at rest is likely to be oxidative in glia, as judged from the well-known incorporation of acetate label into glutamine (Van den Berg, 1973; Hassel et al., 1997; Waniewski and Martin, 1998; Dienel et al., 1999), and proposed from 13C studies in vivo by NMR (Brand et al., 1997; Hassel et al., 1997; Bluml et al., 2002; Lebon et al., 2002). The previously put forth argument that the majority of glial metabolism in resting brain is probably oxidative (Gruetter, 2002) thus appears valid and there is now general consensus that glial energy metabolism has a significant oxidative component on the order of 0.2– 0.1 of that in the glutamatergic compartment, which we were the first to show in vivo (Gruetter et al., 2001). The presence of dominant oxidative metabolism in the astrocyte does not disprove the hypothesis that lactate produced in astrocytes is also a fuel for oxidative metabolism in neurons. The results obtained first by our laboratory and then confirmed by others suggest that in the human brain approximately one sixth of the ATP production from glucose measured by NMR is in the glial compartment. This leaves at least five sixth of the lactate for export to neurons, if assuming the extreme case that phosphorylation of glucose is an exclusively glial process.

4.3. Anaplerosis and the astroglial TCA cycle Astrocytes thus clearly have oxygen metabolism at rest and during activation. Assuming, as implied by the scheme in Fig. 8, that the glutamate –glutamine cycle is the sole contributor to flux through glutamine synthetase, the labeling of the carbon backbone of glutamate and glutamine must be identical at isotopic and metabolic steady-state. However, early studies in rat brain extracts (Lapidot and Gopher, 1994), and in human brain (Gruetter et al., 1998a, 2001) have reported that this is not the case. In this context, the inequality of the label distribution between brain glutamine and glutamate does depend on the relative rate of the glutamate– glutamine cycle relative to other reactions as can be deduced from Eq. (2). Furthermore, all potential contributions of label must be taken into account and thus the effect of a variable Vx must be accounted for. We demonstrated that the inequality of label in glutamate and glutamine implied significant contribution of pyruvate carboxylase to the flux through glutamine synthetase (Gruetter et al., 1998a, 2001). Therefore, other metabolic reactions must contribute substantially to the labeling of glial glutamate, and eventually glial glutamine. One reaction that can lead to a differential labeling of glutamine and glutamate at the different positions of the molecule is the glial enzyme pyruvate carboxylase, which can label the C2 more than the C3 when administering glucose labeled at the 1 and/or 6 position. Pyruvate carboxylase activity is significant in vivo (Lapidot and Gopher, 1994; Gruetter et al., 1998a, 2001; Shen et al., 1999). Although differences exist as to the magnitude of the flux through pyruvate carboxylase, the relative amount of label incorporation into glutamine differs from that into glutamate (Martin et al., 1993; Lapidot and Gopher, 1994; Gruetter et al., 1998a, 2001). As pointed out previously (Gruetter, 2002), even the lowest reported value of 0.04 mmol/g/min (Shen et al., 1999) results in a rate of ATP generation that amounts to , 2/3 of the ATP needed for glutamate uptake and conversion to glutamine. A recent study measured the labeling of glutamate

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and glutamine from 2-13C glucose and concluded that in the rat brain pyruvate carboxylase contributes approximately 30% to the flux of glutamine synthetase (Sibson et al., 2001). In this context it is important to note that using 2-13C glucose labels the C3 of glutamate directly through pyruvate carboxylase and indirectly through the pentose phosphate shunt. The fact that pyruvate carboxylase activity is now accepted as a substantial and significant metabolic flux in astrocytes and that astrocytes thus have substantial oxidative energy metabolism calls into question the underlying mechanism for the proposed 1:1 stoichiometric relationship between glutamate neurotransmission and oxidative glucose metabolism (summarized in Fig. 8), but it does not rule out the proposed predominantly astrocytic location of incremental glucose metabolism during activation as suggested (Magistretti and Pellerin, 1996), which remains an intriguing hypothesis. In fact, the observation that during hypoglycemia, astrocytic glycogen accounts for a majority of the metabolic deficit (see above) implicitly supports the presence of this mechanism. 5. Concluding remarks The new non-invasive method 13C NMR has come a long way: Increases in sensitivity and methodology have paved the way for many new measurements that are now feasible in the live and intact brain, leading to unique insights in anaplerosis, glial and neuronal energy metabolism, metabolic trafficking, brain glycogen metabolism and the regulation of oxidative energy metabolism. It is concluded that considerable care must be exercised when attempting to interpret and model the measured rates of label incorporation. Acknowledgements Supported in part by grants from the US Public Health Service, NIH R21DK58004 (RG), R21NS451119 (RG), R01NS42005 (RG), R01NS38672 (RG), P41RR08079, M01RR00400, and the Whitaker Foundation (RG) and Juvenile Diabetes Research Foundation International (RG). The encouragement and support from colleagues at the Center for MR Research is appreciated, in particular Drs Kamil Ugurbil, Elizabeth R, Seaquist, Wei Chen, Xiao-Hong Zhu. Special thanks to the members of my group, Drs InYoung Choi, Ivan Tkac, Josef Pfeuffer, Gulin Oz, Pierre-Gilles Henry and Melissa J. Terpstra for their tireless efforts and hard work, as well as to Hong-Xia Lei, Sarah L. Crawford, Dee M. Koski and Tian-Wen Yue for their assistance in these experiments.

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Sibson, N.R., Mason, G.F., Shen, J., Cline, G.W., Herskovits, A.Z., Wall, J.E., Behar, K.L., Rothman, D.L., Shulman, R.G., 2001. In vivo (13)C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during. J. Neurochem. 76, 975 –989. Silver, I.A., Erecinska, M., 1997. Energetic demands of the Naþ/Kþ ATPase in mammalian astrocytes. Glia 21, 35–45. Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O., Shinohara, M., 1977. The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28, 897– 916. Sonnewald, U., Gribbestad, I.S., Westergaard, N., Nilsen, G., Unsgard, G., Schousboe, A., Petersen, S.B., 1994. Nuclear magnetic resonance spectroscopy: biochemical evaluation of brain function in vivo and in vitro. Neurotoxicology 15, 579 –590. Sorg, O., Magistretti, P.J., 1992. Vasoactive intestinal peptide and noradrenaline exert long-term control on glycogen levels in astrocytes: blockade by protein synthesis inhibition. J. Neurosci. 12, 4923–4931. Swanson, R.A., 1992. Physiologic coupling of glial glycogen metabolism to neuronal activity in brain. Can. J. Physiol. Pharmacol. 70, S138–S144. Swanson, R.A., Choi, D.W., 1993. Glial glycogen stores affect neuronal survival during glucose deprivation in vitro. J. Cereb. Blood Flow Metab. 13, 162–169. Swanson, R.A., Sagar, S.M., Sharp, F.R., 1989. Regional brain glycogen stores and metabolism during complete global ischaemia. Neurol. Res. 11, 24–28. Swanson, R.A., Seid, L.L., 1998. Barbiturates impair astrocyte glutamate uptake. Glia 24, 365 –371. Tsacopoulos, M., Magistretti, P., 1996. Metabolic coupling between glia and neurons. J. Neurosci. 16, 877 –885. Van den Berg, C., 1973. A model of compartmentation in mouse brain based on glucose and acetate metabolism. In: Balazs, E., Cremer, J. (Eds.), Metabolic compartmentation in the brain. MacMillan, London, pp. 137 –166. Vernadakis, A., 1996. Glia– neuron intercommunications and synaptic plasticity. Prog. Neurobiol. 49, 185 –214. Waniewski, R.A., Martin, D.L., 1998. Preferential utilization of acetate by astrocytes is attributable to transport. J. Neurosci. 18, 5225–5233. Wender, R., Brown, A.M., Fern, R., Swanson, R.A., Farrell, K., Ransom, B.R., 2000. Astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter. J. Neurosci. 20, 6804–6810. Westergaard, N., Sonnewald, U., Schousboe, A., 1995. Metabolic trafficking between neurons and astrocytes: the glutamate/glutamine cycle revisited. Dev. Neurosci. 17, 203–211. Yu, X., Alpert, N.M., Lewandowski, E.D., 1997. Modeling enrichment kinetics from dynamic 13C NMR spectra: theoretical analysis and practical considerations. Am. J. Physiol. 41, C2037–C2048. Yu, A.C., Hertz, E., Hertz, L., 1983. Effects of barbiturates on energy and intermediary metabolism in cultured astrocytes. Prog. Neuropsychopharmacol. Biol. Psychiatry 7, 691–696. Yudkoff, M., Nissim, I., Daikhin, Y., Lin, Z., Nelson, D., Pleasure, D., Erecinska, M., 1993. Brain glutamate metabolism: neuronal–astroglial relationships. Dev. Neurosci. 15, 343–350. Yudkoff, M., Nissim, I., Pleasure, D., 1988. Astrocyte metabolism of [15N]glutamine: implications for the glutamine– glutamate cycle. J. Neurochem. 51, 843–850. Zigmond, M.J., 1999. Fundamental Neuroscience. Academic Press, San Diego, pp. 402– 412.

Ion, transmitter and drug effects on energy metabolism in astrocytes Leif Hertz,a,* Liang Peng,a Christel C. Kjeldsen,b Brona S. O’Dowdc and Gerald A. Dieneld a

College of Basic Medical Sciences China Medical University, Heping District, Shenyang, 110001, P.R. China p Correspondence address: RR 2, Box 245, Gilmour, Ont., Canada K0L 1W0 E-mail: [email protected] b Centre of Psychiatry, Copenhagen County, Denmark c Centre for Magnetic Resonance, University of Queensland, Gehrmann Laboratories, St. Lucia, Queensland 4072, Australia d Department of Neurology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

Contents 1. 2.

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Introduction Mechanisms of metabolic stimulation 2.1. Increase in ADP/ATP ratio 2.2. Increase in intramitochondrial Ca2þ concentration 2.3. Glycogen as a re-chargeable energy reservoir Ion effects on metabolism 3.1. The potassium ion (Kþ) 3.2. The sodium ion (Naþ) Transmitter effects on metabolism 4.1. Second messenger systems 4.2. Individual transmitters Drug effects on metabolism 5.1. Interaction with ion effects 5.2. Interaction with transmitter effects Concluding remarks

Accumulation of Kþ in astrocytes by stimulation of the extracellular Kþ-sensitive site of the Naþ, Kþ-ATPase and by activation of the Naþ, Kþ, 2Cl2 cotransporter plays a major role in ion homeostasis and therefore in energy metabolism in the CNS. Naþ-driven uptake of glutamate also contributes to stimulation of astrocytic energy metabolism by activating the intracellular Naþ-dependent site of the Naþ, Kþ-ATPase, but the energy expenditure for glutamate uptake is probably quantitatively less important, and it may be Advances in Molecular and Cell Biology, Vol. 31, pages 435–460 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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met by oxidative degradation of accumulated glutamate. Several transmitters affect energy metabolism in astrocytes, by Ca2þ-mediated stimulation of mitochondrial dehydrogenases and glutaminase and by activation of the breakdown of glycogen, likely to serve as a rechargeable energy substrate in peripheral processess of the astrocytes, which are too thin to contain any mitochondria. Certain drugs classically assumed to act solely on neurons owe a substantial part of their effect to interactions with effects of ions or transmitters on astrocytes, thereby altering energy metabolism in astrocytes and thus in the brain.

1. Introduction With the recent establishment that the metabolism of cortical astrocytes accounts for a sizeable fraction of energy metabolism, including oxidative metabolism, in the brain in situ (see chapters by Gruetter and by Ha˚berg and Sonnewald) comes the question to what extent astrocyte metabolism is regulated during brain activity. As of yet, this has not been determined in the brain in situ, although it is known that oxidative metabolism in astrocytes in the rat brain is substantially increased during spreading depression (Dienel et al., 2001), a situation in which the extracellular potassium concentration ([Kþ]e) is greatly increased (Vyskocil et al., 1972; Somjen, 1979). This correlation is probably not co-incidental, since there is now overwhelming evidence that regulation of [Kþ]e homeostasis in the brain depends heavily on an initial active, and thus energy-consuming, uptake of Kþ into astrocytes (see chapter by Walz). In addition, it is well established that transmitter glutamate is predominantly accumulated by astrocytes, in co-transport with sodium ions (Naþ), which subsequently must be extruded by active transport (see chapter by Schousboe and Waagepetersen). This represents another, although quantitatively probably less prominent, stimulus of energy metabolism. Evidence has also been obtained in cell culture experiments that transmitters like noradrenaline increase glycolysis (Subbarao and Hertz, 1991; Magistretti et al., 1993) and oxidative metabolism (Subbarao and Hertz, 1991) in astrocytes, but have no corresponding effect in neurons (Subbarao and Hertz, 1990a). The stimulation of oxidative metabolism is probably secondary to an increase in intramitochondrial calcium (Ca2þ), which is secondary to a transmitterinduced increase in free cytosolic Ca2þ ([Ca2þ]i). That a similar stimulation may occur also in the brain in situ is suggested by the repeated observation that administration of a-adrenergic antagonists reduce glucose metabolism, measured by the autoradiographic 2-deoxy-D -[14C]glucose technique, in most brain areas of the rat brain (Savaki et al., 1982; Inoue et al., 1991; French et al., 1995). It is also well established that many transmitters as well as elevated [Kþ]e stimulate breakdown of glycogen (glycogenolysis) in brain and in astrocytes (Hof et al., 1988; Subbarao and Hertz, 1990b; Subbarao et al., 1995; Waagepetersen et al., 2000). In the present chapter we will describe effects of individual ions and transmitters known to stimulate energy metabolism in astrocytes, after we have discussed the general mechanisms involved and the potential role of glycogen metabolism during brain activation. Effects of some drugs, which may interact with ion and transmitter effects on brain metabolism will also be included.

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2. Mechanisms of metabolic stimulation 2.1. Increase in ADP/ATP ratio In neurons an elevated intracellular Naþ concentration following excitation is a major stimulus of Naþ,Kþ-ATPase activity and thus of glucose metabolism due to the resulting decrease in ATP and concomitant increase in ADP and AMP, which stimulate oxidative phosphorylation and phosphofructokinase (PFK) activity and thus glycolysis (see chapter by Roberts and Chih; Hertz and Dienel, 2002). Since normal astrocytes are non-excitable cells, it can a priori be excluded that a similar excitation-induced increase in intracellular Naþ can provide a metabolic stimulus. This does, however, not exclude that postexcitatory stimulation of Naþ,Kþ-ATPase activity might enhance energy metabolism in astrocytes, since the extracellular Kþ concentration ([Kþ]e) in the brain increases as a result of brain activation (see chapter by Walz). Increased [Kþ]e can stimulate the extracellular, Kþ-sensitive site of the Naþ,Kþ-ATPase, because the affinity of the enzyme for Kþ is low enough that maximum activity is not achieved at resting [Kþ]e. Moreover, the intracellular Naþ concentration in astrocytes increases not only during Naþ-coupled uptake of glutamate (and other amino acids) but also during Kþ uptake by a co-transporter, mediating coupled uptake of Kþ, Naþ and 2Cl2 (see chapter by Walz). Thus, as illustrated in Fig. 1, elevated [Kþ]e stimulates energy metabolism (oxidative metabolism as well as glycolysis) via an increase in ADP/ATP ratio, both by a direct stimulation of the extracellular Kþ-sensitive site of the Naþ,Kþ-ATPase and by enhancing co-transporter activity and thus uptake of Naþ, which secondarily stimulates the Naþ,Kþ-ATPase at its intracellular the Naþ-sensitive site. This figure also shows that glutamate-induced uptake of glutamate together with Naþ similarly stimulates energy metabolism by an effect of the accumulated Naþ on the intracellular site of the Naþ,Kþ-ATPase. In addition, ADP-mediated stimulation of energy metabolism can be evoked by transmitters which either activate Naþ,Kþ-ATPase activity (Mercado and Hernandez, 1992; Hajek et al., 1996) or stimulate an energy-requiring process, such as cellular uptake of glutamate in conjunction with Naþ (Hansson and Ro¨nnba¨ck, 1992; Alexander et al., 1997), stimulating the Naþ,Kþ-ATPase at its intracellular Naþ-stimulated site. Transmitters have long been known to stimulate glycogenolysis in brain, and since virtually all glycogen in brain is located in astrocytes (Ibrahim, 1975), glycogenolysis must also be an astrocytic effect (Fig. 1). The functional importance of stimulation of glycogenolysis is indicated by the observation that glycogen turnover, i.e., both glycogenolysis and re-synthesis of glycogen are enhanced during brain activity (Swanson et al., 1992; Dienel and Cruz, 2003). Thus, glycogenolysis and subsequent re-synthesis of glycogen may possibly account for a sizeable fraction of energy metabolism in brain during and after enhanced activity. This conclusion raises the question what role glycogen turnover plays during brain activation. 2.2. Increase in intramitochondrial Ca2þ concentration A decrease in ATP and increase in ADP levels are not the only stimuli regulating energy metabolism in mammalian cells. During the last 10– 15 years, studies in other

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Fig. 1. Schematic illustration of energy-requiring and energy-yielding reactions in astrocytes. Even slightly elevated extracellular Kþ concentrations ([Kþ]e) stimulate the extracellular, Kþ-sensitive site of the Naþ,Kþ-ATPase, stimulating active uptake of Kþ, as well as of the co-transporter, accumulating jointly Naþ,Kþ and 2Cl2 and at slightly higher concentrations they open a voltage sensitive L-channel for Ca2þ, allowing Ca2þ entry into the cell. Extracellular glutamate stimulates uptake of glutamate, co-transported with Naþ, which subsequently stimulates the intracellular, the Naþ-sensitive site of the Naþ,Kþ-ATPase, as does Naþ accumulated by the co-transporter. Stimulation of Naþ,Kþ-ATPase activity leads to conversion of ATP to ADP, and the altered ATP/ADP ratio stimulates both cytosolic glycolysis and mitochondrial oxidative metabolism. In addition increased intracellular Ca2þ ([Ca2þ]i) stimulates oxidative metabolism as well as glycogenolysis. Simultaneously or slightly later glycogen is re-synthesized from glucose, partly fueled by oxidatively derived energy. The transmitter noradrenaline stimulates glycogenolysis by a b-adrenergic effect and glycolysis by an a-adrenergic effect (not shown). It also causes a release of bound Ca2þ from the endoplasmic reticulum by an a-adrenergic effect, leading to an increase in [Ca2þ]i and subsequent increase in glycogenolysis. Thus CNS activation, including the establishment of memory, which is characterized by increases in [Kþ]e and extracellular glutamate and the release of noradrenaline evokes a concerted increase in energy utilization and energy production by glycolysis, oxidative metabolism and glycogenolysis in astrocytes.

tissues than brain have shown that an increase in free intramitochondrial Ca2þ, secondary to a rise in free cytosolic Ca2þ concentration, [Ca2þ]i, within seconds causes a direct stimulation of the mitochondrial dehydrogenases, pyruvate dehydrogenase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase (McCormack and Denton, 1990; Rutter et al., 1996), as well as of glutaminase activity (Halestrap, 1989) and of oxidative phosphorylation (Robb-Gaspers et al., 1998). It is highly likely that a similar response occurs in astrocytes, since astrocytic [Ca2þ]i is increased by a multitude of transmitters (see chapter by Hansson and Ro¨nnba¨ck), including ATP (Peuchen et al., 1996), and noradrenaline (Fig. 1), which cause a release of Ca2þ from the endoplasmic reticulum. It is in agreement with this concept that noradrenaline stimulates pyruvate dehydrogenation (Hertz and Peng, 1992; Chen and Hertz, 1999), a-ketoglutarate dehydrogenation

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(Subbarao and Hertz, 1991) and hydrolysis of glutamine to glutamate (Huang and Hertz, 1995) in mouse astrocytes in primary cultures, and that ATP depolarizes the mitochondrial membrane potential, which may reflect entry of Ca2þ into the mitochondria (Peuchen et al., 1996). Entry of extracellular Ca2þ can also lead to an increase in [Ca2þ]i in astrocytes during exposure to elevated [Kþ]e (Fig. 1), an effect which is due to opening of voltage-sensitive Ca2þ channels (Hertz et al., 1989; Duffy and MacVicar, 1994; Zhao et al., 1996; Thorlin et al., 1998). An influx of Ca2þ occurs also during mechanical stimulation, probably due to opening of stretch-activated Ca2þ channels (e.g., Peuchen et al., 1996). Although it cannot a priori be assumed that increases in [Ca2þ]i evoked by Ca2þ entry and by release from the endoplasmic reticulum exert similar effect (since the subcellular localization differ—see also chapters by Cornell-Bell et al. and by Scapagnini et al.), mechanical stimulation of astrocytes in primary cultures has been shown to cause mitochondrial depolarization (Peuchen et al., 1996). This observation is analogous to the finding that opening of voltage-sensitive Ca2þ channels triggers an increase in free intramitochondrial Ca2þ in epithelial cells (Lawrie et al., 1996). Opening of voltage-activated Ca2þ channels by exposure to elevated [Kþ]e within the range occurring during neuronal stimulation (Subbarao et al., 1995) also causes glycogenolysis, accentuating the question what the functional role of glycogenolysis is in the activated brain.

2.3. Glycogen as a re-chargeable energy reservoir Glycogenolysis may rapidly provide a large amount of energy. Recent experiments have shown that glycogen is present in the resting brain at a higher concentration than previously realized, i.e., , 10 mmol/glucose equivalent per g wet wt (Cruz and Dienel, 2002; Kong et al., 2002b; Dienel and Cruz, 2003). It is often believed that glycogen storage serves the purpose of providing the brain with glucose equivalents for use during failure of glucose delivery. In the rat the ‘resting’ rate of glucose utilization is 0.7 mmol/ min per g wet wt (Sokoloff et al., 1977), meaning that all brain glycogen would be depleted within 15 min, if it were to replace glucose (see, however, chapter by Gruetter for a different conclusion). Moreover, in most cases glucose deprivation is combined with deprivation of O2, increasing the rate of glucose utilization in order to provide sufficient energy by glycolysis alone, so that glycogen stores would last even shorter. However, recent experiments have indicated that glycogen metabolism is much more dynamic than would be expected from an emergency store, as reflected by the observation that both glycogenolysis and glycogen re-synthesis are stimulated during brain activation (Swanson et al., 1992; Dienel et al., 2002; Dienel and Cruz, 2003). Glycogen is also broken down rapidly and subsequently re-synthesized at specific stages of a one-trial avoidance learning task in day-old chicks, i.e., immediately after training (Fig. 2) and again around 55 min after training (O’Dowd et al., 1994). Memory consolidation in this animal model is an energy requiring process, characterized by release of Kþ, glutamate and noradrenaline at specific time points (Hertz et al., 1996). As illustrated in the figure, the degradation of glycogen can occur very rapidly, probably exceeding 1 mmol/min per g wet wt of the brain or, with astrocytes constituting less than 20% of brain cortical volume (see chapter by

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Fig. 2. Content of glycogen in the forebrain of day old chicks after pretraining, involving pecking at a watercoated bead (open square) and representative of the non-aversively trained animal, and after one-trial aversive training, involving pecking at a red aversively tasting bead and a blue neutral bead (filled-in squares, with the training performed seconds before the first point). The aversive training induces a rapid decline in glycogen content (,4 mmol/g wet wt within 2.5 min), which is statistically significant between 0 and 2.5 min, followed by a maintained reduction in glycogen level between 2.5 and 12.5 min (possibly with some oscillation of the level) and complete restoration of aversive-training level of glycogen between 12.5 and 25 min. Not shown in the figure is that there is a second decline in glycogen level around 55 min post-training, likewise followed by complete recovery during a 10 min period.

Wolff and Chao), more than 5 mmol/min per g wet wt of astrocytes. If this value applies to the rat brain, and if astrocytes metabolize glucose at the same rate as the average brain, this value is seven times higher than the rate of glucose utilization (0.7 mmol/min per g wet wt in the rat brain). Storage of glucose as glycogen is an energy requiring process. Synthesis of glycogen requires three high energy phosphate equivalents (one ATP, one UTP, one from cleavage of pyrophosphate) for each glucose equivalent incorporated. However, glycogenolysis provides the advantage that less ATP is required to ‘prime’ glucose equivalents in glycogen than glucose for further metabolism, when there is an abrupt demand for energy. Metabolism of glucose to pyruvate via the glycolytic pathway requires an ‘investment’ of two ATP (to form glucose-6-phosphate and fructose-1,6-bisphosphate), and it yields four ATP for a net production of two ATP. Degradation of glycogen does not require the initial ATP to convert glucose to glucose-6-P, so the net, immediately available energy yield is three ATP, disregarding the ATP consumed in the synthesis of glycogen. If the ATP consumption required for synthesis of glycogen is also taken into account, there is an added cost to degrading glucose via glycogen, which is subsequently re-synthesized. It is therefore likely that activity-induced turnover of glycogen rather than direct utilization of glucose as a metabolic fuel provides an unknown metabolic advantage to astrocytes. Evidence will be presented, suggesting that this advantage may be that glycogen can serve at a re-chargeable energy store in a spatio-temporal manner, i.e., provide energy at specific locations (where oxidatively derived energy may be less readily available) at specific times (when the demand for energy is high), and then subsequently be ‘re-charged’ when there is no urgent demand for energy. Due to the rapidity of the breakdown of glycogen

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and the fact that three molecules of ATP are generated per molecule glucose equivalent degraded glycolytically, compared to two molecules of ATP per molecule glucose, glycogenolysis may be able to supply more ATP more rapidly. The specific location where glycogen is most likely to be of special importance as an energy reservoir is in the most peripheral parts of the astrocytic processes (or filopodia and lamellopodia), which form a specific functional compartment, which accounts for 80% of the total cell surface (see chapters by Derouiche and by Wolff and Chao). Ultrastructural analysis has demonstrated a widespread distribution of glycogen in astrocytic processes throughout the neuropil, and glycogen particles are small enough, with diameters ranging between 10 and 40 nm, depending on staining method (Cataldo and Broadwell, 1986; Wender et al., 2000) to reside within the 50 – 100 nm wide lamellae of even the most peripheral astrocytic processes (Peters et al., 1991). These domains show extensive immunostaining for Cx43, the major astrocytic gap junction protein, and the abundant localization of Cx43 partly reflects the formation of autaptic gap junctions onto other fine processes or onto major branches of the same astrocyte (Rohlmann and Wolff, 1996; Wolff et al., 1998—see also chapter by Wolff and Chao). It is likely that glycogenolysis can spread both intra- and intercellularly through gap junctions (Cruz et al., 1999; Dienel and Cruz, 2003), since gap junctional communication has been found to coordinate vasopressininduced glycogenolysis in rat hepatocytes (Eugenin et al., 1998). Immunoreactivity for the glycogen degrading enzyme glycogen phosphorylase has similarly shown that the enzyme is localized mainly in astrocytes, with diffuse staining throughout the cytoplasm and processes ensheathing capillaries, and in the fine processes and lamellae adjacent to synaptic structures (Richter et al., 1996). Since mitochondria are far too large to be located here, oxidatively generated energy can only reach the peripheral astrocytic regions by diffusion of ATP and/or phosphocreatine. This process may be too slow to keep pace with rapidly developing energy demand, e.g., for uptake of neuronally released Kþ, which accordingly may be responded to by immediate glycogenolysis, as will be discussed below. Subsequently, the energy generated by complete oxidative degradation of pyruvate may partly be utilized for regeneration of glycogen at times when the demand for glucose degradation is less acute. For efficient operation of such a ‘glycogen shunt’ it would be a prerequisite to fuel at least part of the energy demands for glycogen synthesis by oxidatively derived energy. Type I hexokinase, the enzyme converting glucose to glucose6-phosphate (the first metabolic reaction in both glycolysis and glycogen synthesis), has characteristics consistent with this requirement, since it binds reversibly to mitochondria and, as illustrated in Fig. 1, selectively utilizes oxidatively generated ATP to fuel the phosphorylation of glucose (BeltrandelRio and Wilson, 1992; de Cerqueira Cesar and Wilson, 1995). 3. Ion effects on metabolism 3.1. The potassium ion (K þ) 3.1.1. Carrier-mediated effects Neuronal excitation is accompanied by Kþ release, and [Kþ]e in brain increases from its resting level (3 mM) during neuronal excitation, up to a maximum of 12 mM during

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seizures and intense stimulation (see chapter by Walz). An even larger, and completely reversibly increase in [Kþ]e (to . 50 mM) occurs during energy deprivation and ‘spreading depression’, a peculiar phenomenon during which repetitive waves of inhibition of electrical activity, triggered by local application of glutamate, high [Kþ]e, or by strong mechanical stimulation, spreads across the cerebral cortex (Somjen, 2001). As discussed above, excess Kþ is subsequently cleared from the extracellular space, initially mainly into astrocytes, partly by active, Naþ,Kþ-ATPase-mediated Kþ uptake into astrocytes, triggered by the increased [Kþ]e, and partly by activation of the Naþ, Kþ, Cl2 co-transporter (Fig. 1) Any elevation of the concentration of [Kþ]e above its resting level causes a stimulation of Naþ,Kþ-ATPase activity at its extracellular Kþ-sensitive site in cultured astrocytes and in astrocytes isolated from mature brain by gradient centrifugation, but not in corresponding preparations of neurons (e.g., Henn et al., 1972; Grisar et al., 1979; Hajek et al., 1996). In cultured astrocytes maximum Naþ,Kþ-ATPase activity is reached at a [Kþ]e of , 12 mM, the enzyme activity follows Michaelis– Menten kinetics with a Km of 1.9 mM for Kþ, and the maximum activity ðVmax Þ is higher than in neurons (Hajek et al., 1996). In cultured neurons and synaptosomes (Kimelberg et al., 1978) the enzyme has a 3- to 5-fold higher affinity for Kþ compared to astrocytes, and it is therefore operating at maximal velocity (substrate level q Km ) at resting [Kþ]e, and it is accordingly not stimulated by above-normal [Kþ]e. Therefore, at elevated [Kþ]e, Kþ uptake will mainly occur in astrocytes, whereas neuronal re-accumulation may be favored at normal or lowered [Kþ]e due to the higher affinity of the neuronal Naþ,Kþ-ATPase. In addition, neuronal Kþ accumulation will obviously be activated by stimulation of Naþ,Kþ-ATPase activity during extrusion of excess Naþ, accumulated as a result of the action potential, but this process may be slower than the re-establishment of a resting [Kþ]e. Consistent with the stimulation of Naþ,Kþ-ATPase in astrocytes only by elevated þ [K ]e (as long as the elevation is not high enough to lead to neuronal excitation) an increase of [Kþ]e from 5 to 12 mM increases glucose phosphorylation in mouse astrocytes in primary cultures and in neuronal-astrocytic co-cultures from the rat by 25 –50% (Fig. 3), whereas 12 mM [Kþ]e is not sufficient to elicit an action potential and does not enhance glucose metabolism in neurons in primary cultures (Peng et al., 1994, 1996; Huang et al., 1994; Honneger and Pardo, 1999). The metabolic stimulation in astrocytes is almost completely inhibited by ouabain, an inhibitor of Naþ,Kþ-ATPase activity. Since Kþ release during neuronal stimulation causes a much larger relative increase in [Kþ]e than in intracellular Kþ concentration (due to the smaller volume and the lower concentration), and neuronal activity is sensitive to the membrane potential, which is mainly determined by the ratio between extra- and intracellular Kþ (see chapter by Walz), an intense astrocytic Kþ accumulation may primarily serve the purpose of restoring [K]e, and it may be most active at the most peripheral processes in the neuropil. In accordance with the concept that the energy supply for urgent metabolic processes in distant astrocytic processes partly may be met by glycogenolysis, it has been shown by Raffin et al. (1992) that iodoacetate, an inhibitor of glycolysis and glycogenolysis, affects the clearance of [Kþ]e resulting from neuronal stimulation in the rat brain in vivo. As can be seen from Fig. 4, the halflife for the first half of Kþ clearance from its peak level towards baseline was only slightly, and non-significantly, inhibited by hypoxia, whereas it

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Fig. 3. Effect of elevated extracellular Kþ level ([Kþ]e) on deoxyglucose (DG) phosphorylation in mixed neuronal-astrocytic aggregate cultures and in neuron-enriched aggregate cultures from rat brain. The rates of DG phosphorylation were measured during a 30 min incubation in tissue culture medium with 5.5 mM glucose and a fixed concentration of [3H]DG. Note that both 12 and 30 mM [Kþ]e stimulate the DG phosphorylation rate in the mixed neuronal-astrocytic cultures above the rate obtained with 5 mM [Kþ]e. On the other hand, only 30 mM [Kþ]e has a stimulatory effect in the neuron-enriched cultures, presumably secondary to [Kþ]e-induced excitation. The absence of effect by 12 mM [Kþ]e in the neuronal aggregates suggests an effect on astrocytes, which is consistent with the stimulatory effect of 12 mM [Kþ]e on DG phosphorylation in astrocyte cultures consistently reported by Peng et al. (1994, 1996) and Huang et al. (1994). Vertical bars denote SD. The stimulatory effects of 12 and 30 mM [Kþ]e in mixed-cell cultures and of 30 mM [Kþ]e in neuron-enriched aggregates are statistically significant, as is the difference between the effects of 12 and 30 mM [Kþ]e in the mixed-cell cultures (P , 0:05 or better). From Honneger and Pardo (1999).

was almost doubled by iodoacetate, indicating that the rate of Kþ removal was reduced by almost one half; in contrast, the second half of Kþ clearance to the resting level of [Kþ]e was virtually unaffected by iodoacetate but substantially inhibited by hypoxia. The first conclusion of these observations is that Kþ homeostasis at the cellular level of the brain is active and energy-requiring. The second conclusion is that the initial uptake in astrocytes (and perhaps also in some parts of neurons) mainly is fueled by glycolytic metabolism, probably to a large extent of the local glycogen stores and to a minor extent of the glucose present. In contrast, neuronal re-accumulation at lower [Kþ]e as well as the later part of the astrocytic accumulation (depending upon the rate with which mitochondrially formed ATP and phosphocreatine can reach the uptake sites) is fueled by oxidatively generated energy. Since iodoacetate also abolishes subsequent oxidative metabolism of glucosederived pyruvate, a third conclusion is that either metabolism is slow enough that some pyruvate has remained, or different compounds are oxidized, probably primarily glutamate (see chapter by Schousboe and Waagepetersen).

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Fig. 4. Effect of hypoxia and of inhibition of glycolysis on Kþ clearance from the extracellular space in rat brain. Hypoxia was induced by administration of inspired air with a severely reduced oxygen content, and glycolysis was inhibited by application of a 5 mM solution of iodoacetate (IOA) to the surface of the brain. [Kþ]e was measured with an extracellular Kþ-sensitive electrode implanted approximately 100 mm below the cortical surface. After direct cortical stimulation [Kþ]e initially increased to a peak (1/1 in inset in upper left corner) and then decreased to reach its resting level (0 in inset). The time to reach one half of the peak increase (indicated by arrow at 12 ) was considered as the first one half of Kþ clearance and the time from 12 to 0 as the second one half. The times required for [Kþ]e to decline from 1/1 to 12 and from 12 to 0 were determined under control conditions (gray bars), indicated as 100% in the graph (which should not suggest that they were similar), and compared with the corresponding times during hypoxia (black bars) and during inhibition of glycolysis (white bars). Hypoxia had no significant effect on the first one half of Kþ clearance but almost doubled the time needed for the second half of Kþ clearance. In contrast, inhibition of glycolysis by aid of iodoacetate almost doubled the first one half of Kþ clearance, but had no significant effect on the time needed for the second half of Kþ clearance. Significant differences from control conditions are indicated by asterisks.

3.1.2. Channel-mediated effects The [Kþ]e which is required to cause Ca2þ channel-mediated glycogenolysis and stimulate glycogen phosphorylase in brain tissue (Ververken et al., 1982; Cambray-Deakin et al., 1988; Hof et al., 1988) is within the range occurring in the brain in vivo (i.e., well below 12 mM). Spreading depression with its very high [Kþ]e is accompanied by a reduction of glycogen level by one third and complete restoration after 10 min (Lauritzen et al., 1990). However, after 5 min of sensory stimulation, when the glycogen level fell by one quarter, the glycogen level had not been restored 15 min after cessation of stimulation (Cruz and Dienel, 2002). Elevation of [Kþ]e also stimulates glycogenolysis in well differentiated astrocytes in primary cultures (Subbarao et al., 1995). This is illustrated in Table 1, where glycogenolysis is indicated as the percentage release of previously incorporated [14C]glucose during 1 – 10 min of exposure to 10– 30 mM [Kþ]e. It can be seen that 40– 45% of the label is released after at least 1 min exposure to a [Kþ]e of 10 mM or more, and that this is about the maximum amount releasable by elevated [Kþ]e. The release is fast, as shown by the fact that slightly more than one half is completed after exposure for only 12 min; however, since recently incorporated radioactivity may be preferentially released, it is not possible to calculate the amount released, based on the pool size. More than 75% of the response was abolished by Ca2þ depletion (even though no Ca2þ-chelating agent had been added) and a similar reduction was evoked by 100 nM

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Table 1 Kþ-stimulated glycogenolysis in primary cultures of mouse astrocytes, expressed as percentage release of radioactivity previously incorporated from [3H]glucose, measured under control conditions and after medium modification [Kþ]e (mM)

Time (min)

Condition

Glycogenolysis control

Medium modification

Glycogenolysis modified

10 20 30

1 10

Control þ Kþ Control þ Kþ Control þ Kþ

42.5 ^ 8.3 43.8 ^ 7.7 26.5 ^ 2.2

2Ca2þ þ Kþ Nifedipine, 100 nM þ Kþ Diazepam, 20 nM þ Kþ

9.3 ^ 1.6 12.5 ^ 7.8 41.9 ^ 3.1

1 2

Individual cultures were incubated with tissue culture medium containing [6-3H]glucose (20 mCi/ml) at a glucose concentration of 3 mM during a 30 min period as described by Quach et al. (1982) using cultures from the same batch (containing virtually the same amount of cell protein) for the experimental conditions to be compared, i.e., especially those in the same horizontal row. After medium removal and thorough wash the cultures were re-fed with similar, non-labeled medium or similar, non-labeled medium modified as indicated in the table, both containing the indicated Kþ concentration. After the indicated time the medium was removed and the cultures washed with ice-cold isotonic NaCl solution and harvested in a similar solution. Glycogen was isolated on a filterpaper, and after removal of non-glycogen label by washing it was dissolved in boiling distilled water, and radioactivity was determined and expressed relative to that in similarly treated cultures from the same batches, which had not been exposed to an elevated Kþ concentration and in which no glycogenolysis had occurred. All values are means ^ SEM values. Data are from Subbarao et al. (1995).

nifedipine (Subbarao et al., 1995), a blocker of the L-channel for Ca2þ. Thus, the glycogenolytic effect is secondary to opening of voltage-dependent L-channels.

3.1.3. Co-transporter-mediated effects An increase of [Kþ]e in the range occurring in the brain in vivo also stimulates the activity of a co-transporter jointly accumulating Kþ, Naþ and Cl2. Simultaneous uptake of Kþ, Naþ and Cl2 by the Kþ, Naþ, 2Cl2 co-transporter is metabolically driven by the electrochemical gradient of Naþ (and to a minor extent of Cl2). It therefore must lead to stimulation of the Naþ,Kþ-ATPase at its intracellular Naþ-sensitive site (Fig. 1). This results in subsequent exchange of intracellular Naþ with extracellular Kþ, i.e., the co-transporter mediates in essence a net uptake of Kþ and Cl2, which for osmotic reasons is accompanied by water uptake, i.e., cell swelling, when the cell membrane is permeable for water (see chapter by Chen and Spatz). In cultured mouse astrocytes co-transporter activity is stimulated by high [Kþ]e (Walz and Hertz, 1984). This stimulation is probably a result of Kþ-stimulated Ca2þ entry through L-channels (Fig. 1), since co-transporter activity is dependent upon the presence of Ca2þ and is inhibited by 0.5 mM nifedipine (Su et al., 2000). Thus, stimulation of the rate of oxygen consumption in microdissected or cultured astrocytes by elevated [Kþ]e (Hertz, 1966; Hertz et al., 1973; Hertz and Hertz, 1979) may at least partly reflect stimulation of co-transporter activity. This conclusion is supported by the observation that the stimulation of oxidative metabolism, is inhibited by furosemide, a co-transporter inhibitor (Hertz, 1986). Stimulation of co-transporter activity by high [Kþ]e is probably also the principal reason for a Kþ-induced increase in O2 consumption in brain slices, which is

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Ca2þ-dependent (Hertz and Schou, 1962) and rather insensitive to ouabain (suggesting that a Naþ gradient, necessary to drive the co-transport, persists for a considerable amount of time after inhibition of Naþ extrusion, although its magnitude becomes gradually reduced). This stimulation of O2 consumption in brain slices is accompanied by astrocytic cell swelling (Hertz and Kjeldsen, 1985; Hertz and Dienel, 2002), and it is abolished by ethacrynic acid, another co-transporter inhibitor. This inhibition as well as the resistance to ouabain is illustrated in Table 2, which shows a rate of O2 consumption slightly above 100 mmol/g wet wt during incubation in a ‘balanced salt solution’ (Hertz and Kjeldsen, 1985) with 6 mM glucose. Under control conditions an increase in the Kþ concentration to 50 mM causes a 70% increase in rate of O2 uptake (Ashford and Dixon, 1935; Dickens and Greville, 1935; Hertz and Schou, 1962), but in the presence of 0.5 mM ethacrynic acid this stimulation is abolished, whereas the rate of O2 consumption at normal Kþ concentration is unaffected. Ouabain (1025 and 1024 M) reduces the Kþ-induced stimulation, but it does not abolish it. These findings suggest that the Kþ-induced stimulation of O2 consumption is mainly an astrocytic phenomenon, probably due to inactivation of neuronal Naþ channels during maintained depolarization. In contrast, electrical stimulation of brain slices, as developed by McIlwain (1951) is likely to stimulate the neuronal population (due to the pulsatile rather than maintained depolarization), and it is inhibited by tetrodotoxin and several other drugs that have little effect on the Kþ-mediated stimulation of oxygen uptake (for review, see Hertz, 1977). As illustrated in Fig. 5, spreading depression, which is accompanied by very high [Kþ]e, and must represent one of the strongest possible demands for Kþ clearance, is accompanied by a 40% stimulation of oxidative metabolism of acetate (Dienel et al., 2001; Dienel and Cruz, 2003), a substrate that is exclusively taken up and therefore oxidized by astrocytes (O’Dowd, 1995; Waniewski and Martin, 1998; Hertz and Dienel, 2002). Table 2 Rates of oxygen consumption (mmol/g wet wt per h) in rat brain slices incubated in a balanced salt solution with 6 mM glucose under control conditions, in the presence 0.5 mM ethacrynic acid, an inhibitor of the Naþ, Kþ, 2Cl2 co-transporter, and in the presence of ouabain (1025 and 1024 M) Condition

Rate of O2 consumption balanced medium

Rate of O2 consumption 50 mM Kþ

Percent stimulation

Control 0.5 mM ethacrynic acid 1025 M ouabain 1024 M ouabain

111.1 ^ 4.04 114.0 ^ 1.20 137.0 ^ 5.26 86.7 ^ 4.40

188.7 ^ 4.47 114.0 ^ 3.47 174.3 ^ 3.62 112.4 ^ 6.35

50.7 0.1 27.2 29.6

Brain cortex slices of 0.5 mm thickness (first slices only) were prepared from adult Wistar rats as described by Hertz and Kjeldsen (1985) and incubated in balanced bicarbonate-buffered saline medium, containing 6 mM glucose and 5 mM KCl and prepared with or without the drugs indicated and equilibrated with a CO2/O2 mixture (5%/95%) in a closed chamber equipped with a Clark oxygen electrode and completely filled with medium. The oxygen tension was recorded, and the rate of O2 consumption, expressed per g initial wet wt, calculated from the total O2 content in the fluid volume and the rate of decline in O2 tension during the experiment, which was found to be constant for at least 30 min. After a stable decline in O2 tension had been recorded for 10 –15 min, a concentrated KCl solution was added to a final concentration of 50 mM, and the decline in O2 tension followed for another 10 2 15 min. Results are means ^SEM.

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Fig. 5. Metabolic imaging of unilateral spreading cortical depression. An intravenous pulse of [14C] tracer was injected at 20 min after induction of unilateral spreading depression by topical application of KCl to the dura of left cerebral cortex of the conscious rat. The labeling period was 5 min for both tracers, and autoradiographs were prepared from serial coronal sections. Spreading depression caused heterogeneous increases in labeling of the left compared to the untreated right cerebral cortex with both [14C]DG and [1-14C]acetate; red indicates high metabolic rate, with progressively lower rates represented by yellow, green, blue, and black. The dark area in the left cortex is the KCl application site; the cortical tissue below the KCl site had very low uptake of all tracers, presumably due to the very high KCl levels. Labeling with DG and acetate was highest near the KCl application site, and tended to be higher than average in the most dorsal and most ventral layers of left cerebral cortex. Note that labeling by acetate was heterogeneous in gray matter in both hemispheres, and corpus callosum (white matter) had lower levels compared to gray matter structures. Modified from Hertz and Dienel (2002).

Blockade of voltage-dependent Ca2þ channels does not prevent the initial wave of spreading depression, but after application of blockers of either the L-, N- or P/Q-type of voltage-dependent Ca2þ channels, application of KCl to the cortical surface elicited one, or at most a few, waves of spreading depression, rather than the usual repetitive waves (Richter et al., 2002). This inhibition was interpreted as indicating an influence of voltagegated Ca2þ channels on cortical excitability. However, inhibition of restorative processes mediated by the co-transporter, and restoring intracellular Kþ would be in perfect agreement with the unaffected initial response and secondary development of refractoriness. It would be of interest to establish the effect of Ca2þ channel blockers on glucose and acetate metabolism during spreading depression. 3.1.4. K þ-induced stimulation of enzymes involved in glucose metabolism Several enzymes involved in glucose metabolism are stimulated by elevated Kþ concentrations (see, e.g., Hertz and Dienel, 2002), including pyruvate carboxylase (Ruiz-Amil et al., 1965; McClure et al., 1971). Accordingly pyruvate carboxylation increases with a rise in the extracellular Kþ concentration from 2 to 25 mM in cultured astrocytes (Kaufman and Driscoll, 1992).

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3.2. The sodium ion (Naþ) 3.2.1. Carrier-mediated effects The Naþ, Kþ, 2Cl2 co-transporter is not the only carrier that is metabolically driven by the Naþ gradient across the astrocytic cell membrane. Many amino acids are accumulated by a Naþ-driven active uptake, none more important for energy metabolism than glutamate, which is avidly accumulated by the astrocyte-specific EAAT1 and EAAT2 (see chapter by Schousboe and Waagepetersen). Administration of glutamate accordingly stimulates the rate of O2 consumption in cultured astrocytes (Eriksson et al., 1995; Hertz and Hertz, 2003). Since glutamate is oxidatively degraded in astrocytes (Yu et al., 1982; McKenna et al., 1996; Hertz and Hertz, 2003), the increase in O2 consumption may reflect oxidation of glutamate as an alternate metabolic substrate. It is in agreement with this conclusion that it repeatedly has been observed in several different laboratories that deoxyglucose phosphorylation is unaltered or slightly reduced in the presence of glutamate (Hertz et al., 1998; Peng et al., 2001; Chen and Liao, 2001; Qu et al., 2001). However, a glutamate-mediated stimulation of glucose phosphorylation in primary cultures of astrocytes was reported by Pellerin and Magistretti (1994) and by Sokoloff et al. (1996), possibly reflecting a lack of ability of the cultures used by these authors to degrade glutamate oxidatively. It is in agreement with this interpretation that Peng et al. (2001), who found no glutamate-induced stimulation of glucose phosphorylation, did observe such a stimulation after administration of the non-metabolizeable D -aspartate, which utilizes the same carrier as glutamate. 3.2.2. Channel-mediated effects Exchange between intracellular Hþ and extracellular Naþ, brought about by monensin, stimulates not only glucose phosphorylation (Yarowsky et al., 1986), but also glucose oxidation in astrocytes (Peng et al., 2001), presumably by activation of the intracellular Naþ-sensitive site of the Naþ,Kþ-ATPase. The evoked alkalosis might also stimulate glucose metabolism, but the stimulation is inhibited by ouabain, indicating that it is secondary to enhanced Naþ,Kþ-ATPase activity (Yarowsky et al., 1986; Peng et al., 1994).

4. Transmitter effects on metabolism 4.1. Second messenger systems 4.1.1. The phosphoinositide second messenger system Phospholipase C catalyzes formation of inositoltrisphosphate (IP3) and diacylglycerol (DAG) from the membrane lipid phosphatidylinositide 4,5-bisphosphate (PIP2) (see chapter by Hertz, Chen et al.). IP3 stimulates protein kinase C, activating downstream signal transduction, and DAG causes release of Ca2þ from intracellular stores on the endoplasmic reticulum, leading to an increase in [Ca2þ]i (see chapter by Scapagnini et al.), which subsequently can lead to activation of glycogen phosphorylase, the Naþ, Kþ, 2Cl2 co-transporter and/or direct stimulation of energy metabolism, caused by an elevation of intramitochondrial Ca2þ, as illustrated in Fig. 1.

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4.1.2. The adenylyl cyclase second messenger system The activated and released a subunit of the G-protein Gs stimulates adenylyl cyclase, leading to the formation of cAMP from ATP. In turn, cAMP binds to the regulatory subunits of inactive protein kinase A, releasing the free catalytic subunits, which are then able to phosphorylate and activate their target proteins, which include phosphorylase kinase. The activated, phosphorylase kinase converts an inactive form of glycogen phosphorylase, phosphorylase b to its active form, phosphorylase a. The free catalytic subunits of protein kinase C are also able to enter the cell nucleus, where they phosphorylate the transcription factor CRE-binding protein (CREB), leading to the activation of cAMP-inducible genes containing the regulatory sequence cAMP responsive element (CRE) and thus to synthesis of specific proteins.

4.2. Individual transmitters 4.2.1. Noradrenaline Adrenergic receptors are expressed on cerebral astrocytes (see chapter by Hansson and Ro¨nnba¨ck), indicating that astrocytes represent a major target for activation of locus coeruleus, the nucleus of origin for noradrenergic fibers to the brain (Stone and Ariano, 1989; Stone et al., 1992). Rather than releasing transmitter from conventional synapses many of these fibers release noradrenaline from varicosities from which it can diffuse to all adjacent cells, including astrocytes and endothelial cells. The ‘classical’ adrenergic receptor activating the phosphoinositide second messenger system is the a1-adrenergic receptor, but astrocytes also express phospholipase C-linked a2-adrenergic receptors. Both the a1-adrenergic receptor agonist phenylephrine and the a2-adrenergic receptor agonist clonidine stimulate formation of labeled CO2 from 1-[14C]glutamate, indicating activation of the a-ketoglutarate dehydrogenase complex (aKGDH), as summarized in Table 3 (Subbarao and Hertz, 1991). The same two subtype agonists also stimulate hydrolysis of glutamine to glutamate (Huang and Hertz, 1995). However, only the a2-adrenergic agonist clonidine was found to significantly stimulate the pyruvate dehydrogenase complex (PDH) (Table 3), as indicated by its stimulation of the formation rate of labeled CO2 from 1-[14C]pyruvate (Chen and Hertz, 1999). Thus, an increase in [Ca2þ]i is a necessary, but not sufficient requirement for stimulation of mitochondrial dehydrogenases by stimulation of the phophoinositide second messenger system. Naþ, Kþ-ATPase activity in astrocytes is also increased by clonidine, but not by phenylephrine (Hajek et al., 1996). Stimulation of glycolysis (Subbarao and Hertz, 1991) and of glutamate uptake (Hansson and Ro¨nnba¨ck, 1992; Alexander et al., 1997) is, in contrast, dependent upon stimulation of a1-adrenergic receptors. The reason for dissimilar effects of different phospholipase C-linked transmitter agonists may be differences in subcellular localization of increased [Ca2þ]i and/or differences in downstream signaling; such differences do exist, as indicated by an ability of the a2-adrenergic agonist dexmedetomidine, but not of the a1-adrenergic agonist phenylephrine to induce phosphorylation at the MAP kinases ERK1/2 (see chapter by Peng). Stimulation of glycogenolysis by noradrenaline in non-cultured cerebral cortical tissue (Magistretti, 1988) and in retina (Ghazi and Osborne, 1989) has indicated a metabolic

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Table 3 Transmitters affecting glucose and glutamine metabolism in primary cultures of mouse astrocytes Transmitter

Subtype

Messenger

Noradrenaline Noradrenaline Noradrenaline Serotonin Serotonin Dopamine Histamine Histamine Adenosine ATP VIP Secretin Substance P Vasopressin

b a1 a2 5-HT2A 5-HT2B D(1)-like H1 H2 A2B P2Y VPAC – NK V1b/V3

cAMP Ca2þ Ca2þ Ca2þ Ca2þ cAMP Ca2þ cAMP cAMP Ca2þ cAMP cAMP Ca2þ Ca2þ

PDH

0 X 0 0

aKGDH

PAG

Glycolysis

Glycogenolysis

0 X X

0 X X

0 X 0

X 0 X X X X X X X X X X X

0

Stimulation of the PDH was measured by determination of rate of 14CO2 production from [1-14C]pyruvate; stimulation of the a-ketoglutarate dehydrogenase complex (aKGDH) by determination of rate of 14CO2 production from [1-14C]glutamate; stimulation of phosphate-activated glutaminase (PAG) by incubation with [U-14C]glutamine followed by HPLC analysis and counting of radioactivity in the glutamate and glutamine fractions; stimulation of glycolysis by measurement of lactate formation; and stimulation of glycogenolysis in our own experiments by release of radioactivity from preloaded glycogen, as described in the legend of Table 1 and in experiments by other investigators as indicated in the respective publications. X indicates significant stimulation and 0 that no stimulation was observed. Information for messenger of subtype-specific receptors are from chapter by Hansson and Ro¨nnba¨ck; from Jin et al. (2001) (D(1)-like receptor); from Dickenson and Hill (1994) (H1 receptor); from Dartt (1989) (VPAC receptor); from Olde et al. (1998) (secretin receptor); and from Ueda (1999) (NK receptor).

effect on astrocytic energy metabolism in non-cultured tissue, and the activation of glycogen phosphorylase was found to be mediated by b-adrenergic receptors (Ververken et al., 1982; Table 3). The glycogenolytic effect at specific stages of imprinting in day-old chicks is also a b-adrenergic effect, as indicated by its inhibition by propranolol (O’Dowd, 1995). Studies using cultured astrocytes have confirmed a b-adrenergic stimulation of glycogenolysis and indicated an additional effect of a2-adrenergic stimulation by showing that glycogenolysis can be stimulated by isoproterenol and clonidine, but not by phenylephrine (Subbarao and Hertz, 1990b). The effects can be explained by a stimulatory effect of Ca2þ on glycogen phosphorylase and by the well known activation of phosphorylase by protein kinase A. Since glycogen is not only rapidly degraded but also re-synthesized during and after brain activation and at specific stages of learning (Fig. 2), it becomes of importance to establish whether noradrenaline also enhances glycogen synthesis. Magistretti and coworkers have demonstrated that noradrenaline exerts long-term control of glycogen levels in astrocytes and within 9 h leads to a large, protein synthesis-dependent induction of glycogen synthase and increase in glycogen, an effect mediated by protein kinase A and its phosphorylation of CREB (Sorg and Magistretti, 1992; Pellegri et al., 1996). However,

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such a long-term effect is unable to explain re-synthesis of glycogen concurrently with or relatively soon after glycogenolysis. We have therefore measured the effect of noradrenaline, phenylephrine, clonidine and isoproterenol on incorporation of label from [U-14C]glucose into glycogen, realizing that this is not a ‘clean’ experiment, because of simultaneous stimulation of glycogenolysis by most of the agonists. The results, as percentage of incorporation in the absence of any adrenergic agonist (control), are shown in Table 4. It can be seen that noradrenaline and the b-adrenergic agonist, isoproterenol exert an inhibition, which is statistically significant in the case of isoproterenol, and can be explained by simultaneous glycogenolysis. This inhibition is counteracted by the b-adrenergic antagonist, alprenolol. In spite of the glycogenolytic activity by the a2adrenergic agonist clonidine, clonidine tended to cause an increase in incorporation of labeled glucose into glycogen, and the a2-adrenergic antagonist yohimbine significantly decreased this incorporation, further supporting the stimulation of glycogen synthesis by a2-adrenergic agonists. Phenylephrine had no effect (Table 4). 4.2.2. Serotonin (5-HT) Serotonergic fibers spread from the raphe nuclei across the entire brain in much the same way as noradrenergic fibers spread from locus coeruleus. Like noradrenaline, serotonin increases glycogenolysis in brain tissue and in retina (Magistretti, 1988; Ghazi and Osborne, 1989). It has been reported that glycolysis in retina is 5-HT1 receptormediated, but glycogenolysis in astrocytes can be stimulated by activation of simultaneously expressed high-affinity 5-HT2B (Kong et al., 2002a) and lower affinity Table 4 Transmitter effects on incorporation of [6-3H]glucose (3 mM) for 30 min into glycogen Transmitter

Percentage incorporation

Control Noradrenaline (NA) Phenylephrine Clonidine Isoproterenol NA þ Alprenolol NA þ Yohimbine

100 ^ 3.0 85.9 ^ 6.8 91.0 ^ 8.5 111.3 ^ 15.0 62.8 ^ 10.7 97.2 ^ 3.9 65.5 ^ 5.7

Individual cultures were incubated with tissue culture medium containing [6-3H]glucose (20 mCi/ml) at a glucose concentration of 3 mM during a 60-min period in tissue culture medium with or without the noradrenergic agonists (0.1 mM) and antagonists (1 mM) indicated, using cultures from the same batch (containing virtually the same amount of cell protein) for all experimental conditions. After the indicated time the medium was removed and the cultures washed with ice-cold isotonic NaCl solution and harvested in a similar solution. Glycogen was isolated on a filter paper, and after removal of non-glycogen label by washing, it was dissolved in boiling distilled water, and radioactivity per culture was determined and expressed relative to that in similarly treated cultures which had not been exposed to an elevated Kþ concentration. All values are means ^ SEM values.

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5-HT2A receptors (Table 3), and the concentration dependence of the glycogenolytic effect is similar to that for a biphasic increase in [Ca2þ]i (Chen et al., 1995). In contrast to clonidine, but similar to phenylephrine, serotonin does not stimulate pyruvate dehydrogenation (Table 3; Chen, 1996).

4.2.3. Dopamine In primary cultures of mouse cerebrocortical astrocytes 10 mM dopamine causes glycogenolysis (Table 3), which is more marked than that evoked by the corresponding concentration of serotonin (L. Peng, unpublished experiments). Intraventricularly injected dopamine increases glycogenolysis in the mouse brain (Leonard, 1975). However, it does not influence glycogenolysis in retina (Ghazi and Osborne, 1989).

4.2.4. Histamine Histamine resembles noradrenaline and serotonin by stimulating glycogenolysis in brain tissue (Magistretti, 1988) and in primary cultures of astrocytes (Arbones et al., 1990). As in the case of noradrenaline, both a Gs-coupled, cAMP-linked receptor subtype, the H2 receptor, and a phospholipase C-linked, Gi/o-coupled receptor, the H1 receptor, stimulate glycogenolysis (Table 3). No data are available whether histamine stimulates mitochondrial dehydrogenases.

4.2.5. Purinergic receptors Both P1 receptors, activated by adenosine, and phospholipase C-linked P2Y ATP receptor mediate glycogenolysis in primary cultures of astrocytes (Magistretti, 1988; Sorg et al., 1995). The Gs-coupled, cAMP-linked A2b receptor mimics its b-adrenergic counterpart by promoting long-term glycogen synthesis in astrocytes (Allaman et al., 2002).

4.2.6. Peptidergic receptors Vasoactive intestinal peptide (VIP) was among the very first agents shown to promote glycogenolysis in cultured astrocytes, and it was suggested that this effect was exerted via stimulation of adenylyl cyclase activity (Magistretti et al., 1983). VIP also induces long-term glycogen re-synthesis in a similar manner as noradrenaline (Sorg and Magistretti, 1992), and it stimulates glycogenolysis in non-cultured cerebrocortical tissue (Magistretti, 1988) and in retina (Ghazi and Osborne, 1989). Other neuropeptides which stimulate glycogenolysis include substance P (Medrano et al., 1994) and secretin (Sorg and Magistretti, 1992). It is unknown whether vasopressin (AVP) stimulates glycogenolysis in astrocytes, but it does not enhance the activity of mitochondrial dehydrogenases (Table 3).

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5. Drug effects on metabolism 5.1. Interaction with ion effects 5.1.1. Barbiturates Barbiturates inhibit Kþ uptake into cultured astrocytes (Hertz, 1979), apparently especially at elevated [Kþ]e (Hertz, 1986), and they depress Kþ-stimulated oxidative metabolism in astrocytes quite potently, whereas they have only little effect on O2 consumption and production of labeled CO2 from [U-14C]glucose at normal [Kþ]e (Hertz et al., 1986). Since they also inhibit Kþ-stimulated respiration in brain slices (for review, see Hertz, 1977), it is likely that they interfere with co-transporter-mediated Kþ uptake. These observations should not lead to the impression that barbiturate effects are mainly exerted on astrocytes, since they cause a very large decrease in production of labeled CO2 from [U-14C]glucose in cerebellar granule neurons, a glutamatergic preparation (Peng and Hertz, 2002), and since in the brain in vivo they mainly affect neuronal metabolism (see chapter by Gruetter). 5.1.2. Benzodiazepines Astrocytes express the so-called mitochondrial-type benzodiazepine binding sites (see chapter by Be´langer et al.), which are important for the synthesis of neurosteroids from cholesterol (see chapter by Melcangi et al.). The pharmacological profile of these sites is quite different from that of neuronal benzodiazepine receptors. For example the neuronal benzodiazepine antagonist flumazenil has only low affinity for these sites, whereas PK11195, which binds with very low affinity to neurons, has a high affinity for the mitochondrial-type benzodiazepine binding sites (Bender and Hertz, 1987). In addition to these sites astrocytes also express benzodiazepine receptors, which partly mimic the neuronal benzodiazepine receptors linked to GABAA receptors (Backus et al., 1988). However, in astrocytes activation of these receptors causes depolarization, rather than hyperpolarization (see chapter by Hansson and Ro¨nnba¨ck). An interaction between Ca2þ channel ligands and benzodiazepines in astrocytes had long been anticipated (Bender and Hertz, 1985), when it was shown that clinically used benzodiazepines like diazepam increase depolarization-induced Ca2þ uptake through L-channels into cultured astrocytes during exposure to Kþ concentrations too low to exert maximum channel opening on their own (Zhao et al., 1996), probably reflecting an additional benzodiazepine-mediated partial depolarization. A benzodiazepine-mediated increase in [Ca2þ]i has been confirmed in freshly isolated astrocytes (Fraser et al., 1995), and membrane-associated peripheral-type benzodiazepine receptors have been described in heart, liver, adrenal gland, testis, hemopoietic cells, and, not least, cells of the cardiovascular system (Bolger, 1993; Woods and Williams, 1996). Because glycogenolysis is stimulated by Ca2þ entry through voltage-gated channels, the glycogenolytic effects of submaximally effective Kþ concentrations is also enhanced by benzodiazepines (Subbarao et al., 1995). This effect is illustrated in the bottom line of Table 1, showing that glycogenolysis, which after 12 min of exposure to 30 mM [Kþ]e under control conditions amounts to 27% of previously incorporated label, in the presence of 20 nM diazepam is increased to 42% of the incorporated radioactivity.

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5.2. Interaction with transmitter effects 5.2.1. Therapeutics It is likely that many clinically used drugs, that are receptor agonists or antagonists exert effects on astrocytes. An example is the anti-depressant drug fluoxetine (Prozac), which is a specific serotonin re-uptake inhibitor (SSRI), but has sufficient activity for at least the 5-HT2B receptor that clinically relevant concentrations of fluoxetine cause a direct stimulation of glycogenolysis in cultured astrocytes, which do not express the serotonin transporter (Chen et al., 1995; Kong et al., 2002a). In spite of its promotion of glycogenolysis, fluoxetine causes an inhibition of the production of labeled CO2 from U-[14C]glucose (Chen, 1996). Other serotonin-specific reuptake inhibitors, e.g., paroxetine (Paxol) have a lower affinity for the 5-HT2 receptor, and do not by themselves stimulate glycogenolysis at usual therapeutic concentrations. Chronic treatment of cultured astrocytes with fluoxetine can nevertheless be used as a model of the effect of a drug specifically stimulating 5-HT2 receptors, which in any case will be stimulated during in vivo treatment with any serotonin-specific reuptake inhibitor (due to inhibition of neuronal serotonin transporters). Chronic treatment with 1 mM fluoxetine initially downregulates 5-HT2 receptor-mediated glycogenolysis in cultured astrocytes (Chen et al., 1995), but subsequently upregulates it (Kong et al., 2002a). The upregulation appears after 2– 3 weeks of treatment, which is similar to the lag time for the onset of fluoxetine’s therapeutic effect. Like fluoxetine, the anti-migraine drug methysergide displaces serotonin from its binding site on astrocytes in primary cultures (Hertz et al., 1979), but it remains to be demonstrated whether it has any effect on glycogenolysis.

5.2.2. Drugs of abuse Both amphetamine (Nowak, 1988) and 3,4-methylenedioxymethamphetamine (‘ecstasy’) (Darvesh et al., 2002) cause an enhancement of glycogenolysis in the rodent brain, which was suggested at least partly to result from a concomitant increase in body temperature. A single subcutaneous dose of 10– 40 mg/kg of 3,4-methylenedioxymethamphetamine caused a dose-dependent reduction of glycogen, which reached 40%, lasted for more than 1 h, and could be inhibited by a 5-HT2 antagonist. However, 3,4methylenedioxymethamphetamine does cause a temperature-independent effect on glycogen breakdown in astrocytes, as shown by the observation that active glycogen phosphorylase in primary cultures of astrocytes is increased by 70% in the presence of 5 mM 3,4-methylenedioxymethamphetamine (Poblete and Azmitia, 1995). Metabolic effects have been observed after chronic administration of cocaine (1 and 3 mM) to immature mouse astrocyte cultures (simulating human drug exposure during the last third of gestation), in which the usual stimulatory effect on a-ketoglutarate dehydrogenase activity in response to noradrenaline became abolished (Peng and Hertz, 1992). After treatment for 21 days this effect persisted during ‘withdrawal’, i.e., discontinuation of drug treatment, for the entire period investigated (more than one month from the time of withdrawal). The implications of this finding are scary, considering the wide-spread abuse of cocaine by pregnant women.

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6. Concluding remarks The demonstration by Su et al. (2000) that the Naþ, Kþ, 2Cl2 co-transporter is activated by opening of L-channels for Ca2þ (Fig. 1) has been crucial for the understanding of effects on oxidative metabolism by [Kþ]e of the magnitude found extracellularly during brain activity It can now be concluded that activation of this transporter requires both high enough [Kþ]e to activate voltage-gated Ca2þ as well as Naþ,Kþ-ATPase activity to extrude accumulated Naþ(Fig. 1). There is also no longer any doubt that Kþ accumulation in astrocytes by this mechanism and by stimulation of the extracellular Kþ-sensitive site of the Naþ,Kþ-ATPase plays a major role in ion homeostasis (see chapter by Walz) and therefore in energy metabolism in the CNS. Any claim that Kþ is passively distributed in astrocytes should by now be regarded as obsolete. Although other processes, probably especially Naþ-driven uptake of glutamate also contribute to Naþ,Kþ-ATPase-mediated stimulation of astrocytic energy metabolism by activating the intracellular Naþ-dependent site of the Naþ,Kþ-ATPase, they are probably of quantitatively minor importance compared to the regulation of Kþ homeostasis. In addition to ion-stimulated energy metabolism, emerging evidence indicates an important role of transmitter effects on energy metabolism in astrocytes, both by stimulation of mitochondrial dehydrogenases and glutaminase (glutamine is also an energy substrate for brain [see chapter by Schousboe and Waagepetersen]) and by activating glycogenolysis. Although it has been realized for some time that glycogen stores turn over dynamically during neuronal activation (Swanson et al., 1992; O’Dowd et al., 1994), it is only recently that attention has been drawn to the possibility that glycogen may serve as a re-chargeable energy substrate in peripheral processes of the astrocytes, which are too thin to contain any mitochondria (Dienel and Cruz, 2003). With the increasing realization of the importance of astrocytes in brain energy metabolism comes emerging evidence that drugs classically assumed to act solely on neurons may owe a substantial amount of their effect to actions on astrocytes, including astrocytic energy metabolism. The list of such drugs is likely to grow in the future. Many of them may interact with effects of ions or transmitters and thereby affect energy metabolism in astrocytes and thus in the brain.

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Somjen, G.G., 1979. Extracellular potassium in the mammalian central nervous system. Ann. Rev. Physiol. 41, 159 –177. Somjen, G.G., 2001. Mechanisms of spreading depression and hypoxic spreading depression-like depolarization. Physiol. Rev. 81, 1065–1096. Sorg, O., Magistretti, P.J., 1992. Vasoactive intestinal peptide and noradrenaline exert long-term control on glycogen levels in astrocytes: blockade by protein synthesis inhibition. J. Neurosci. 12, 4923–4931. Sorg, O., Pellerin, L., Stolz, M., Beggah, S., Magistretti, P.J., 1995. Adenosine triphosphate and arachidonic acid stimulate glycogenolysis in primary cultures of mouse cerebral astrocytes. Neurosci. Lett. 188, 109–112. Stone, E.A., Ariano, M.A., 1989. Are glial cells target of the central noradrenergic system? A review of the evidence. Brain Res. Rev. 14, 297–309. Stone, E.A., John, S.M., Zhang, Y., 1992. Studies of the cellular localization of biochemical responses to catecholamines in the brain. Brain Res. Bull. 29, 285– 288. Su, G., Haworth, R.A., Dempsey, R.J., Sun, D., 2000. Regulation of Na(þ)–K(þ) –Cl(2) co-transporter in primary astrocytes by dibutyryl cAMP and high [K(þ)]0. Am. J. Physiol. Cell. Physiol. 279, 1710–1721. Subbarao, K.V., Hertz, L., 1990a. Noradrenaline induced stimulation of oxidative metabolism in astrocytes but not in neurons in primary cultures. Brain Res. 527, 346 –349. Subbarao, K.V., Hertz, L., 1990b. Effects of adrenergic agonists on glycogenolysis in primary cultures of astrocytes. Brain Res. 527, 346 –349. Subbarao, K.V., Hertz, L., 1991. Stimulation of energy metabolism in astrocytes by adrenergic agonists. J. Neurosci. Res. 28, 399 –405. Subbarao, K.V., Stolzenburg, J.-U., Hertz, L., 1995. Pharmacological characteristics of potassium-induced glycogenolysis in astrocytes. Neurosci. Lett. 196, 45–48. Swanson, R.A., Morton, M.M., Sagar, S.M., Sharp, F.R., 1992. Sensory stimulation induces local cerebral glycogenolysis: demonstration by autoradiography. Neuroscience 51, 451–461. Thorlin, T., Eriksson, P.S., Ro¨nnba¨ck, L., Hansson, E., 1998. Receptor-activated Ca2þ increases in vibrodissociated cortical astrocytes: a nonenzymatic method for acute isolation of astrocytes. J. Neurosci. Res. 54, 390–401. Ueda, H., 1999. In vivo molecular signal transduction of peripheral mechanisms of pain. Jpn. J. Pharmacol. 79, 263 –268. Ververken, D., Van Veldhoven, P., Proost, C., Carton, H., De Wulf, H., 1982. On the role of calcium ions in the regulation of glycogenolysis in brain cortical slices. J. Neurochem. 38, 1286– 1295. Vyskocil, F., Kriz, N., Bures, J., 1972. Potassium-selective microelectrodes used for measuring the extracellular brain potassium during spreading depression and anoxic depolarization in rats. Brain Res. 39, 255–259. Waagepetersen, H.S., Westergaard, N., Schousboe, A., 2000. The effects of isofagomine, a potent glycogen phosphorylase inhibitor, on glycogen metabolism in cultured mouse cortical astrocytes. Neurochem. Int. 36, 435 –440. Walz, W., Hertz, L., 1984. Intense furosemide-sensitive potassium accumulation into astrocytes in the presence of pathologically high extracellular potassium levels. J. Cereb. Blood Flow Metab. 4, 301–304. Waniewski, R.A., Martin, D.L., 1998. Preferential utilization of acetate by astrocytes is attributable to transport. J. Neurosci. 18, 5225– 5233. Wender, R., Brown, A.M., Fern, R., Swanson, R.A., Farrell, K., Ransom, B.R., 2000. Astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter. J. Neurosci. 20, 6804–6810. Wolff, J.R., Stuke, K., Missler, M., Tytko, H., Schwarz, P., Rohlmann, A., Chao, T.I., 1998. Autocellular coupling by gap junctions in cultured astrocytes: a new view on cellular autoregulation during process formation. Glia 24, 121–140. Woods, M.J., Williams, D.C., 1996. Multiple forms and locations for the peripheral-type benzodiazepine receptor. Biochem. Pharmacol. 52, 1805–1814. Yarowsky, P., Boyne, A.F., Wierwille, R., Brookes, N., 1986. Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry. J. Neurosci. 6, 859–866. Yu, A.C.H., Schousboe, A., Hertz, L., 1982. Metabolic fate of (14C)-labelled glutamate in astrocytes. J. Neurochem. 39, 954–966. Zhao, Z., Hertz, L., Code, W.E., 1996. Effects of benzodiazepines on potassium induced increase in free cytosolic calcium concentration in astrocytes and neurons in primary cultures. Can. J. Physiol. Pharmacol. 74, 273 –277.

Role of astrocytes in homeostasis of glutamate and GABA during physiological and pathophysiological conditions Arne Schousboep and Helle S. Waagepetersen Department of Pharmacology, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark. p Correspondence address: Tel.: þ 45-3530-6330; fax: þ 45-3530-6021. E-mail: [email protected](A.S.)

Contents 1. 2.

3.

4.

Introduction Glutamate 2.1. Release 2.2. Uptake 2.3. Metabolism GABA 3.1. Release 3.2. Uptake 3.3. Metabolism Concluding remarks

Glutamatergic and GABAergic neurotransmission is terminated by high affinity uptake of the released neurotransmitters into the neuronal or astroglial entities of the synapses. In case of glutamate astroglial transport prevails over neuronal transport, whereas for GABA the opposite is the case. Regardless of this, the astroglial contribution to removal of the transmitter is of crucial importance for function. Malfunction, which may occur, e.g., as a result of energy failure, therefore has serious consequences, such as neuronal degeneration. In this context, the metabolism of the transmitters is also of interest, since astroglial cells control the availability of precursors for biosynthesis of glutamate and GABA in glutamatergic and GABAergic neurons, respectively. The energy balance plays an important role in these processes as well.

Advances in Molecular and Cell Biology, Vol. 31, pages 461–474 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction Generally the homeostatic mechanisms operating in relation to neurotransmission consist of enzymatic processes regulating biosynthesis and biodegradation of the neurotransmitters as well as plasma membrane transporters, which conduct uptake of the transmitters into nerve endings. In the case of excitatory and inhibitory neurotransmission, mediated by the amino acids glutamate and g-aminobutyrate (GABA), respectively, the homeostasis is more complicated, since astrocytes play a prominent role in glutamatergic and GABAergic neurotransmission with regard to both membrane transporters and the metabolic machinery involved (Hertz et al., 1999; Waagepetersen et al., 1999b; Schousboe, 2003). It is thought provoking that the role of astrocytes is particularly prominent in the neurotransmission processes utilizing glutamate and GABA, since neurotransmission mediated by these two transmitters account for at least 90% of all synaptic neurotransmission in the central nervous system. In this context it may be of interest to note important quantitative and functional differences between glutamatergic and GABAergic neurotransmission. From studies of rates of release of neurotransmitter glutamate and GABA it may be concluded that the amount of released glutamate exceeds that of GABA by at least a factor of 10 (Gram et al., 1988; Palaiologos et al., 1988; Waagepetersen et al., 2001b). In keeping with this, the capacity of glutamate transporters in the central nervous system or cultured neural cells is far higher than that of GABA transporters (Balcar and Johnston, 1973; Schousboe et al., 1977a,b; Larsson et al., 1981; Drejer et al., 1982). Another important difference with regard to the transporters is their distribution between neurons and astrocytes, where astrocytic glutamate transporters by far outnumber neuronal glutamate transporters (Hertz, 1979; Schousboe, 1981; Hertz et al., 1999; Danbolt, 2001), whereas in the case of GABA neuronal transporters dominate (Schousboe and Kanner, 2002; Schousboe, 2003). These aspects of glutamate and GABA homeostasis are schematically presented in Fig. 1 (adapted from Hertz and Schousboe, 1987), which shows preferential uptake of released glutamate into glia and preferential uptake of released GABA into neurons. A mainly neuronal re-uptake of GABA, but not of glutamate, results in the ability of GABA synapses to operate primarily by recycling of the neurotransmitter, whereas glutamatergic neurotransmission is dependent upon continuous de novo synthesis of neurotransmitter from either astrocytically accumulated glutamate or from glucose. Due to the fact that key enzymes in these processes (glutamine synthetase and pyruvate carboxylase) are expressed only in astrocytes (see Hertz et al., 1999), glutamatergic neurons are totally dependent on metabolic interactions with surrounding astrocytes for supply of precursors for synthesis of transmitter glutamate (Westergaard et al., 1995; Hertz et al., 1999). Interruption of the glial components of these interactions almost immediately abolishes glutamatergic neurotransmission (Keyser and Pellmar, 1994). The present review is aimed at a discussion of these aspects of glutamate and GABA homeostasis during normal function of the CNS and during conditions of energy failure induced by hypoxia, hypoglycemia or ischemia. An additional discussion focusing on the beneficial or harmful consequences of interactions between neurons and astrocytes during focal ischemia is provided in the chapter by Ha˚berg and Sonnewald.

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Fig. 1. Illustration of evoked release and uptake of glutamate and GABA in GABAergic or glutamatergic neurons and astrocytes. The sizes of the arrows provide an estimate of the relative magnitudes of the respective fluxes. It can be seen that neuronally released glutamate to a major extent is accumulated into astrocytes, whereas most of the released GABA is reaccumulated into neurons.

2. Glutamate 2.1. Release Depolarization of glutamatergic neurons, typically by activation of different types of glutamate receptors, leads to influx of Ca2þ and subsequent release of vesicularly stored glutamate (McMahon and Nicholls, 1991). Depending upon the depolarizing signal the release may under certain conditions consist not only of vesicular glutamate but also to a considerable extent of glutamate released by reversal of the glutamate transporters, i.e., originating from the cytoplasmic, ‘metabolic’ pool of glutamate (Belhage et al., 1992; Bernath, 1992; Jensen et al., 2000; Waagepetersen et al., 2001a; Bak et al., 2003). Vesicular release is energy dependent and is therefore likely to be decreased during energy failure (Nicholls and Attwell, 1990). On the contrary, energy failure will lead to a massive release of glutamate via a reversal of the glutamate carriers, as indeed observed during ischemia and hypoglycemia (Benveniste et al., 1984; Hagberg et al., 1985; Sandberg et al., 1986; Phillis et al., 2000). This is because the plasma membrane glutamate transporters are dependent on energy and co-transport of Naþ along an intact Naþ gradient for optimal function (Danbolt, 2001) and during energy deprivation serve to release glutamate along its concentration gradient. It should be noted that although the glutamate content is larger in the neuronal compartment than in the glial compartment (Ottersen et al., 1992), a considerable fraction of the glutamate released by reversal of the transporters during energy failure could be of astrocytic origin, because the vast majority of glutamate transporters are localized on astrocytes (Lehre et al., 1995; Lehre and Danbolt, 1998; Levy, 2002). Direct evidence supporting the concept that the metabolic glutamate pool may be quantitatively more important than the neurotransmitter pool in ischemia-induced overflow of glutamate in hippocampus is provided by the demonstration that phenylsuccinate, which selectively inhibits biosynthesis of transmitter glutamate

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(Palaiologos et al., 1988), blocks Kþ-stimulated overflow of glutamate in the brain in vivo but not the overflow occurring during a 20 min period of global cerebral ischemia (Christensen et al., 1991). It should also be noted that release of glutamate evoked by combined deprivation of glucose and oxygen both in vivo and in vitro can be reduced by threo-b-benzyloxyaspartate (TBOA), a non-transportable inhibitor of the glutamate transporters (Phillis et al., 2000; Anderson et al., 2001; Waagepetersen et al., 2001a; Bonde et al., 2003). 2.2. Uptake As mentioned above, glutamate uptake is an energy dependent process, which is mediated by different plasma membrane carriers, five of which have been cloned and named EAAT1– 5 (Gegelashvili and Schousboe, 1998; Danbolt, 2001). Of these, EAAT1 (GLAST) and EAAT2 (GLT), which have a preferential if not exclusive glial expression (Danbolt, 2001; Levy, 2002), are by far the most important for maintenance of low extracellular glutamate levels, and they are able to create an extracellular/intracellular glutamate gradient of 1/105. Since the cycling time of the glutamate transporters is relatively long (Wadiche et al., 1995), binding to the extremely abundant glutamate transporters in the glial plasma membrane is likely to be of fundamental importance for the immediate clearance of glutamate in the synaptic cleft following synaptic release of vesicular glutamate (Lehre and Danbolt, 1998). However, although the binding of glutamate to the carrier is not in itself energy dependent, the capacity for binding is exhausted in case the transport cycle is interrupted by energy failure and collapse of the transmembrane Naþ gradient (Wadiche et al., 1995). This obviously will have serious consequences for neuronal function due to the excitotoxic action of glutamate (Choi and Rothman, 1990; Schousboe and Frandsen, 1995). 2.3. Metabolism After receptor interaction, astrocytically accumulated neurotransmitter glutamate can be oxidatively degraded in the tricarboxylic acid (TCA) cycle (Fig. 2), amidated to glutamine, or converted into other metabolites, e.g., glutathione. Glutamate dehydrogenase (GDH) and several different aminotransferases, such as aspartate- and alanineaminotransferases, are capable of catalyzing the reversible conversion of glutamate to its corresponding a-ketoacid, thereby initiating the oxidative degradation of the carbon skeleton (Fig. 2). In astrocytes the entrance of exogenous glutamate into the TCA cycle occurs most likely via the action of GDH (Yu et al., 1982; Schousboe et al., 1993; Westergaard et al., 1996). GDH is a strictly mitochondrial enzyme, located in the inner mitochondrial membrane, whereas the aminotransferases exist in cytosolic as well as mitochondrial isoforms. Both aspartate and lactate are formed during glutamate metabolism via the TCA cycle in astrocytes (Sonnewald et al., 1993b), the latter after exit of glutamate-derived malate from the TCA cycle (Sonnewald et al., 1996; Waagepetersen et al., 2002). This is a quantitatively important pathway (Hertz and Hertz, 2003), necessitating de novo synthesis of glutamate from glucose (see below).

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Fig. 2. Schematic illustration of the TCA cycle and closely connected reactions, i.e., those catalyzed by, pyruvate dehydrogenase (1), pyruvate carboxylase (2), glutamate dehydrogenase (3), aspartate aminotransferase (4) and malic enzyme (5). The pathways accounting for the net synthesis of aspartate from glutamate are indicated by bold arrows.

There is a large increase in the synthesis of aspartate from glutamate during hypoglycemia (Bakken et al., 1998), when glutamate becomes an alternative energy substrate. For every molecule of glutamate which is converted to aspartate, 9– 12 molecules of ATP are produced in a truncated TCA cycle (3 during GDH-mediated conversion of glutamate to a-ketoglutarate [but none during transamination], 3 during conversion of a-ketoglutarate to succinyl coenzyme A, 1 during conversion of succinyl coenzyme A to succinate, 2 during conversion of succinate to fumarate and 3 during conversion of malate to oxaloacetate as outlined in Fig. 2). In contrast, lactate production from glutamate via the TCA cycle decreases during hypoglycemia as well as hypoxia (Bakken et al., 1998). The balance between the extent of oxidative consumption of glutamate and synthesis of glutamine is dependent on the extracellular concentration of glutamate, with relatively more glutamate being oxidized at higher glutamate concentrations (McKenna et al., 1996a). Glutamine is synthesized exclusively in astrocytes (and other glial cells) by an ATP dependent amidation of glutamate, catalyzed by the cytosolic enzyme glutamine synthetase (Norenberg and Martinez-Hernandez, 1979; D’Amelio et al., 1990; Tansey et al., 1991). In spite of the energy dependence of the glutamine synthetase reaction it is not sensitive to hypoxia or hypoglycemia in cultured astrocytes (Bakken et al., 1998). Glutamine, a non-neuroactive amino acid, which is the predominant glutamate precursor, is released from astrocytes and taken up in neurons, where it is rapidly and extensively metabolized to glutamate (Bradford et al., 1978; Rothstein and Tabakoff, 1984; Szerb and

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O’Reagan, 1985; Hogstad et al., 1988; Shank et al., 1989). Thus, the net flow of glutamate from neurons to astrocytes is counterbalanced by a glutamine flow from astrocytes to neurons, a phenomenon, which is referred to as the glutamate-glutamine cycle, shown in Fig. 3 (Berl and Clarke, 1983). Recently, transporters for glutamine out of astrocytes and into neurons have attracted considerable interest (Boulland et al., 2002). A neuronal high affinity glutamine transporter has been cloned and shown to be preferentially located on glutamatergic neurons (Varoqui et al., 2000), confirming the importance of glutamine as a glutamate precursor. The supply of a glutamate precursor to the neurons is necessary due to the fact that neurons lack a quantitatively important anaplerotic pathway, since they do not express the enzyme pyruvate carboxylase (Yu et al., 1983; Shank et al., 1985; Kaufman and Driscoll, 1992; Cesar and Hamprecht, 1995), an enzyme which is crucial for net synthesis of TCA cycle intermediates and their derivatives, including glutamate and glutamine. However, on account of the considerable oxidative metabolism of glutamate in astrocytes the transport of glutamine from astrocytes to neurons is not stochiometrically equivalent to the uptake of neuronally released glutamate, and in addition some glutamine is oxidized in neurons (Hertz and Schousboe, 1986; Yudkoff et al., 1988; Hertz et al., 1992a; Westergaard et al., 1995). In order to compensate for the ensuing deficit in neuronal glutamate precursor, additional glutamate must be generated from other sources, probably mainly or exclusively glucose. The de novo synthesis of glutamate from glucose requires pyruvate carboxylation. There is a relatively high pyruvate carboxylase activity in astrocytes (Yu et al., 1983; Kaufman and Driscoll, 1992; Gamberino et al., 1997), and a high rate of pyruvate carboxylation has been established in the brain in vivo, including human brain (see chapter by Gruetter). Since net synthesis of glutamate occurs in astrocytes but not in neurons, either a TCA cycle intermediate, glutamate itself or glutamine must be transported from astrocytes to neurons. Glutamate itself is not suitable on account of its transmitter activity, but it is likely that at least some glutamate is converted in astrocytes to glutamine, which is then carried across to neurons in the glutamate-glutamine cycle.

Fig. 3. The GABA-glutamate/glutamine cycle illustrating the flux of the neurotransmitters glutamate and GABA from the neuronal to the astrocytic compartment and the corresponding glutamine flux in the opposite direction. GLN, glutamine; GLU, glutamate; GS, glutamine synthetase; PAG, phosphate activated glutaminase.

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The TCA cycle intermediate, a-ketoglutarate has also been shown to serve as precursor for the releasable neurotransmitter pool of glutamate, although not to the same extent as glutamine (Kihara and Kubo, 1989; Shank et al., 1989; Peng et al., 1991). a-Ketoglutarate as well as other TCA cycle intermediates, such as citrate, malate and succinate are released to a larger extent from astrocytes than from neurons. Citrate is the intermediate released at by far the highest rate and the extent of release is sensitive to hypoxia (Sonnewald et al., 1991; Mu¨ller et al., 1994; Westergaard et al., 1994a,b). This may be explained by the importance of the energy requiring process, pyruvate carboxylation for the synthesis of releasable citrate (Westergaard et al., 1994b; Waagepetersen et al., 2001c). Evidence for a role of citrate as a neuronal glutamate precursor could, however, not be obtained (Westergaard et al., 1994b). The possible role of malate as a glutamate precursor seems limited, considering a very modest uptake rate of malate into neurons (Shank and Campbell, 1984; Hertz et al., 1992b) compared to the rate of stimulated glutamate release (Drejer et al., 1982). Furthermore, the incorporation of 14C from [14C]malate into glutamate appeared inadequate for malate to be a significant precursor for glutamate (Shank and Campbell, 1984; Hertz et al., 1992b). Astrocytic TCA cycle and amino acid metabolism is complicated by the existence of mitochondrial heterogeneity presumably within each single cell (Schousboe et al., 1993; Sonnewald et al., 1993a, 1998; McKenna et al., 1996b; Waagepetersen et al., 1999a). This is particularly evident for the metabolic pathways leading to synthesis of citrate and glutamine (Schousboe et al., 1993; Waagepetersen et al., 2001c). Pyruvate carboxylation seems extremely important for synthesis of a releasable pool of citrate. The sequence of reactions leading to synthesis of this pool of citrate is separated from that operating for the synthesis of glutamine (Waagepetersen et al., 2001c). These two energy requiring processes, i.e., pyruvate carboxylation and glutamine synthesis, might a priori both be susceptible to hypoxia but a differential effect of hypoxia on release of citrate and glutamine might be explained by the above mentioned compartmentation (Sonnewald et al., 1994; Waagepetersen et al., 2001c). Moreover, synthesis of releasable glutamine, i.e., the neurotransmitter precursor pool, is compartmentalized from synthesis of a main intracellular pool of glutamine (Waagepetersen et al., 2001c). This compartmentation of the biochemical machinery might serve an important role decreasing the vulnerability of astrocytes during energy failure.

3. GABA 3.1. Release In analogy with other neurotransmitters, the inhibitory neurotransmitter, GABA, is released by exocytosis from GABAergic nerve endings following depolarization (Sihra and Nicholls, 1987). However, depolarization may additionally lead to release from the cytoplasmic pool via reversal of the plasma membrane carriers (Bernath, 1992; Belhage et al., 1993). This means that the release process, like that for glutamate (see above), is sensitive to energy failure, which favors release from the cytoplasmic pool. In keeping with this, ischemia has been shown to result in an increase in the extracellular

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concentration of GABA, measured by microdialysis (Hagberg et al., 1985—see also chapter by Ha˚berg and Sonnewald). 3.2. Uptake The maintenance of a low extracellular GABA concentration is brought about by plasma membrane GABA carriers, four of which have been cloned and named GAT1– 4 (Schousboe and Kanner, 2002). It should be noted that the nomenclature for the GABA transporters is confusing, since the numbering differs between transporters cloned from mice and rats (Schousboe and Kanner, 2002). These transporters are mainly expressed in GABAergic neurons, and GAT1 is the most abundant transporter. However, glial cells also express GAT1 in addition to GAT2 and GAT4 (Schousboe and Kanner, 2002). That the GABA transporters are important for the maintenance of low extracellular GABA concentrations is demonstrated by repeated findings that application of inhibitors of GABA transporters, using the microdialysis technique, leads to an increase in the extracellular concentration of GABA (Fink-Jensen et al., 1992; Richards and Bowery, 1996; Juha´sz et al., 1997). Since the GABA carriers are Naþ dependent and electrogenic, the uptake process is also highly energy dependent (Schousboe, 1981). The ability of inhibitors of GABA transport to increase extracellular GABA levels is particularly pronounced for those inhibitors which act primarily on glial GABA uptake (Juha´sz et al., 1997; White et al., 2002), probably reflecting that GABA which has been taken up into presynaptic nerve endings is primarily re-used as a transmitter, whereas GABA accumulated by astrocytes is either oxidatively degraded or returned to neurons in a rather complex GABA-glutamine-glutamate shuttle (see below). It has therefore been suggested (Schousboe et al., 1983) that inhibitors of glial GABA uptake might be particularly attractive as anticonvulsant agents. It should, however, be noted that other factors may also play important roles in this context, as the antiepileptic drug Tiagabine, which exclusively inhibits GAT1, is only slightly more potent as an inhibitor of glial GABA uptake compared to inhibition of neuronal GABA uptake (White et al., 2002). It may also be of interest to note that seizure sensitive gerbils have a higher expression of GAT1 than their seizure resistant counterparts, which has been interpreted as a compensatory mechanism to provide a higher release of GABA by reversal of the carrier during depolarization (Kang et al., 2001). However, it would be in keeping with the anticonvulsant activity of astrocytic GABA uptake inhibitors, if the excessive expression of GAT1 in these animals mainly were astrocytic (Schousboe et al., 1983; White et al., 2002). 3.3. Metabolism Although astrocytes lack the enzyme responsible for synthesis of GABA from glutamate they do play an important role in GABA metabolism (Schousboe, 1980; Waagepetersen et al., 1999b). Thus, glutamine also functions as a precursor for GABA via glutamate (Reubi et al., 1978; Battaglioli and Martin, 1990, 1991; Sonnewald et al., 1993c), whereas there is no evidence that the TCA cycle intermediates, which can support

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synthesis of transmitter glutamate (see above), also can serve as precursors for GABA synthesis (Hertz et al., 1992b). This suggests that the only route for de novo formation of GABA is via formation of glutamine in astrocytes, followed by its transfer to GABAergic neurons, hydrolysis to glutamate, and decarboxylation to GABA by glutamate decarboxylase, GAD (Waagepetersen et al., 1999b). This series of metabolic processes occurs in a glutamate-glutamine cycle, which has been expanded to a GABA-glutamateglutamine cycle (Van den Berg and Garfinkel, 1971; Berl and Clarke, 1983). This cycle is illustrated in Fig. 3. It involves several steps requiring a high energy level, such as the glutamine synthetase reaction in the astrocytic compartment, and a need for entry of at least part of the newly synthesized glutamate into an operational TCA cycle in the neuronal compartment (Waagepetersen et al., 1999b, 2001b). This means that although GAD is capable of operating under hypoxic conditions, maintenance of a functional GABA pool in GABAergic neurons is hampered by oxygen deprivation. The finding that TCA cycle intermediates are not precursors for GABA is in agreement with a lower quantitative demand for precursors in GABAergic neurons, since the net flow of glutamate into astrocytes from neurons is much higher than that of GABA (Hertz and Schousboe, 1987) as illustrated in Fig. 1. Oxidative degradation of GABA in astrocytes, facilitated by a high activity of GABAtransaminase (Schousboe et al., 1977a), is also dependent on oxidative metabolism, since the subsequent catabolism requires oxidation of generated succinic acid semialdehyde by succinic acid semialdehyde dehydrogenase to succinate. As shown in Fig. 2, succinate can be further metabolized in the TCA cycle to malate, which can exit the cycle to form pyruvate, a process which preferentially takes place in astrocytes (Sonnewald et al., 1996; Waagepetersen et al., 1999b, 2002; Lieth et al., 2001). The finding that extracellular GABA levels increase during energy failure (Hagberg et al., 1985) is therefore likely to reflect not only the failure of the GABA carriers to maintain a steep extra/intracellular concentration gradient (see above) but also a decreased glial as well as neuronal catabolic capacity. Alternatively malate may remain in the TCA cycle and eventually be converted to a-ketoglutarate (Fig. 2), followed by formation of glutamate and glutamine in astrocytes and transfer of glutamine to neurons, where it can be hydrolyzed to glutamate, which is then decarboxylated by GAD to form GABA. Thus, a possible return of astrocytically accumulated GABA to neurons follows a much more complex pathway than return of glutamate in the glutamate-glutamine cycle, and it is directly dependent upon oxidative metabolism.

4. Concluding remarks The metabolic relationships between glutamine, glutamate and GABA have always been a cornerstone in studies of metabolic interactions between different neural cells. The very concept of metabolic compartmentation in brain was developed from the discovery of an anomalous precursor product relationship between glutamate and glutamine in whole brain, and the operation of a GABA-glutamate-glutamine cycle was deduced from the formation of GABA in one metabolic compartment and its metabolism, at least partly, in a different compartment (Berl and Clarke, 1983). Studies of kinetics for uptake of amino acids

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and of metabolic fluxes in primary cultures of astrocytes and of different types of neurons were instrumental in establishing present-day understanding of these interactions, as outlined in the present chapter. During recent years, modern techniques, such a nuclear magnetic resonance spectroscopy, have enabled quantitative determination of metabolic fluxes in the intact brain during both physiological and pathophysiological conditions (see chapters by Ha˚berg and Sonnewald and by Gruetter), and they have been instrumental in the development of the concept that de novo synthesis, especially of glutamate, is a quantitatively important pathway, even compared with return of astrocytically accumulated glutamate and GABA via the glutamate-glutamine and the GABA-glutamate-glutamine cycle (a concept that had been proposed earlier, based on the high rate of oxidative degradation of glutamate in cultured astrocytes). What we still do not know is the dynamics of these interactions during increased brain activity, e.g., whether brain stimulation might be associated with enhanced de novo synthesis of glutamate from glucose, and cessation of brain activity with oxidative degradation of glutamate and glutamine. Rapid technical developments of in vivo methods for determination of metabolic fluxes in brain, may provide answers to this question within the coming years. Acknowledgements The expert secretarial assistance by Ms Hanne Danø is highly appreciated. The work has received financial support from the Danish MRC (grants 22-00-1011 and 1747), as well as the Lundbeck and NOVO Nordisk Foundations.

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Astrocytic receptors and second messenger systems Elisabeth Hanssonp and Lars Ro¨nnba¨ck Institute of Clinical Neuroscience, Go¨teborg University, Medicinaregatan 5, SE 405 30 Go¨teborg, Sweden p Correspondence address: Tel.: þ 46-31-773-3363; fax: þ 46-31-773-3330. E-mail: [email protected](E.H.)

Contents 1. 2.

3.

4.

5.

6.

Introduction Astrocytic G-protein- and/or ion channel-coupled membrane receptors 2.1. Glutamate receptors 2.2. Gamma-aminobutyric acid receptors 2.3. Glycine receptors 2.4. Noradrenaline receptors 2.5. Dopamine receptors 2.6. Serotonin receptors 2.7. Histamine receptors 2.8. Acetylcholine receptors 2.9. Purine receptors 2.10. Endothelin receptors 2.11. Opioid receptors 2.12. Peptide receptors 2.13. Protease receptors 2.14. Prostanoid receptors 2.15. Chemokine receptors Astrocytic cytokine and growth factor receptors 3.1. Cytokine receptors 3.2. Growth factor receptors Astrocytic intracellular receptors 4.1. Nuclear receptors 4.2. Mitochondrial receptors Functional consequences of astrocytic receptor activation 5.1. Physiological activity 5.2. Neurotrauma 5.3. Development Concluding remarks

Advances in Molecular and Cell Biology, Vol. 31, pages 475–501 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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Abbreviations A1, 2B, 3,: adenosine analog1, 2B, 3; ADNF: activity-dependent neurotrophic factor; ADP: adenosine phosphate; AMPA: a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid; ATP: adenosine triphosphate; B1-2: bradykinin1-2: bFGF: basic fibroblast growth factor; Ca2+: calcium; [Ca2+]i: intracellular calcium; cAMP: cyclic adenosine monophosphate; CCR1: chemokine receptor 1; CCR3: chemokine receptor 3; CCR5: chemokine receptor 5; CNTF: ciliary neurotrophic factor; CT-1: cardiotrophin-1; CXCR2, chemokine receptor 2; CXCR4: chemokine receptor 4; D1-5: dopamine subtypes1-5: DAG: diacylglycerol; EGF: epidermal growth factor; EP3: prostaglandin receptor EP3 subtype; ET: endothelin; FP: prostaglandin receptor FP; Gi: inhibitory G protein; Go: o for other G protein; Gq: subtype for a G protein; Gs: stimulating G protein; GABA: gamma-aminobutyric acid; GFAP: glial fibrillary acidic protein; GluR: glutamate receptor; GM-CSF: granulocyte/macrophage colony-stimulating factor; GR: glucocorticoid receptor; H: histamine; 5-HT: 5-hydroxytryptamine; iGluR: ionotropic glutamate receptor; IL: interleukin; IL-1b: interleukin-1beta; INF: interferon; IP: inositolphosphate; IP3: inositoltrisphosphate; KA: kainate; LIF: leukemia inhibitory factor; M1-2: muscarinic receptor1-2; MAPK: mitogenactivated protein kinase; M-CSF: macrophage colony-stimulating factor; mGluR: metabotropic glutamate receptor; MR: mineralcorticoid receptor; mRNA: messenger ribonucleic acid; NK1-3: neurokinin1-3; NMDA: N-methyl-D-aspartic acid; NMDA R1: N-methyl-D-aspartic acid receptor 1; NMDA R2: N-methyl-D-aspartic acid receptor 2; NPY: neuropeptide Y; NT1-3: neurotrophin1-3; OSM: oncostatin M; P1: purine receptor 1; P2: purine receptor 2; P2Y1-2, subclasses P2Y1-2 of purine receptor 2; P2X7: subclass P2X7 of purine receptor 2; PAR: protease-activated receptor; PAR-1: protease-activated receptor 1; PAR-2: protease-activated receptor 2; PAR-3: protease-activated receptor 3; PAR-4: protease-activated receptor 4; PCR: polymerase chain reaction; PGD2: phosphogluconate dehydrogenase2; PGDF: platelet-derived growth factor; PGE2: prostaglandin E2; PGF2a, prostaglandin F2alpha; PIP2: phosphatidyl inositol 4,5-biphosphate; PKA: protein kinase A; PKC: protein kinase C; PLA2: phospholipase A2; PLC: phospholipase C; PLD: phospholipase D; PTBR: peripheral-type benzodiazepine receptor; RNA: ribonucleic acid; Subst P: substance P; TGF-a: transforming growth factor-alpha; TGF-b: transforming growth factor-beta; TNF-a: tumor necrosis factor-alpha; TP: thromboxane receptor TP; TPA: tissue plasminogen activator; TXA2, thromboxane aprotinin A2; TXB: thromboxane; VIP: vasoactive intestinal peptide. Astrocytes, the most numerous glial cells in the CNS, express a large number of receptors for neurotransmitters, peptides, purines, cytokines and other neuroactive substances, many of them previously thought to be present only on neurons. These astrocytic receptors are coupled to G proteins or ion channels, to intracellular protein kinases, or associated with mitochondria or cell nuclei. Even if most evidence for the existence of this extensive repertoire of functional astrocytic receptors comes from in vitro studies, convincing studies have demonstrated the presence of astrocytic receptor groups also in the intact nervous system. The astrocytic receptors and the signaling systems with which they are associated, set the stage for extensive neuronal – glial signaling, which in turn is essential for neuroplasticity, both in the intact nervous system and after trauma and in degenerative

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disease. Pharmacological manipulation of astrocytic receptor systems might provide a new strategy to reinforce the reparative processes within the CNS after injury or in disease.

1. Introduction Glial cells support neuronal activity and create opportunities for the dynamics and plasticity of the central nervous system (CNS). After injury, metabolic disturbance, or toxic influence, and in degenerative disease, glial cell reactions protect neurons and facilitate rebuilding. When driven out of physiological control, however, glial reactions can be destructive and cytotoxic. Glial functions of physiological importance include astroglial glutamate uptake, transport of low molecular weight substances in the astroglial gap junction-coupled network, and astroglial volume regulation, with secondary effects on the size and shape of the extracellular space, and consequently, on volume transmission. Extensive inter- and intracellular signaling regulates and integrates these and other processes, and astrocytic receptors, both membrane-bound and intracellular, are of utmost importance. Calcium (Ca2þ)-mediated signaling is one of the mechanisms by which CNS cells communicate with, and modulate the activity of, adjacent cells after stimulation by specific receptors. Astrocytic Ca2þ signals can propagate within the astroglial network as well as to neighboring cells in the CNS. Information on signals, in the form of ions, molecules, proteins, and peptides, which mediate receptor-triggered responses, is crucial for our understanding of how different cell types communicate with each other. After receptor stimulation, transmitters induce transient elevations of internal Ca2þ levels and other second messengers and change ion fluxes in astrocytes. Up to now, most studies of glial cell reactions and signaling involving the receptors in one way or another have been performed and evaluated in primary cultures. However, some studies have also been performed in more ‘in vivo-like systems’, such as brain slices. Due to the complex morphology of the CNS, with tightly interwoven cellular networks and a large array of substances active in cell signaling, the use of cell cultures enriched with defined cell types have made it easier to evaluate the role of individual substances at the single cell level. Most astrocytic receptors, e.g., glutamate receptors and receptors for monoamines, such as noradrenaline, dopamine, serotonin and histamine, are coupled to heterotrimeric G proteins or ion channels, with some receptor subtypes for a given transmitter being coupled to G proteins and other to ion channels. However, other membrane receptors are coupled to intracellular protein kinases, and a third group of astrocytic receptors are nuclear, associated with either mitochondria or cell nuclei.

2. Astrocytic G-protein- and/or ion channel-coupled membrane receptors 2.1. Glutamate receptors Glutamate is the major excitatory neurotransmitter in the CNS and glutamate receptors are expressed in many different locations throughout the CNS (Hollmann and Heinemann, 1994; Nakanishi, 1994). They play an important role in neuronal plasticity, neuronal

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development, and neurodegeneration. Glutamate receptors can be divided into metabotropic and ionotropic receptors. 2.1.1. Metabotropic glutamate receptors Eight different metabotropic glutamate receptors (mGluRs) have been identified and cloned, mGluR1 –mGluR8. The mGluR1 and the mGluR5 are coupled via G proteins with phospholipase C (PLC). They are activators of the inositoltrisphosphate (IP3)-mediated intracellular signaling pathway and release calcium from intracellular calcium ([Ca2þ]i) stores (Pin and Duvoisin, 1995—see also chapter by Shuai et al.). Glutamate evokes a nonoscillatory [Ca2þ]i response in single-cell [Ca2þ]i recordings in mGluR1-expressing cells and an oscillatory [Ca2þ]i response in mGluR5-expressing cells (Nakanishi et al., 1998). The difference results from a single amino acid substitution, aspartate in mGluR1 and threonine in mGluR5, in the G protein-interacting carboxy-terminal domains. Protein kinase C (PKC) phosphorylation of the threonine of mGluR5 is responsible for inducing [Ca2þ]i oscillations in mGluR5-expressing cells and cultured glial cells, because phosphorylation of threonine by PKC abolishes the [Ca2þ]i increase by interfering with the signal transduction between the receptor and the intracellular effector, IP3, and the subsequent dephosphorylation regenerates the [Ca2þ]i increase by restoring the signal transduction. In contrast, aspartate of mGluR1 is not phosphorylated by PKC, resulting in a non-oscillatory, single-peak [Ca2þ]i increase. However, in general, astrocytes fail to express mGluR1, whereas immunoreactivity for mGluR5 as well as for the mGluR5 transcript and protein has been detected on astrocytes from the hippocampus, hypothalamus, and cerebral cortex (Romano et al., 1995; Van den Pol et al., 1995; Nakahara et al., 1997; Muyderman et al., 2001a), as well as from glial fibrillary acidic protein (GFAP)-positive astrocytes in the cerebral cortex, both in brain slices (Muyderman et al., 2001a) and in vivo (Steinha¨user and Gallo, 1996). The mGluR5 expression is upregulated by specific growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor-a (TGF-a) (Miller et al., 1995). The other six subtypes, mGluR2 – 4 and mGluR6 –8, are all coupled to the inhibition of adenylate cyclase. Using in situ hybridization techniques, mGluR3 has been detected on astrocytes from different brain regions (Ohishi et al., 1993; Fotuhi et al., 1994). It has been speculated that glutamate released at the synaptic clefts may either stimulate or reduce the proliferation rate of surrounding astrocytes through activation of distinct mGluR subtypes. Metabotropic GluR5 enhances proliferation and mGluR3 reduces proliferation in cultured astrocytes (Ciccarelli et al., 1997). Metabotropic GluR3 has been found to be co-localized with the water channel Aquaporin 4 and upon activation by glutamate, can act as an osmoregulation sensor (Shigemoto et al., 1999). Messenger ribonucleic acid (mRNA) for mGluR2, mGluR4 and mGluR6 – 8 has not been detected on astrocytes (Tanabe et al., 1993; Testa et al., 1994). 2.1.2. Ionotropic glutamate receptors Ionotropic glutamate receptors (iGluRs) include three main groups: N-methyl-D aspartic acid (NMDA), consisting of NMDA R1 and NMDA R2A – D subunits, and

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non-NMDA ionotropic receptors, which can be subdivided into a-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) receptors. They are all ligand-gated ion channels through which Naþ, Kþ, and Ca2þ can pass. The iGluR subunits GluR1 – 4 form AMPA-sensitive receptors, and GluR5 –7 form KA-sensitive receptors. Stimulation of the AMPA/KA receptors leads to depolarization and an influx of Ca2þ across the plasma membrane (Jabs et al., 1994; Porter and McCarthy, 1995). Four AMPA receptor subunits (GluR1 – 4) and five KA receptor subunits (GluR5 –7 and KA1 – 2) have been cloned. Expression of GluR4 has been detected on astrocytes from different brain regions. For the other iGluRs, it has been hypothesized that subpopulations of astrocytes express some of the receptors, preferentially GluR1 and GluR3 (for a review, see Porter and McCarthy, 1997). In many studies on astrocytes in culture, no expression of NMDA receptors was recorded. However, other data suggest that astrocytes in slices express NMDA receptors (for a discussion and references, see Porter and McCarthy, 1997). Moreover, the radial glial cells, namely, the Bergmann glia in the cerebellum and the Mu¨ller cells in the retina, which are not converted into conventional astrocytes after birth, express the NMDA R1 and NMDA R2A or B subunits (Lo´pez et al., 1997).

2.2. Gamma-aminobutyric acid receptors Gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the CNS, can mediate its effects through three different GABA receptors: GABAA, GABAB, and GABAC. Gamma-aminobutyric acidA and GABAC are intrinsic ligand-gated Cl2 channels, whereas the GABAB receptor is coupled via G proteins to its effectors, Kþor Ca2þ-permeable plasmalemmal channels (Johnston, 1994; Nakayasu et al., 1995). In astrocytes, GABA depolarizes the membrane. By contrast, in neurons, it hyperpolarizes the membrane. The difference is due to different intracellular Cl2 levels in the two cell types. Astrocytes have a more positive Cl2 equilibrium potential than do neurons due to a higher intracellular Cl2 concentration, which may be due to the activity of two inwardly directed Cl2 transporters, the Naþ – Kþ – Cl2-cotransporter and the Cl2/HCO2 3 exchanger (Von Blankenfeld and Kettenmann, 1991; Fraser et al., 1994). Depolarization of the astrocytes takes place mainly through the GABAA receptors, which have been found on most types of astrocytes (for a review, see Porter and McCarthy, 1997). Activation of membrane-associated benzodiazepine receptors on cultured astrocytes (expressed in additional to the mitochondrial-type benzodiazepine receptors (see Section III.b)) by conventional benzodiazepines or by endogenous agonists, called endozepines, causes an increase in [Ca2þ]i which mainly, if not exclusively is due to an enhancement of Ca2þ entry through L-channels (in dibutyryl cyclic AMP-treated cultures), possibly triggered by benzodiazepine mediated enhancement of astrocytic GABAA receptors and resulting depolarization (Zhao et al., 1996; Gandolfo et al., 2001—see also chapter by Hertz, Peng et al.). Gamma-aminobutyric acidC receptors on astrocytes have not yet been described (Verkhratsky and Steinha¨user, 2000). Gamma-aminobutyric acidB receptors induce [Ca2þ]i increases, which may be due to Ca2þ entry via voltage-gated channels or to plasmalemmal Ca2þ entry (Nilsson et al., 1993).

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2.3. Glycine receptors Functional glycine receptors have been detected on astrocytes. These receptors mediate a Cl2 conductance (Betz et al., 1994) and show similarities with the GABAA receptor. 2.4. Noradrenaline receptors The monoamines noradrenaline and dopamine share a common biosynthetic pathway that starts with the amino acid tyrosine. Tyrosine is converted to L -dopa by tyrosine hydroxylase and, in noradrenergic neurons, further transformed to noradrenaline by dopamine b-hydroxylase. The noradrenergic fibers originate from the locus coeruleus and spread through the entire cerebral cortex, hippocampus, and cerebellum, where they terminate in varicosities rather than in genuine synapses (Lindvall and Bjo¨rklund, 1984). Astrocytes express adrenergic receptors both in vitro and in vivo (Ho¨sli and Ho¨sli, 1982; Salm and McCarthy, 1989; Shao and Sutin, 1992). The proportion of astrocytes surrounding noradrenergic varicosities is fairly high and, consequently, the noradrenergic system is believed to exert a large part of its effect on astrocytes (Stone and Ariano, 1989). There are several basic subtypes of adrenergic receptors, a1, a2, b1, and b2. They are coupled to different signal transduction systems and have distinct pharmacological profiles, and they have all been localized on astrocytes (for a review, see McCarthy et al., 1995; Verkhratsky et al., 1998). The expression of a1 adrenoceptors has been demonstrated with radiolabeled a1 adrenoceptor agonists on astrocytes (Shao and Sutin, 1992). The a1 receptor activates a pertussis toxin-insensitive protein, Gq, which in turn triggers PLC to generate IP3 and diacylglycerol (DAG) from phosphatidyl inositol 4,5-bisphosphate (PIP2) in the plasma membrane (Berridge and Irvine, 1984). Diacylglycerol elicits a cascade of intracellular events, such as the gating of ion channels, mobilization of Ca2þ, and the activation of intracellular kinases such as protein kinase A (PKA) and PKC, respectively, leading to alterations in the degree of protein phosphorylation (Nilsson et al., 1991). In astrocyte cultures from cerebral cortex the mobilization of Ca2þ leads to an increase in [Ca2þ]i, which frequently is oscillatory (Muyderman et al., 2001a). The a2 receptor is negatively linked via an inhibitory G protein (Gi) to the enzyme adenylate cyclase and thus decreases cyclic adenosine monophosphate (cAMP) production (Hansson, 1988). However, stimulation of a2 receptors on astrocytes is also associated with increases in IP3 (Enkvist et al., 1996) and in [Ca2þ]i (Zhao et al., 1992) in astrocytes in primary cultures, which in turn can lead to stimulation of glucose metabolism (see chapter by Hertz, Peng et al.). Alternatively, and dependent on the subtype of the a2 receptor, stimulation of a2 receptors can lead to opening of Ca2þ channels in the cell membrane, causing a non-oscillating increase in [Ca2þ]i (Muyderman et al., 2001b). The presence of b adrenoceptors on astrocytes has been demonstrated by binding of b adrenoceptor antagonists (Salm and McCarthy, 1989; Shao and Sutin, 1992). The b receptor can be divided into three subgroups, b1, b2 and b3, although the b3 receptor (Evans et al., 1999) has not been localized to astrocytes so far. The b1 and b2 receptors are linked via the stimulating G protein (Gs) to the enzyme adenylate cyclase and increase the cAMP production (Hansson, 1985). Immunoreactivity for b adrenoceptors

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has been found in both gray and white matter, indicating that both protoplasmic and fibrous astrocytes have b receptors, preferentially the b2 subtype (Aoki, 1992; Liu et al., 1992). The b2 adrenoceptors have been detected on astrocytic processes surrounding axons, dendrites, and immunoreactive synapses, which suggests that astrocytes may be able to respond to noradrenaline released from synaptic terminals (Aoki, 1992). On the other hand, neuronally released noradrenaline may diffuse and activate receptors at significant distances from the adrenergic synapses and thus take part in volume transmission (Fuxe and Agnati, 1991). Stimulation of b adrenoceptors by isoproterenol can also elicit [Ca2þ]i elevations in astrocytes, a non-oscillating response that is found in relatively few cells compared with the reaction to receptors coupled to the inositolphosphate (IP) system, and is due to opening of Ca2þ channels in the cell membrane (Muyderman et al., 2001a).

2.5. Dopamine receptors The dopaminergic system in the CNS plays a crucial role in the regulation of physiological actions, such as the control of locomotion, cognition, emotion, and neuroendocrine secretion. These actions are mediated by five distinct G protein-coupled receptor subtypes, which are classified into two main groups (Jaber et al., 1996). The first, D1-like dopamine receptor subtype (D1 and D5) activates adenylate cyclase, enhancing cAMP formation, while D2-like dopamine receptor subtype (D2, D3, and D4) inhibits adenylate cyclase, decreasing cAMP formation, and also activates Kþ channels. Dopamine receptors have been demonstrated on groups of astrocytes in astroglial primary cultures cultured from striatum, cerebral cortex, and the spinal cord (Hansson et al., 1984; Hansson, 1985; Ho¨sli and Ho¨sli, 1986). With in situ hybridization techniques and polymerase chain reaction (PCR), dopamine D2 receptor mRNA was found to be expressed by astrocytes from striatum (Bal et al., 1994). It has, however, also been shown that dopamine is able to elicit transient increases in [Ca2þ]i in cultured or bulk-separated astrocytes, which can be blocked by D1 and D2 receptor-specific antagonists (Reuss et al., 2000; Khan et al., 2001). Our previous results, that there is regional heterogeneity in the expression of D1 receptor mRNA in cultured astrocytes from different brain regions, with a maximum response to dopaminergic agonists in striatal astrocytes, a lower effect in cortical astrocytes, and no effect in cerebellar astrocytes, have since been confirmed by PCR and Southern blot hybridization (Zanassi et al., 1999). At the electromicroscopic level, it was observed that cortical interneurons are surrounded by astrocytic processes, which strongly express D2 receptors and communicate with dopaminergic interneurons in the same region, as indicated by an increase in astrocytic [Ca2þ]i by exposure to dopaminergic agonists (Khan et al., 2001). This finding confirms earlier observations of ‘cross-talk’ between astrocytes and neurons, made in a cocultivation system, where the sensitivity of cultured striatal astrocytes to application of dopamine was enhanced by co-culturing with neurons from substantia nigra, one of the natural projection areas from substantia nigra (Hansson and Ro¨nnba¨ck, 1988).

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2.6. Serotonin receptors Serotonin [5-hydroxytryptamine (5-HT)] is an important neurotransmitter in the CNS and is synthesized from tryptophan. Disturbances in the 5-HT system have been demonstrated in several mental and neurological disorders, such as depression, anxiety, schizophrenia, and migraine. 5-HT-containing neuronal cell bodies are concentrated in the raphe nuclei, from where fibers project to virtually all parts of the CNS. Not all fibers establish synaptic contacts with target neurons, but instead some mediate a volume transmission, a process, which is slower than synaptic signaling (Jacobs and Azmitia, 1992). The 5-HT receptor family consists of seven major classes, based on structural and pharmacological properties, the 5-HT1 – 7 receptors, which are further divided into several subtypes (Hoyer and Martin, 1997). The 5-HT3 receptor is a ligand-operated cationic channel, but the others (i.e., 5-HT1 – 2 and 5-HT4 – 7) are coupled to G proteins. 5-HT1 and 5-HT4 are coupled to adenylate cyclase, whereas 5-HT2 receptors are coupled to PLC, thus regulating IP3 production (Hoyer et al., 1994). In cultured astrocytes, 5-HT receptors were originally detected by Hertz and Schousboe (Hertz et al., 1979). Several different subtypes of 5-HT receptors have been found on astrocytes: the 5-HT1A (Azmitia et al., 1996), the 5HT2A (Nilsson et al., 1991; Deecher et al., 1993; Hagberg et al., 1998), the 5-HT2B (Hirst et al., 1998; Sande´n et al., 2000; Kong et al., 2002), the 5-HT5A (Carson et al., 1996), and the 5-HT6,7 receptors (Hirst et al., 1997). When the 5-HT1A receptor is stimulated, the astrocytes respond by releasing S-100b and attaining a mature morphology, with a shift from a flattened to a process-bearing morphology (Whitaker-Azmitia et al., 1990). Stimulation of the 5-HT2 receptor on astrocytes results in glycogenolysis (Poblete and Azmitia, 1995) and increases the [Ca2þ]i levels in astrocytes (Nilsson et al., 1991—see also chapter by Hertz, Peng et al.). 2.7. Histamine receptors Three different types of histamine receptors have been detected: histamine1 (H1) receptor, which is coupled to PLC and mobilization of [Ca2þ]i, histamine2 (H2) receptor, which increases adenylate cyclase activity, and histamine3 (H3) receptor, which controls histamine turnover and release (Leurs et al., 1995). Astrocytes express both H1 and H2 receptors (Ho¨sli et al., 1984; Inagaki and Wada, 1994; Carman-Krzan and Lipnik-Stangelj, 2000). The H1 receptor-mediated [Ca2þ]i increases occur mainly in cultures of the so-called type 2 astrocytes, but have also been demonstrated in subpopulations of conventional astrocytes, occasionally called type-1 astrocytes (McCarthy and Salm, 1991; Inagaki and Wada, 1994). In an experimental study, cultured rat cerebellar astrocytes responded with [Ca2þ]i increases after stimulation with histamine, an effect which was antagonized by H1 receptor blocker (Jung et al., 2000). 2.8. Acetylcholine receptors Progress in studying the role of acetylcholine in neuronal – glial interactions has not been as fast as it has in the case of glutamate and GABA and of the aminergic receptors.

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The reason for this may be that acetylcholine is less widely used in central synaptic transmission than are either glutamate or GABA. Two distinct families of acetylcholine receptors have been described: ionotropic nicotinic cholinoreceptors and metabotropic muscarinic cholinoreceptors. Nicotinic cholinoreceptors contain an integral cationic channel and metabotropic muscarinic cholinoreceptors are coupled to G proteins (Changeux, 1995). There is some evidence that nicotinic receptors exist on astrocytes (Ho¨sli and Ho¨sli, 1988; Sharma and Vijayaraghavan, 2001), and muscarinic receptors are widely found on astrocytes. There are five major subtypes of metabotropic muscarinic cholinoreceptors, M1 – M5. Type 1, 3, and 5 are coupled to PLC controlling the IP3 turnover, and type 2 and 4 are coupled to adenylate cyclase and decrease cAMP activity (Caulfield, 1993). In vivo, about 20% of the astrocytes in the rat brain label for muscarinic receptors (Van der Zee et al., 1993). These astrocytes are mainly found in the cerebral cortex and in the corpus callosum. In vitro, most cultured astrocytes possess muscarinic receptors (Hamprecht et al., 1976; Repke and Maderspach, 1982). The two muscarinic receptor subtypes found on astrocytes have been identified as muscarinic receptor1 (M1) and muscarinic receptor2 (M2) (Murphy et al., 1986). Activation of M1 stimulates PLC (Murphy et al., 1986), which leads to an IP3 increase and a Ca2þ mobilization (Enkvist et al., 1989; Araque et al., 2002). Stimulation of muscarinic receptors also leads to phospholipase D (PLD) activation (Gustavsson et al., 1993).

2.9. Purine receptors Glial cells represent a very important source of purines in the CNS, both under physiological conditions and in pathological states. Astrocytes are the main source of cerebral purines. They release the adenine-based purines adenosine and adenosine triphosphate (ATP) and the guanine-based purines guanosine and guanosine triphosphate. There are several specific purine receptors for adenosine and adenine nucleotides (adenosine phosphate (ADP) and ATP), which are called purine receptor 1 (P1) and purine receptor 2 (P2), respectively (Burnstock, 1978). The P1 purinoceptors are divided into two classes, A1 and A2, on the basis of the abilities of their adenosine agonists to either increase (A2) or decrease (A1) cAMP production, a distinction originally made on the basis of experiments using cultured astrocytes (Van Calker et al., 1979). The P1 receptors are characterized as G protein-coupled receptors. The A2 receptor has been divided into A2A and A2B, both of which subclasses stimulate the Gs protein and thus increase cAMP. The more recently demonstrated A3 receptor is coupled to Gi or Go proteins and thus inhibits cAMP production. This receptor also stimulates IP3 formation. The P2 receptors are divided into the subclasses P2X and P2Y (Burnstock and Kennedy, 1985). The P2X receptor complex comprises a ligand-gated ion channel (i.e., it is an ionotropic receptor), which is opened by receptor activation and promotes influx of Naþ and Ca2þ, and efflux of Kþ, followed by membrane depolarization and fast synaptic transmission (Bean, 1992). These receptors comprise a family of seven members that have been cloned and named P2X1 – 7 (Fredholm et al., 1997). The P2Y receptors are G proteinlinked receptors (i.e., metabotropic receptors), which are coupled to PLC and activation of IP3, with [Ca2þ]i mobilization. Seven P2Y receptors have also been cloned and named

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P2Y1 – 7 (Fredholm et al., 1997). Astrocytes express several types of purinoceptors, mainly A1, A2B, A3, P2Y1, P2Y2, and P2X7 (for a review, see Di Iorio et al., 1998; Ciccarelli et al., 2001).

2.10. Endothelin receptors Astrocytes represent a major target for endothelins, a family of peptides, released by several cell types in the brain (Kuwaki et al., 1997), that have potent and multiple effects on signal transduction pathways, such as activation of the IP3 system followed by [Ca2þ]i transients, and activation of mitogen-activated protein kinases (MAPKs), phospholipase A2 (PLA2), and PLD. Endothelins constitute a peptide family composed of at least three isoforms termed endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3) (Inoue et al., 1989). They have been found to have multiple biological activities in both vascular and non-vascular tissues (Yanagisawa et al., 1988). Endothelin mRNA expression is widely distributed in the brain (Giaid et al., 1991). Endothelin-induced cellular reactions are mediated via three major receptor subtypes, displaying different sensitivity to the various endothelin isoforms. The most abundant form in the brain is the non-selective endothelinB (ETB) receptor, which is equally sensitive to all three endothelins. The endothelinA (ETA) receptor, preferentially sensitive to ET-1 and ET-2, but not to ET-3, is much less expressed; the endothelinC (ETC) receptor has been described only in non-mammalian tissues (Sokolovsky, 1995). All three subtypes are coupled to heterotrimeric G proteins and they are significantly involved in the regulation of [Ca2þ]i (Goldman et al., 1991; Blomstrand et al., 1999) and release of arachidonic acid (Wu-Wong et al., 1996). EndothelinA receptors mediate vasoconstriction, whereas ETB receptors mediate vasodilation. In the brain, ETA receptors are mostly expressed in vascular cells (Hori et al., 1992), whereas ETB receptors are mainly expressed on glial cells (Lazarini et al., 1996; see also chapter by Chen and Spatz). However, expression of both ETA and ETB receptor mRNA has been detected in astrocytes in culture (Ehrenreich et al., 1993; Schinelli et al., 2001). The endothelin-induced [Ca2þ]i responses in astrocytes have been reported to have heterogeneous kinetic properties, varying from simple peaks to polyphasic peaks (Blomstrand et al., 1999). 2.11. Opioid receptors The opioid receptor family consists of three pharmacologically distinct subtypes, m, d, and k (Leslie, 1987). They are heterogeneously distributed through the CNS and produce a multitude of behavioral, neuroendocrinological, and autonomic effects. All three receptors are G protein-coupled, and all three have been cloned (Uhl et al., 1994). The classic opioid agonist, morphine acts primarily on m receptors. The first endogenous opioid peptides discovered were the enkephalins, which act preferentially on the d receptors. Other endogenous opioids, the b endorphins and the dynorphins, affect m and k receptors (Borsodi and To´th, 1995). Protein and/or mRNA for m, d, and k receptors have been found in astrocyte cultures derived from different rat brain regions. There is heterogeneity of

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opioid receptor expression in astrocytes from different brain regions, but the sum of the three subtypes is largest in brain cortex. The expression of d and k receptors on astrocytes appears to be relatively similar, whereas m receptors are scarce (Ruzicka et al., 1995). Spinal astrocytes also express d and m receptors, although most spinal receptors are on neurons (Cheng et al., 1997). Application of d- and k-selective agonists decrease forskolin-stimulated levels of cAMP in astrocytes (Rougon et al., 1983; Eriksson et al., 1990). However, increases in [Ca2þ]i have also been demonstrated after stimulation of k receptors (Eriksson et al., 1993) and d receptors (Thorlin et al., 1998).

2.12. Peptide receptors Some transmitter peptides have already been described, i.e., endothelins, enkephalins, b endorphins and dynorphins, and others will be described below (growth factors, cytokines). However, the receptors for these are generally not regarded as peptide transmitter receptors. The term ‘peptide receptors’ defines receptors for a relatively well delineated group of neuroactive peptides, some of which are also hormones (e.g., vasopressin and somatostatin), and many of which were first known for their action in the gastrointestinal system (e.g., secretin). The first studies of peptide receptors on astrocytes were performed by Hamprecht and coworkers (Van Calker et al., 1980), who showed that astrocytes in primary cultures have receptors for vasoactive intestinal peptide (VIP), secretin, and somatostatin, which regulate cAMP. Later, Cholewinski et al. (1988) reported that some peptides, namely, oxytocin, vasopressin, bradykinin, and tachykinin, activate IPs and increase [Ca2þ]i. Furthermore, angiotensin II receptors and atrial natriuretic peptide receptors have been found on astrocytes (Sumners et al., 1994). Two different receptors for VIP have been identified: one with a low-affinity binding site coupled to activation of adenylate cyclase (Gozes et al., 1991), and the other with a high-affinity site, which is coupled to IP3 generation and changes in [Ca2þ]i (Fatatis et al., 1994). Receptors for neurotensin and neuropeptide Y (NPY) have been identified on the basis of ligand binding and electrophysiology (Gimpl et al., 1993; Ho¨sli et al., 1995). Two subtypes of bradykinin receptors, B1 and B2, have been described. In cultured astrocytes, mainly the B2 receptor has been reported to trigger [Ca2þ]i elevations, but a minor subpopulation of astrocytes may also contain B1 receptors (Stephens et al., 1993). Finally, glucagon receptor stimulation leads to increases in cAMP (Cockram et al., 1995). For more extensive information on peptide receptors can be referred to Deschepper (1998). The role of vasopressin in regulation of fluid homeostasis in the CNS is discussed in the chapter by Chen and Spatz. Substance P belongs to the family of neurokinins, and three subtypes of receptors have been identified, neurokinin 1 (NK1), neurokinin 2 (NK2), and neurokinin 3 (NK3) (Dam et al., 1988). They are G protein-coupled metabotropic receptors. Binding of the NK1 receptor to substance P results in phosphoinositide hydrolysis, and substance P increases phosphatidylinositide turnover in cerebellar astrocytes (Marriott and Wilkin, 1992). Both brain astrocytes and spinal astrocytes express NK-1 receptors, but the receptor density is much higher on the spinal cells (Palma et al., 1997). Besides stimulating formation of IP3, substance P also potentiates the inducing effects of interleukin-1b (IL-1b) and tumor

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necrosis factor-a (TNF-a) on secretion of interleukin-6 (IL-6) by spinal cord astrocytes (Palma et al., 1997). These effects may play a role for the influence of astrocytes on persistent pain, which is described in the chapter by Raghavendra and DeLeo. 2.13. Protease receptors Proteases, also called ‘proteinases’, are believed to be involved in development and in repair processes in the CNS, and they regulate several functions of the CNS by activating protease-activated receptors (PARs). PARs are cell surface receptors that mediate cellular signaling through heterotrimeric G proteins (Wang et al., 2002). Some of the receptors, PAR-1, PAR-2, PAR-3, and PAR-4, are functionally active on astrocytes. Stimulation of these receptors on astrocytes with trombin, trypsin, or peptides induces maintained [Ca2þ]i elevations (Wang et al., 2002). 2.14. Prostanoid receptors Evidence from in vitro studies suggests that the predominant cell responsible for brain prostanoid formation is the astrocyte (Murphy et al., 1988), although neurons also produce prostanoids (Inagaki and Wada, 1994). Astrocytes in primary cultures have been shown to produce prostaglandins (PGDs) and thromboxanes (TXBs), including phosphogluconate dehydrogenase2 (PGD2), prostaglandin E2 (PGE2), and prostaglandin F2a (PGF2a), as well as thromboxane aprotinin A2 (TXA2) and thromboxane 2 (TXB2). The release of prostanoids from astrocytes in primary cultures is stimulated by ATP, substance P, interleukin-1b (IL-1b) and tissue plasminogen activator (TPA) (Inagaki and Wada, 1994). Prostanoid receptors present on astrocytes are the PGF2a receptor (FP receptor), thromboxane A2 receptor (TP receptor), and PGE receptor EP3 subtypes (EP3 receptor) (Kitanaka et al., 1996a). The EP3 receptor is known to be G protein-coupled (Satoh et al., 1999), and a thromboxane analog increases [Ca2þ]i in primary cultures of astrocytes (Kitanaka et al., 1996b). 2.15. Chemokine receptors Chemokines are a family of cytokines, which originally were described in the immune system, where they regulate motility of immune cells. Chemokines are synthesized in the CNS by astrocytes, microglia, and neurons, and a chemotactic response of these cells to chemokines may play a role during brain development (Rezaie et al., 2002). This is consistent with a role for chemokines not only in the immune system but also in development and migration of many cell types in the absence of inflammation, e.g., in cell growth, angiogenesis, hematopoiesis, free radical production, apoptosis, neoplasia, wound healing, tissue repair, and interactions with pathogens, including viruses (Bacon and Harrison, 2000). The chemokines have been classified into the four subfamilies a, b, g, and d, or CXC, CC, C, and CX3C (Zlotnik and Yoshie, 2000). In contrast to other cytokines, chemokines signal via G protein-coupled receptors, i.e., 7TM receptors of the serpentine superfamily, and elicit biological activities at nanomolar concentrations

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(Murphy et al., 2000). Astrocytes express a number of chemokine receptors, including CCR1, CCR3, CCR5, CXCR2, and CXCR4, some of which have been shown to have a functional expression (Dorf et al., 2000—see also chapter by Nakagawa and Schwartz).

3. Astrocytic cytokine and growth factor receptors 3.1. Cytokine receptors Neurotrophic factors (growth factors acting on neurons and/or glial cells) and cytokines (factors originally described in hematopoietic cells and cells of the immune system, where they regulate development and function) are of fundamental importance in the CNS, and astrocytes express functional receptors for many of them (Otero and Merrill, 1994—see also chapter by Nakagawa and Schwartz). Growth factors and cytokines play a huge role in the CNS, where cytokines can be either pro-inflammatory, neuropoietic, i.e., acting as growth factors, and/or anti-inflammatory. Their receptors are not G protein-coupled, but directly associated with cascades of protein kinases, often receptor protein-tyrosine kinases. Receptor activation is followed by distinct protein kinase pathways (e.g., the Ras, Raf, MAP kinase pathway) and eventual translocation of a phosphorylated kinase to the cell nucleus, leading to transcriptional induction of immediate early genes. Interleukins (IL) constitute a functionally heterogenous group, with some ILs being pro-inflammatory, other neuropoietic or anti-inflammatory. Interleukin-6 (IL-6) belongs to the family of neuropoietic cytokines, a family that includes ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), IL-6, interleukin-11 (IL11), and cardiotrophin-1 (CT-1) (Taga and Kishimoto, 1997). All IL-6-type cytokine family members act via receptor complexes that contain the glycoprotein gp130. Some astrocytes express gp130, but respond weakly to IL-6 alone (Van Wagoner and Benveniste, 1999). Interleukin-6 exerts neurotrophic and neuroprotective effects, but it can also function as a mediator of inflammation, demyelination, and astrogliosis (Gadient and Otten, 1997). Astrocytes play a key role in brain inflammation in response to trauma, ischemia and infection and, along with microglia, belong to the most important cell types for local regulation of inflammatory reactions and tissue repair in the CNS (Ridet et al., 1997). Astrocytes respond to the pro-inflammatory interleukin IL-1 and express IL-I receptor ˇ arman-Krzˇan, 2001). The type I receptor is biologically active, while type 1 (Jurıˇc and C type II receptor functions as a decoy. IL-receptors display an equivalent number of both receptor types (Pousset et al., 2001). Interleukin-1 receptor expression is regulated by multiple control mechanisms, such as IL-1 itself, anti-inflammatory cytokines, such as interleukin-2 (IL-2), IL-4, IL-10, IL-13, and interferon (INF), growth factors, such as platelet-derived growth factor (PDGF), and glucocorticoids (Pousset et al., 2001). Binding of IL-1b to the IL-1 receptor type 1 is followed by recruitment and phosphorylation of receptor associated kinases and eventually by translocation of nuclear factor-kB (NF-kB) to the nucleus (Mercurio and Manning, 1999. IL-1b also activates a distinct MAP kinase cascade that results in DNA binding of activator protein-1 (AP-1) (Minden and Karin, 1997). Effects of peripherally generated IL-1b on CNS IL-1 receptors are discussed in the

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chapter by Mercier and Hatton and the role of IL-1b in persistent pain in the chapter by Raghavendra and DeLeo. A second pro-inflammatory cytokine is tumor necrosis factor (TNF). Tumor necrosis factor-a (TNF-a) is a polypeptide, first identified as an inducer of necrosis of some tumor cells, but today considered as an endogenous modulator of many normal physiological parameters. The signaling for biological effects of this cytokine is exerted through cell surface receptors, activating similar cascades as IL-1, although cell-type and speciesspecific differences exist in the relative potency of these two cytokines. The presence of receptors for TNF-a has been identified both on cultured astrocytes (Ara´nguez et al., 1995) and on astrocytes in the brain (see chapter by Nagakawa and Schwartz). Autocrine/paracrine signaling via P2 receptors modulates both IL-1b- and TNF-amediated activation of NF-kB and AP-1 in human fetal astrocytes (Liu et al., 2000). 3.2. Growth factor receptors Members of several families of growth factors are produced by astrocytes, which also express corresponding receptors (see chapter by Nakagawa and Schwartz). There is recent evidence that growth factors can be released from astrocytes as a result of stimulation with G protein-coupled receptors, e.g., a2-adrenergic receptors, in a ‘transactivation’ process (see chapter by Peng). There are several factors released by astrocytes, including transforming growth factor-b (TGF-b, macrophage colony-stimulating factor (M-CSF), and granulocyte/macrophage colony-stimulating factor (GM-CSF), which can induce ramification and upregulation of KþDR in microglia (Schilling et al., 2001). Astrocytes are also capable of suppressing phagocytosis (De Witt et al., 1998) and production of the pro-inflammatory cytokine interleukin-12 (IL-12) by microglia (Aloisi et al., 1997). 4. Astrocytic intracellular receptors 4.1. Nuclear receptors Several receptors, including those for many hormones (e.g., glucocorticoid receptors (GR)), translocate from the membrane to the nucleus after activation. In the brain, glucocorticoids bind to two types of receptors, a high-affinity mineralcorticoid receptor (MR) and a lower-affinity GR with affinities for endogenous glucocorticoids that differ by an order of magnitude (De Kloet, 1991). They are both expressed in astrocytes (Bohn et al., 1994). After treatment with glucocorticoids, an increase in [Ca2þ]i concentration has been observed with a Ca2þ wave propagation (Simard et al., 1999). This may appear peculiar, because the glucocorticoid receptors are not G-protein coupled, but the explanation appears to be that glucocorticoids enhance the response to such transmitters as ATP and bradykinin. Astrocytes express receptors for different steroid hormones as described in the chapter by Melcangi et al. Neither these receptors, nor those for polypeptide hormones, such as growth hormone receptors will be discussed here. The same applies to thyroid hormone receptors (see chapter by Gomes and Rehen).

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4.2. Mitochondrial receptors The astrocytic, peripheral-type, benzodiazepine receptor is mainly localized on the mitochondrial membrane (see, however, also Section 2.2). 1,4-Benzodiazepines bind to two distinct classes of receptors. The central benzodiazepine receptor is membraneassociated and coupled to GABAA receptor-gated Cl2 channels on neurons, and possibly also on astrocytes (Section 2.2). It is generally assumed to be responsible for all antianxiety, sedative, and anti-convulsant actions of benzodiazepines (Richards et al., 1987). The peripheral-type benzodiazepine receptor (PTBR), by contrast is localized on astrocytes and absent on neurons (McCarthy and Harden, 1981) and, as in other tissues, it appears to be present chiefly on the outer mitochondrial membrane (Itzhak et al., 1993). A nuclear localization has, however, also been found recently (Kuhlmann and Guilarte, 2000). The PTBR is involved in the intracellular transport and metabolism of cholesterol and the production of neurosteroids (Rao et al., 2001). The PTBR has been demonstrated to increase after brain injury in both activated microglia and astrocytes (Kuhlmann and Guilarte, 2000), and in acute hyperammonemia (Felipo and Butterworth, 2002—see also chapter by Be´langer et al.).

5. Functional consequences of astrocytic receptor activation 5.1. Physiological activity Since astrocytes are intimately associated with the synapse, enwrapping many pre- and postsynaptic terminals, and since they are in close contact with the capillaries, they are in a position to shuffle nutrients and metabolites between the blood supply and the active neuron. Astrocytes can also transfer information to neighboring neurons and one single astrocyte can have contact with multiple neurons (Ventura and Harris, 1999). The coordination of neuronal and glial activity requires that appropriate signals pass from one cell type to another. Astrocytic functions are probably to a considerable extent regulated by monoaminergic neurons, from which transmitters are released not only in the privacy of synapses, but also from varicosities, from where they reach their targets (e.g., neurons, astrocytes, oligodendrocytes, abluminal surface of endothelial cells) by diffusion. In astrocytes, the released monoamines exert a multitude of functional effects, including regulation of energy metabolism (see chapter by Hertz, Peng et al.) and of channel opening. However, monoamines are not the only transmitters which influence astrocytic activities. As demonstrated in this chapter, glutamatergic and GABAergic transmission can no longer be regarded as an exclusive neuronal phenomenon, since astrocytes express a multitude of different subtypes of receptors for both glutamate and GABA. The same applies to purinergic receptors. A common feature of all of these receptors is that they are membrane-associated and G protein coupled. Activation of G protein-coupled receptors is primarily linked to stimulation of PLC, with associated increases in IP3 and DAG, as well as in [Ca2þ]i, and/or to increases/decreases in cAMP. The prototypical receptor mediating an increase in cAMP is the b-adrenergic receptor, and a large fraction of b-adrenergic receptors in the CNS are

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found on astrocytes (Stone and Ariano, 1989). Whereas many developmental effects of cAMP have been described in astrocytes, we know relatively little about the consequences of cAMP increases, except for such events as stimulation of glycogenolysis (see chapter by Hertz, Peng et al.) and effects on ion channels. With respect to activation of PLC the situation is different: During the last decade it has become firmly established that astrocytes carry out long-distance signaling, mediated by changes in [Ca2þ]i. Increases in [Ca2þ]i not only spread in an astrocytic network, connected by gap junctions, but also across long distances from one network to another, with which it is not anatomically coupled, by aid of ATP, released from activated astrocytes (see below). Astrocytes are able to synthesize and release many neuroactive compounds, both at rest and perhaps especially during exposure to various stimuli, including receptor activation and astrocytically mediated increases in [Ca2þ]i. They release compounds such as taurine, GABA, glutamate, and aspartate (Kimelberg et al., 1990—see also chapter by CornellBell et al.), as well as purine nucleotides and nucleosides (Di Iorio et al., 1998). The released transmitters are able to either enhance or inhibit activity in adjacent neurons. Since on one hand neuronally released transmitters can activate astrocytic [Ca2þ]i signals, and on the other hand these signals can activate or deactivate adjacent neurons, astrocytes mediate a neuronal – astrocytic – neuronal impulse transmission system. This system is not even dependent upon anatomical contiguity, since [Ca2þ]i signals can be transferred by ATP release. Therefore, synaptic sensitivity could be modulated by astrocytes even with no cellular bridges in between the synaptic regions involved. The synthesis and storage of a wide variety of peptides within the CNS is well established. Specific roles for the peptides are less well known. However, angiotensin II and NPY have been shown to modulate noradrenergic transmission (see Sumners et al., 1994), and interactions also take place between cAMP-activated b receptors on astrocytes and VIP receptors, which results in a decrease in the cAMP level (Hansson and Ro¨nnba¨ck, 1988). Angiotensin II and atrial natriuretic peptide are involved in the CNS control of water and Naþ balance and in blood pressure/baroreflex regulation (Sumners et al., 1994). The involvement of receptors on astrocytes, brain endothelial cells and choroid plexus epithelial cells in regulation of fluid balance within the CNS by vasopressin is discussed in the chapter by Chen and Spatz. Signaling by cytokines and growth factors to and from astrocytes is generally believed to be of importance mainly during development and after injury to the nervous system

Fig. 1. The astrocytes (red in the figure) occupy a strategic position in the central nervous system (CNS). The cells are coupled in networks, and come in contact with blood vessels (to the left in the figure) and synaptic regions. The astrocytes are known to support neuronal activity (neurons are depicted in white to pale blue). They create opportunities for the dynamics and plasticity in the CNS, both in physiological and in pathological conditions. The astrocytes express a large number of receptors and signaling systems. These receptors and their second messengers play specific roles in the signaling between individual astrocytes and in the communication between astrocytes and other cells. In this figure, astroglial receptors detected thus far are marked. The figure is a theoretical illustration, since we do not yet know in detail how the receptors are arranged on the cells. m, mu opioid receptor; d, delta opioid receptor; k, kappa opioid receptor. For further information and abbreviations, see text.

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(see Hansson and Ro¨nnba¨ck, 2003). However, it is completely feasible that these compounds also may subserve normal physiological activity. That this is the case in the regulation of the secretion of hypothalamic hormones is discussed in the chapter by Mercier and Hatton. 5.2. Neurotrauma In neurotrauma, including cerebral ischemia, glutamate is released at the synapse and by reversal of astrocytic glutamate uptake, and the astrocytes are stimulated, which results in increased [Ca2þ]i and Ca2þ wave propagations and in an increased release of ATP. Many studies have addressed the question of the relevance for extracellular ATP in glial functions. Besides their involvement in [Ca2þ]i signaling described above, activation of purinergic receptors is functionally coupled to prostanoid formation in astrocytes (Bruner et al., 1994). There is evidence supporting the importance of a glial arachidonic acid cascade in certain pathophysiological states, including an autocrine effect, since pharmacological studies suggest that astrocytes may also be among the target cells for prostanoids in the CNS (Ito et al., 1992). The purines participate in many vital intracellular processes, including astrocytic energy metabolism (formation of ATP and GTP) and the synthesis of cholesterol and of nucleic acids. In CNS disorders, such as seizures, trauma, ischemia, and hypoxia, both neurons and astrocytes greatly increase their release of purine nucleotides and nucleosides. Purinomimetic drugs, such as propentophylline, may become of importance in the treatment of the glial contribution to persistent pain (see chapter by Raghavendra and DeLeo). Injury, as well as neurodegenerative disease, have profound effects on cytokines. In CNS injury, the pro-inflammatory cytokines TNF-a is upregulated and released by the astrocytes (Ben-Adani et al., 2001) and microglia (Kim et al., 2000; Hansson and Ro¨nnba¨ck, 2003). The release promotes gliosis (Selmaj et al., 1991), inhibits astrocytic glutamate uptake (Fine et al., 1996) and induces apoptosis, particularly in oligodendrocytes (Hisahara et al., 1997). The cAMP-mediated action of VIP appears to reduce the release of TNF-a, at least by the microglia (Kim et al., 2000). Neurodegenerative diseases such as Alzheimer’s disease and parkinsonism, are associated with large increases in both TNF-a and IL-1b (see chapters by Barger and by Przedborski and Goldman). 5.3. Development It is well established that agents which in the mature organism function as transmitters may play a major developmental role as ‘morphogens’ during ontogenesis (Lauder, 1988). This applies to ‘classical’ transmitters like serotonin and noradrenaline acting via stimulation of adenylyl cyclase or PLC (Narumi et al., 1978; Mobley et al., 1986; Lauder, 1988). It has also been speculated whether opioids directly modify brain development at the cellular level or whether they act indirectly by causing alterations in hormone levels, respiration, or nutritional status. There is an upregulation of astrocytic d receptors during the mitotic phase (Thorlin et al., 1999), and this type of expression during critical periods may support the theories of direct regulatory mechanisms.

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VIP is thought to stimulate astrocytes to generate neurotrophic factors, the most potent and neuroprotective protein of which appears to be activity-dependent neurotrophic factor (ADNF) (Brenneman and Gozes, 1996). ADNF then acts directly on neurons to promote glutamate responses and morphological development. ADNF also decreases oxidative stress (Guo and Mattson, 2000) and causes secretion of neurotrophin 3 (NT-3), which regulates the NMDA receptor subunits 2A and 2B (Blondel et al., 2000).

6. Concluding remarks It is of utmost importance that we learn to understand the cellular signaling underlying the plasticity of the CNS both in physiological regulatory activity and in functional disturbances resulting from damage and disease. It is now well known that neurons signal not only to each other, but also to the glial cells, informing the astroglial networks about neuronal activity and enabling them to support the neurons metabolically and trophically, according to their specific requirements. The astroglial cells also signal back to the neurons and modulate synaptic activity. ATP is an important signal substance, mediating contact between neurons, astroglial cells and microglial cells. During physiological conditions, microglia synthesize and release trophic factors; however, as soon as there is a functional disturbance of any kind in the CNS, microglia react, proliferate, and express protective or, in some situations, cytotoxic properties. These reactions are to a large extent mediated via receptors for cytokines and growth factors on astrocytes. Microglial cells also participate in restorative work, again often in conjunction with astrocytes. By identifying intercellular signaling, which mediates protective and restorative processes and reinforcing such signaling following tissue damage or during degeneration, it may eventually be possible to limit the extent of permanent damage in nervous tissue. Acknowledgements The work performed in the authors’ lab was supported by grants from the Swedish Research Council (Project no. 33X-06812 and 21X-13015), Edith Jacobson’s Foundation, and the Swedish Council for Working Life and Social Research. Fig. 1 was designed by Eva Kraft, Go¨teborg, Sweden.

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Transactivation in astrocytes as a novel mechanism of neuroprotection Liang Peng Professor, College of Basic Medical Sciences, China Medical University, No. 92 Beier Boad, Heping Distric, Shenyang, P.R. China E-mail: [email protected]

Contents 1. 2. 3. 4.

5. 6.

Introduction EGFR transactivation pathways HB-EGF and EGFR expression in brain Is transactivation in astrocytes receptor selective? 4.1. EGF transactivation in astrocytes 4.2. a2-Adrenergic receptors 4.3. Metabotropic glutamate receptors 4.4. Thrombin 4.5. ATP 4.6. Other transmitters Cytoprotective effect of HB-EGF Concluding remarks

Epithelial growth factor receptor (EGFR) transactivation in response to stimulation of a G protein-coupled receptor (GPCR) is a novel signaling pathway in cross-talk from cell to cell and from receptor to receptor. The presently reviewed material suggests that transactivation may be specific for the EGFR and not a phenomenon common to many growth factor receptors. However, different GPCRs can lead to EGFR transactivation, albeit probably utilizing different transduction pathways in the donor cells, as exemplified by similar effects of the group I metabotropic glutamate receptor agonist, [RS ]-3,5-dihydroxyphenylglycine, and the a2A-adrenergic agonist dexmedetomidine. Both drugs were found to act on astrocytes, and these cells are likely to be a major source of soluble heparin-binding EGF-like growth factor (HB-EGF), since GPCRs related to EGF activation and HB-EGF are expressed at high level in astrocytes in primary cultures and in the brain in vivo, and since expression of the HB-EGF gene may mainly occur in astrocytes in neurodegenerative diseases. Advances in Molecular and Cell Biology, Vol. 31, pages 503–518 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction Transactivation is a process in which signaling to a G-protein-coupled receptor (GPCR) leads to release (‘shedding’) of a growth factor, which in turn stimulates receptor tyrosine-kinases (RTK) on the same or adjacent cells and evokes a response that is indistinguishable from that which can be directly obtained by direct RTK stimulation, e.g., phosphorylation of mitogen-activated protein (MAP) kinases, such as extracellular-signal regulated kinase (ERK) 1 and 2, and subsequent down-stream signaling. Transactivation may play a major role in not only astrocytic functions but also in interactions between astrocytes and neurons, including astrocyte-mediated neuroprotection. Both astrocytes and neurons respond to neurotransmitters and growth factors by specific receptors (see chapters by Hansson and Ro¨nnba¨ck and by Nakagawa and Schwartz), and both cell types also express and release a number of neurotrophic factors, such as heparin-binding EGF-like growth factor (HB-EGF), ciliary neurotrophic factor, nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3 (Rudge et al., 1992), and transforming growth factor-beta (TGF-b) (Bruno et al., 1998). This arrangement sets the stage for extremely complicated communications from cell to cell, and from receptor to receptor, which may be crucial for the precise regulation of astrocytic effects on proliferation, differentiation, function, and survival, not only of astrocytes but also of their neuronal neighbors. Cross-talk at the molecular level between different signaling pathways, especially between GPCRs and RTKs, was first uncovered in transfected cell lines (Daub et al., 1997), and has later been demonstrated in primary cultures of different cell types (Peavy et al., 2001; Kalmes et al., 2000; Grewal et al., 2001; Saito et al., 2002; Uchiyama-Tanaka et al., 2001). Recently, we have established that dexmedetomidine, a specific agonist of the a2-adrenergic receptor, stimulates the transactivation pathway in primary cultures of mouse astrocytes by shedding of HB-EGF. This conclusion was based on inhibition of dexmedetomidine-induced ERK phosphorylation in astrocytes by tyrphostin AG 1478, a specific inhibitor of the EGFR tyrosine kinase (Peng et al., 2002). Since HB-EGF has neuroprotective properties (see below), and since released HB-EGF in the brain in situ is likely to act not only on astrocytes but also on adjacent neurons, such a transactivation may be one of the underlying mechanisms of the well-established neuroprotective effect by dexmedetomidine (Jolkkonen et al., 1999). There is also evidence that dexmedetomidine in the brain may decrease neuronal glutamate release and/or enhance glutamate removal (Jolkkonen et al., 1999; Talke and Bickler, 1996; Huang and Hertz, 2000; Li and Eisenach, 2001). However, its neuroprotective effects can be established even in the absence of reduced extracellular glutamate concentration (Engelhard et al., 2002). Many neuroscientists have been interested for a long time in the potential use of growth factors as neuroprotective agents (Gozes, 2001). However, clinical application of growth factors with large molecular size is, at best, difficult, because these polypeptides are unable to cross the blood – brain-barrier (Hefti, 1994). Instead, using a small molecule, such as the a2-adrenergic agonist dexmedetomidine, to induce HB-EGF would provide a novel therapeutic strategy for treatment of neurodegenerative and ischemic brain diseases. However, other transmitters, e.g., agonists of certain

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metabotropic receptors, have also been found to have neuroprotective properties (Bruno et al., 1998, 2000), and it cannot be excluded that neuroprotection by transactivation and resulting release of growth factor(s) may be a phenomenon of more general importance than presently realized. It is the purpose of the present chapter to discuss this possibility.

2. EGFR transactivation pathways Transactivation following GPCR stimulation has been observed with many different kinds of GPCRs and in many different cell types. The best studied RTK transactivation by GPCRs is activation of the EGFR. Activation of this receptor represents the paradigm for cross-talk between GPCRs and RTKs (Gschwind et al., 2001), but a priori it cannot be excluded that other growth factor receptors may be activated in a similar manner. The involvement of EGFR in transactivation was demonstrated by Ullrich’s group in 1997, who showed that MAPK activity induced by the GPCR agonists endothelin-1, lysophosphatic acid and thrombin was reduced by tyrphostin AG1478 or in a dominantnegative EGFR receptor mutant (Daub et al., 1997). For a period of time, it was not clear whether a RTK ligand was involved, since the onset of transactivation is rapid, and no free ligand was detected in the extracellular medium. However, by now, HB-EGF shedding has been successfully demonstrated with several different GPCR agonists (or activators of second messengers) and in many different cell types. Phorbol ester-induced HB-EGF shedding from monkey kidney Vero-H cells (stable transfectants of Vero cells, overexpressing human pro-HB-EGF) was first demonstrated immunocytochemically by Goishi et al. (1995), and it has been confirmed by Western blotting, using antibodies specific to the tail fragment of pro-HB-EGF in the cell lysate or to HB-EGF in the medium and cell lysate (Umata et al., 2001). The shedding is dependent on metalloproteinases and therefore blocked in mutants lacking the metalloproteinase domain (Izumi et al., 1998). Phorbol esters activate protein kinase C (PKC), which accordingly can trigger HB-EGF shedding. However, in NbMC-2 prostate epithelial cells it has been shown that this can also be achieved independently of PKC activation by an increase in free cytosolic Ca2þ concentration evoked by administration of a Ca2þ ionophore and dependent on the presence of extracellular calcium (Dethlefsen et al., 1998). Secondarily, released HB-EGF stimulates phosphorylation of EGFR, a response which in primary cultures of vascular smooth muscle (VSMCs) stimulated by thrombin was shown to be blocked by heparin (which inactivates HB-EGF) and neutralizing HBEGF antibody, as well as by a metalloproteinase inhibitor (Kalmes et al., 2000). Transactivation of EGF can be induced by GPCR-mediated activation of either Gi/oand Gq-coupled receptors (Table 1). In the conventional signaling pathway of GCPRs, receptor stimulation leads to exchange of GDP, bound to the alpha subunit of the Gprotein, with GTP. This exchange triggers dissociation of the ‘activated’ alpha subunit and initiation of the typical receptor response, e.g., hydrolysis of PIP2 to generate the two second messengers inositol trisphosphate (IP3), which stimulates release of Ca2þ from intracellular stores, and diacylglycerol (DAG), which stimulates PKC activity. The importance of these mechanisms in the signaling mechanisms of HB-EGF shedding and EGFR transactivation are not completely clear, and could be different in different cell

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Table 1 Cell types in which transactivation has been demonstrated, the G protein-coupled receptors involved, and the types of G protein activated by receptor stimulation G protein

Cell type

a2A-Adrenergic receptor

Gi/o (Antaa et al., 1995)

a2B-adrenergic receptor

Gi/o (Antaa et al., 1995)

mGluR5 Thrombin receptor

Gq (Miura et al., 2002) Gq and Gi/o (Gardner et al., 2002; Debeiret et al., 1996)

P2Y 5-HT2A receptor Angiotensin II receptor

Gq and Gi/o (Lin and Chuang, 1994) Gq and Gi (Miyoshi et al., 2001; Maayani et al., 2001) Gq (Keys et al., 2002)

Astrocytes (Peng et al., 2003) Retinal Mu¨ller cells (Peng et al., 1998)a Transfected cell line (Pierce et al., 2001) Renal tubular cell line; proximal tubule cells (Cussac et al., 2002a,b) Astrocytes (Peavy et al., 2001) Astrocytes (Daub et al., 1997) VSMC (Kalmes et al., 2000) Transfected cell line (Daub et al., 1997) Astrocytes (Daub et al, 1997) Renal mesangial cells (Grewal et al., 2001)

Beta2-adrenergic receptor Estrogen receptor

Gi (Zamah et al., 2002) GPR 30 (Filardo et al., 2002)

Endothelin-1 receptor Bradykinin receptor

Gq (Takigawa et al., 1995) Gq (Graness et al., 1998)

VSMC: vascular smooth muscle cells. a The involvement of transactivation has not been directly established in these cells.

VSMC (Saito et al., 2002; Voisin et al., 2002) Glomerular mesangial cells (Uchiyama-Tanaka et al., 2001) Transfected cell lines (Goishi et al., 1995; Umata et al., 2001) Cardiac fibroblasts (Kim et al., 2002) Breast cell lines (Filardo, 2002; Filardo et al., 2002) Tranfected cell line (Daub et al., 1997) PC12 cells (Zwick et al., 1997)

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Receptor

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types and after stimulation of different receptors. Consistent with the dependence of HBEGF shedding on either PKC activity or Ca2þ entry in some cell types, it has been demonstrated (i) that phosphorylation of EGFR by bradykinin in PC12 cells, a neuronal cell line developed from a pheochrocytoma, is dependent upon the presence of extracellular Ca2þ (Zwick et al., 1997); and (ii) that serotonin (5-HT) in renal mesangial cells induces a concentration-dependent EGFR phosphorylation (by stimulation of 5-HT2A receptors), which can be attenuated by a PKC inhibitor, GF109203X, but not by PD98059, an inhibitor of MEK (MAP kinase/ERK kinase), and thus of ERK phosphorylation (Grewal et al., 2001). Whether GPCR-induced and metalloprotease-mediated proteolytic cleavage of HB-EGF plays a key role in these cases needs, however, to be further studied. It has also been reported that calmodulin is involved in phorbol ester-induced EGF transactivation in a monkey kidney cell line, COS-1 (Tebar et al., 2002). In a renal tubular cell line and in primary cultures of proximal tubule cells, a2B-adrenergic receptor stimulation by dexmedetomidine causes a release of arachidonic acid, which is PKCindependent, but abolished by prior treatment with pertussis toxin (indicating the involvement of a GPCR) and leads to ERK phosphorylation, which is reduced by tyrphostin AG 1478, showing the dependence on EGF shedding (Cussac et al., 2002a,b). However, this pathway is specific to the a2B-adrenergic receptor, since agonist stimulation of a2A/D and a2C receptors has no effect on archidonic acid release (Audubert et al., 1999). Using a2-adrenergic receptor stimulation of two types of transfected COS-7 cells (another monkey kidney cell line), one serving as a HB-EGF donor and expressing a2A-adrenergic receptors, and the other expressing no a2A-adrenergic receptors and serving as a HB-EGF acceptor, many details of a2A-adrenergic receptor-mediated transactivation in these cells have been elegantly elucidated in co-culture experiments (Pierce et al., 2001). a2A-Adrenergic stimulation was found to cause ERK phosphorylation by two different, major pathways (Pierce et al., 2001). One of these leads directly to ERK phosphorylation in the stimulated cell, the second is the two-stage transactivation pathway (Fig. 1). In its first stage the beta,gamma subunits of the activated, heterotrimeric Gi protein lead, via activation of cytosolic Src tyrosine kinases, to dynamin-dependent, metalloprotease-catalyzed shedding of HB-EGF from its extracellular transmembranespanning HB-EGF precursor; in the second stage the shedded HB-EGF transactivates EGFR in the same and adjacent cells in conventional manners, i.e., the EGFR is phosphorylated by a RTK and internalized. This leads to the recruitment of Grb2– Sos1 complexes to the activated RTK and the exchange of GDP for GTP on the low molecular weight G protein, Ras, catalyzed by Ras guanine nucleotide exchange factor, Sos1. Ras activation, in turn, initiates the phosphorylation cascade consisting of Raf, MEK, and ERK. Recent work has demonstrated that beta-arrestins, i.e., cytosolic proteins that mediate desensitization and internalization of GPCRs, can increase the phosphorylation of ERK (in angiotensin II-stimulated COS-7 cells), presumably by functioning as adaptors and scaffolds for MAP kinase activation, bringing sequentially acting kinases into proximity with each other and with the receptor (Tohgo et al., 2002). In addition, phosphatidylinositol-3-kinase (PI3K) can be activated by EGFR stimulation of astrocytes (Zelenaia et al., 2000), but the pathways connecting PI3K activation with MAP phosphorylation are uncertain and may vary between cell types and from transmitter to transmitter. The PI3K pathway may be important for the neuroprotective effect of

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Fig. 1. Schematic representation of key signaling mechanisms presumably involved in transactivation by exposure of astrocytes (A) to the a2-adrenergic agonist dexmedetomidine. The beta, gamma subunits of the activated, heterotrimeric Gi protein lead to shedding of HB-EGF from its extracellular transmembrane-spanning HB-EGF precursor, catalyzed by a metalloprotease (metpr). The shedded HB-EGF transactivates EGFR in the same (A) and adjacent cells, presumably including neurons (N), in conventional manners, i.e., the EGFR is phosphorylated by a RTK and internalized. This leads to the recruitment of Grb2–Sos1 complexes to the activated RTK and the exchange of GDP for GTP on the low molecular weight G protein, Ras. Ras activation of Ras, in turn, initiates the phosphorylation cascade consisting of Raf, MEK, and the MAP kinases (MAPK) ERK. The proposed signaling sequences are based upon determinations by Pierce et al. (2001) of a2-activated signaling pathways in a transfected cell line and the observation by Peng et al. (2003) that inhibition of HB-EGF stimulation in astrocytes reduces ERK phosphorylation in these cells.

HB-EGF (Brunet et al., 2001), because of its ability to phosphorylate the membrane phospholipid phosphatidylinositide bisphosphate (PIP2) to phosphatidylinositide trisphosphate (PIP3). PIP3 activates the protein-serine/threonine kinase Akt, which then phosphorylates and de-activates a number of pro-apoptotic proteins, including Bad, a Bcl-2 family member, which induces cell death by stimulating release of cytochrome c from mitochondria (Yamaguchi and Wang, 2001; Xue et al., 2000). However, ERK1/2 phosphorylation has also been linked to cytoprotection (Kim et al., 2000).

3. HB-EGF and EGFR expression in brain HB-EGF is a 22 kDa, O-glycosylated protein, which was isolated from conditioned medium of a human macrophage-like cell line in 1991 (Higashiyama et al., 1991). It is a member of the EGF family, which consists of EGF, transforming growth factor-alpha (TGF-a) and heparin-binding EGF (HB-EGF). A number of membrane-anchored growth factors, including members of the EGF family, have dual functions. As membrane glycosylated proteins, they may be important for cell-to-cell adhesion. After cleavage by proteases, they are released into the extracellular fluid as soluble growth factors. In addition to HB-EGF, TGF-a may act in this fashion. In a Chinese hamster ovary cell line transfected with rat pro-TGF-a gene, TGF-a shedding can be activated with serum factors and tumor-promoting phorbol esters via both PKC-dependent and PKCindependent pathways (Pandiella and Massague, 1991). TGF-a specifically binds to HER1. In brain, HB-EGF promotes proliferation of astrocytes and stem cells,

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neurogenesis (Jin et al., 2002; Michalsky et al., 2002) and survival and differentiation of postmitotic neurons (Kornblum et al., 1999). HB-EGF is expressed in both neurons and astrocytes (Nakagawa et al., 1998). In agreement with its function during early developmental stages, the level of HB-EGF expression is much higher in brains of young animals than in adult brain (Opanashuk et al., 1999). Gene expression of HB-EGF is upregulated in cerebral ischemia (Grewal et al., 2001; Tanaka et al., 1999) and by excitotoxicity and brain injury (Opanashuk et al., 1999). In cultured cerebral cortical neurons, expression of HB-EGF is increased to 150% of control level after hypoxia (Jin et al., 2002). Although gene expression of TGF-a also is quite high in brain, it is not upregulated by ischemia (Grewal et al., 2001). The presence of EGF has only rarely been reported in brain (Scalabrino et al., 1999), and in contrast to HB-EGF, there seems to be no reports that EGF gene expression is increased in ischemic brain. The mitogenic, and non-mitogenic (including neuroprotective) effects of HB-EGF could be stage- and cell type-specific, and dependent upon the type of EGF receptor expressed by the particular cells at the specific period of time. The EGF receptor is widespread in brain and plays a major role both under physiological and pathological conditions, including carcinogenesis (Prenzel et al., 2001). There are four EGF receptors, HER1, HER2, HER3 and HER4 (Prenzel et al., 2001). According to studies in transfected cell lines that express either an individual receptor or a combination of receptors, HB-EGF binds only to HER1 and HER4 (Raab and Klagsbrun, 1997), although binding of HB-EGF to those two receptors may indirectly active other HER family member (Raab and Klagsbrun, 1997). Expression of EGF receptors varies among different types of neurons (Gerecke et al., 2001). Astrocytes express HER1 and maybe small amounts of HER4 (Pinkas-Kramarski et al., 1997; Kornblum et al., 1999). HER4 has been detected at high level in the postsynaptic density, indicating the possibility of its involvement in synaptic plasticity (Huang et al., 2001). Head injury induces an increase of HER4 expression in neurons, but not in astrocytes (Erlich et al., 2000), suggesting a role of neuronal HER4 under pathological conditions. Nevertheless, HB-EGF-promoted survival of cultured dopaminergic neurons in neuronal-astrocytic co-cultures appears to be exerted by stimulation of astrocytic EGFR (Farkas and Krieglstein, 2002).

4. Is transactivation in astrocytes receptor selective? 4.1. EGF transactivation in astrocytes Several different G-protein coupled transmitters appear to be capable of inducing EGF transactivation in astrocytes (Table 1), and the question arises whether HB-EGF shedding is a common underlying mechanism. Conceivably, transactivation might also stimulate release of other neurotrophic factors, such as basic fibroblast growth factor (basic FGF) or transforming growth factor-beta (TGF-b), which belongs to a different growth factor family than TGF-a. Several growth factors other than those acting on the EGFR provide neuroprotection. Below, it will be discussed whether they may act by transactivation.

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4.2. a2-Adrenergic receptors The a2-adrenergic receptor comprises three subtypes, a2A/D, a2B and a2C, which all are coupled to pertussis-sensitive Gi/o-coupled receptors (Bylund and Chacko, 1999). In brain, only a minor fraction of a2-adrenergic receptors may be presynaptic (Hein et al., 1999). Since the major target cells in the CNS expressing a2-adrenergic receptors include astrocytes, we tested effects of dexmedetomidine on ERK phosphorylation in these cells. Indeed, dexmedetomidine induced EGF transactivation in astrocytes, demonstrated by inhibition of ERK phosphorylation with tyrphostin AG1478 (Peng et al., 2002). It confirms the involvement of HB-EGF shedding that phosphorylation of ERK is also inhibited by heparin, which inactivates HB-EGF. In retina, a2-adrenergic agonists stimulate basic FGF expression in photoreceptors and protects photoreceptors against light damage (Wen et al., 1996); ERK phosphorylation induced by a2-adrenergic receptor activity has been observed in Mu¨ller cells, an astrocytelike cell of the retina (Peng et al., 1998). However, no experiments are available that unequivocally indicate that transactivation is involved. Unlike EGFR agonists there is also no extensive literature suggesting transactivation by shedding of basic FGF in other cell types. However, it cannot be excluded that the a2-adrenergic receptor stimulation may have caused EGFR transactivation, and that released HB-EGF secondarily has stimulated release of basic FGF. Moreover, as in the brain, reduction of glutamate excitotoxicity may also play a role in the neuroprotective properties of a2-adrenergic agonists (Baptiste et al., 2002). 4.3. Metabotropic glutamate receptors There are three groups of metabotropic glutamate receptors (mGluRs). Group I mGluRs, i.e., mGluR1 and 5, are linked to Gq protein, and activation of these receptors stimulate hydrolysis of PIP2, leading to release of DAG and IP3 and an increase in free cytosolic Ca2þ concentration; Group II mGluRs, i.e., mGluR2 and 3, and Group III, i.e., mGluR4, 6, 7 and 8, are coupled to Gi/o protein, and are linked to opening/closing of ion channels and inhibition of adenylyl cyclase (Bruno et al., 1997). Several of these receptors are expressed in astrocytes (see chapter by Hansson and Ro¨nnba¨ck). The mechanisms of the neuroprotective effects differ among the three groups of mGluRs. Agonists of the presynaptically located mGluR III are thought to protect neurons against excitotoxicity by inhibition of glutamate release from presynaptic terminals (Gasparini et al., 1999; Bruno et al., 2000). The neuroprotective effect of mGluR II agonists requires synthesis of new protein (Bruno et al., 1997, 1998). Stimulation of mGluR II receptors induces a severalfold increase of TGF-b release from glial cultures, but it is unknown whether EGF transactivation is involved in this release (Bruno et al., 1998). It has, however, been clearly demonstrated in cultured astrocytes that the activity of mGluR5 is associated with EGF transactivation (Peavy et al., 2001). Like the a2-adrenoceptor-induced EGF transactivation in transfected COS-7 cells, phosphorylation of EGFR by mGluR5 activity in astrocytes is dependent on Src family tyrosine kinase, but unlike a2-adrenergic transactivation in COS-7 cells, it is not dependent on G-protein beta,gamma subunits. Thus the transactivation pathway in the donor cells is either cell type-specific or

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GCPR-specific. The EGF transactivation in astrocytes by mGluR5 agonists may be responsible for activity-induced upregulation of EGF, EGFR and mGluR in these cells. It will be very interesting to establish definitively whether activation of mGluR5 actually stimulates HB-EGF shedding from astrocytes and whether the transactivation pathways in astrocytes are GCPR-specific. 4.4. Thrombin Thrombin is a serine proteinase that at low doses has been reported to have protective effect in brain injury. It binds to a GPCR called protease-activated receptor (PAR). Astrocytes express three subtypes of thrombin receptor, PAR-1, PAR-2, and PAR-4 (Wang et al., 2002). Stimulation with thrombin induces an increase of intracellular calcium concentration in astrocytes (Ubl and Reiser, 1997), and enhances PKC activity in rat glioma C6 cells (Kaufmann et al., 1996). In 1321 astrocytoma cells, thrombin increases Ras and MAPK activation (Post et al., 1996). Not surprisingly, it was shown that thrombin stimulates EGF transactivation by EGFR phosphorylation in astrocytes (Daub et al., 1997). There is no information whether thrombin induces HB-EGF shedding in astrocytes, but it is known to be the case in smooth muscle cells (SMCs) (Kalmes et al., 2000). 4.5. ATP ATP functions both as a trophic factor and a transmitter (see chapter by Hansson and Ro¨nnba¨ck), and ATP-induced EGF transactivation has been demonstrated in primary cultures of astrocytes (Daub et al., 1997). Astrocytes express both ionotropic (P2X) and metabotropic (P2Y) receptors for purine nucleotides. There are seven subtypes of P2X receptors. With specific antibodies, astrocytes were found to stain for P2X1 – P2X4, P2X6, and P2X7 receptors, but not for P2X5 (Kukley et al., 2001). ATP induces a significant increase of intracellular Ca2þ concentration in astrocytes by both influx of extracellular Ca2þ and G-protein-mediated Ca2þ release from intracellular stores (Shiga et al., 2001). P2Y receptors are GPCR. Stimulation of P2Y receptor in astrocytes leads to an IP3induced Ca2þ release from intracellular Ca2þ stores, PKC activation, ERK phosphorylation and cell proliferation (Neary et al., 1999; Lenz et al., 2000). Interestingly, P2X7 seems also to be involved in MAPK activation in astrocytes (Panenka et al., 2001). Also, extracellular ATP stimulates the release of heparin-binding epidermal and platelet-derived growth factors from cultured Mu¨ller cells (see chapter by Bringmann et al.). 4.6. Other transmitters Some GPCRs, that have been demonstrated on astrocytes in the brain in vivo, have been shown to stimulate EGF transactivation in other tissues and cells, but have not been tested in astrocytes. The b2-adrenergic receptor, the angiotensin II receptor and the estrogen receptor may be of particular interest. DNA synthesis induced by b2-adrenergic receptor activity in cardiac fibroblasts is dependent on EGF transactivation (Kim et al., 2002). Since the b2-adrenergic receptor traditionally is believed to be exclusively linked to Gs

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protein, its activation should not be expected to lead to EGF transactivation. However, b2-adrenergic linkage to Gi protein has recently been reported in cardiac myocytes, where PKA-mediated phosphorylation of the b2-adrenergic receptor regulates its coupling to Gs and Gi, respectively (Zamah et al., 2002). The angiotensin II receptor is coupled to Gq protein, and mediates IP3-induced Ca2þ release from intracellular Ca2þ stores. Stimulation of protein synthesis by angiotensin II in vascular SMCs is mediated by EGF transactivation (Voisin et al., 2002). Furthermore, angiotensin II-induced EGF transactivation in vascular SMCs was inhibited by a metalloprotease inhibitor, indicating the involvement of EGF shedding, whereas the concomitant increase of intracellular Ca2þ concentration was unaffected (Saito et al., 2002). Even more relevant in the present context, in glomerular mesangial cells, EGF transactivation might be necessary for angiotensin II-stimulated TGF-b release (Uchiyama-Tanaka et al., 2001), a phenomenon which might be similar to that observed in astrocytes stimulated with a group II mGluR agonist. Estrogen increases cAMP and IP3, elevates free intracellular Ca2þ concentration, causes MAPK phosphorylation and stimulates EGF transactivation in normal and cancer breast cell lines via a GPCR (Filardo, 2002; Filardo et al., 2002). In astrocytes, estrogen induces extension of processes and expression of glial fibrillary acidic protein (Garcia-Segura et al., 1989). It has also been suggested that estrogen is involved in astrocyte-directed neuronal plasticity (Garcia-Segura et al., 1999) and that it may be neuroprotective (Dhandapani and Brann, 2002). An estrogen-producing enzyme, aromatase is induced in astrocytes after brain injury (see also chapter by Melcangi et al.). It is an important question whether or not these effects of estrogen are mediated by a typical steroid receptor or whether they are unrelated to the conventional effects of estrogen. 5. Cytoprotective effect of HB-EGF HB-EGF shedded from the cell surface and released into the extracellular space may exert both autocrine and paracrine cytoprotective effects. It is protective against apoptosis and necrosis in intestinal epithelial cells (Michalsky et al., 2001) and against intestinal ischemia/reperfusion injury, probably by down-regulation of cytokine-induced nitric oxide synthase (Lara-Marquez et al., 2002) and enhancement of hexokinase expression (Bryson et al., 2002). In spite of the up-regulation of HB-EGF gene expression after brain injury, relatively little is presently known about cytoprotective effects of HB-EGF in the CNS. In cultured hippocampal neurons, pretreatment of cells with HB-EGF prevents kainate-induced cell damage without affecting intracellular Ca2þ concentration (Opanashuk et al., 1999). As previously mentioned, its cytoprotective effect on cultured dopaminergic neurons seems to depend on astrocytes (Farkas and Krieglstein, 2002). It is also well established that EGFR activity induced by TGF-a and EGF protects neurons from injury (Staecker et al., 1997; Maiese and Boccone, 1995). 6. Concluding remarks Our understanding of the astrocytic signaling pathways involved in dexmedetomidineinduced neuronal rescue by transactivation in astrocytes is, at best, sketchy, and to a large

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extent based on information about pathways stimulated by a2-adrenergic agonists in other cell types or about the effects of other GPCR agonists in astrocytes. It is known that stimulation of the Gi/o-coupled a2A-adrenergic receptors in astrocytes leads to hydrolysis of PIP2, triggered by GTP binding to alpha units of the G-protein, and a resulting increase in [Ca2þ]i and IP3, and presumably also to stimulation of PKC. In other cells expressing a2A-adrenergic receptors naturally or after transfection, both an increase in [Ca2þ]i and stimulation of PKC activity have been correlated with phosphorylation of EGFR and/or release of HB-EGF. However, at least in transfected COS-7 cell, shedding of HB-EGF may be more directly mediated by the cytosolic tyrosine kinase Src following its activation mediated by G-protein beta,gamma subunits. Since the signaling pathway(s) may be GPCR and/or cell type-specific, they must be established in astrocytes. What is known, is that dexmedetomidine-induced stimulation in astrocytes causes release of growth factor(s), capable of causing tyrphostin AG1478-sensitive ERK1/2 activation and that the effect is at least partly neutralized by heparin. It is likely that HB-EGF is the only or major growth factor involved, but no attempt has been made to identify the released growth factor(s), which constitutes a high-priority goal. It has been assumed in this review that neuroprotection by released HB-EGF is exerted by paracrine stimulation of neurons, but it cannot be excluded that another autocrine loop exists, in which an action of HB-EGF on astrocytes leads to release of additional growth factor(s), which then may act on neurons. This might explain the apparent involvement of additional growth factors during exposure of retina to a2-adrenergic agonists. Also, in spite of an almost unanimous conclusion that dexmedetomidine has neuroprotective capability in the brain in vivo, no attempts have been made to clarify the participating signaling pathways in culture preparations highly enriched in neurons. Such cultures are readily available and would allow verification of the involvement of Ras– Raf and MEK in the phosphorylation of ERK1/2 and also examination of the possible importance of PI3K and IP3 in neuroprotection and the transduction processes linking PI3K activation to HBEGFR stimulation. The neuroprotective role of a2-adrenergic agonists in the retina seems on the verge of becoming of major importance in the treatment of glaucoma. Being a chronic, slowly developing condition, this is a different situation than ischemic brain damage, with a much broader window of opportunity for drug intervention. It is possible, but yet unproven, that the a2-adrenergic agonists may act in part by exerting a Mu¨ller cell-mediated neuroprotection of neuronal elements in the retina. Confirmation whether transactivation is, indeed, involved in retinal neuroprotection by a2-adrenergic agonists is urgently needed.

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Roles of glia cells in cholesterol homeostasis in the brain Jin-ichi Ito and Shinji Yokoyama* Biochemistry, Cell Biology and Metabolism, Graduate School of Medical Sciences, Nagoya City University, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan * Correspondence address: E-mail: [email protected]

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction Cholesterol in brain Lipoproteins and their receptors in brain ApoE synthesis and secretion by astrocytes Production of HDL by astrocytes Brain injury and apoE production Concluding remarks: glia and cholesterol homeostasis in the brain

Abbreviations apo: apolipoprotein; HDL: high density lipoprotein; LDL: low density lipoprotein; BBB: blood – brain barrier; CSF: cerebrospinal fluid; CNS: central nervous system; SR-B1, scavenger receptor B1; ABCA1: ATP binding cassette transporter protein A1; VLDL: very low density lipoprotein; LRP: LDL receptor-related protein; FGF: fibroblast growth factor Astrocytes generate high density lipoprotein (HDL) with apolipoprotein (apo) E and apoJ synthesized by astrocytes themselves and with other, exogenous, apolipoproteins, such as apoA-I. These HDLs are thought to function in the transport of cholesterol between brain cells, whether to supply cholesterol when it is needed, such as during recovery from damage, or to remove it from the cells for its homeostasis. The physiological importance and many pathophysiological roles of brain HDL are discussed in this chapter. 1. Introduction The brain is a cholesterol-rich organ, and increasing evidence suggests that cholesterol plays a number of key roles in the central nervous system (CNS) through regulation of membrane functions and by other mechanisms. What makes cholesterol metabolism in the Advances in Molecular and Cell Biology, Vol. 31, pages 519–534 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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brain special is that brain is an autonomous organ with respect to cholesterol homeostasis, since the blood – brain barrier prevents lipoproteins in circulating plasma from entering the CNS. Therefore, a unique system for cholesterol metabolism is found in the brain, operating through high density lipoprotein (HDL), generated from endogenously synthesized apolipoproteins E and J (apoE and apoJ) by astrocytes and microglia. Brain cells can also react with extracellular apoA-I from unknown sources. ApoE –HDL reactivity increases in the brain in response to acute and chronic injury of the nervous system, which is believed to play a role in brain recovery by supporting neurite outgrowth and synapse formation. Since glia cells are the main source of brain HDL, they play a key role in the maintenance of brain structure and function by cholesterol-dependent mechanisms.

2. Cholesterol in brain Cholesterol is an important lipid component of the plasma membrane. While its hydroxyl group is believed to be localized close to the hydrophilic head groups of phospholipid molecules, its steroid backbone with a flat and rigid structure fills a potential space among the hydrocarbon chains of the phospholipids, regulating their mobility and accordingly a physicochemical property of the membrane. In addition, cholesterol preferably interacts with sphingomyelin molecules to form cholesterol/sphingomyelinrich domains in the plasma membrane (rafts or caveolae) (Radhakrishnan et al., 2000), which offer a special environment for accumulation of specific proteins involved in many specific membrane functions, including signal transduction and intercellular interaction (Schnitzer et al., 1995; Shin and Abraham, 2001). Because cholesterol plays such key roles in regulating membrane functions, it is essential for cells to maintain its homeostasis. Approximately 25% of the total cholesterol in the human body is found in the brain, where it accounts for 15% of the dry weight (Anonymous, 1981). Cholesterol is abundant in the plasma membranes of neurons and astrocytes, and myelin membranes also contain large amounts of cholesterol. Suppression of cholesterol synthesis in cultured neurons significantly lowers their viability and reduces axonal growth, indicating a critical dependence on cholesterol supply (de Chaves et al., 1997; Michikawa and Yanagisawa, 1999). A raft-like domain, rich in cholesterol –sphingomyelin, appears to be present in neurons (Gorodinsky et al., 1994; Gorodinsky and Harris, 1995; Maekawa et al., 1999; Masserini et al., 1999), and to be a preferred localization for tyrosine kinase receptors (Wu et al., 1997). Also, AMPA-type glutamate receptor subunits can be recovered in a low density, Triton-insoluble fraction of rat synaptic membranes, which is considered to represent dendritic rafts (Suzuki et al., 2001). These findings indicate that neurotransmitter-mediated signal transduction at least partly takes place in raft-like domains, and that cholesterol accordingly is important for neuronal responsiveness to neurotransmitters (Scanlon et al., 2001). It has also recently been reported that formation of synapses, development of synaptic vesicles and synthesis of appropriate amounts of synapsin I and synaptophysin are dependent on supply of cholesterol complexed to apoE-containing HDL (Mauch et al., 2001). Therefore, cholesterol and its transport system are essential for the

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very fundamental functions of the CNS (Barres and Smith, 2001; Lang et al., 2001; Mauch et al., 2001; Nagler et al., 2001; Ullian et al., 2001). All somatic cells synthesize cholesterol, and they are also capable of incorporating it by uptake of cholesterol-containing low density lipoprotein (LDL) through the LDL receptor. In contrast, cholesterol is not metabolized in most somatic cells, except for limited partial hydroxylation at a few locations, and its major catabolic sites are the liver and steroidogenic cells. Therefore, peripheral cells require a cholesterol export system for their cholesterol homeostasis. Such a system is also required for whole-body cholesterol homeostasis, as cholesterol has to be transported to its catabolic sites. HDL is believed to play a central role in this transport system (Yokoyama, 2000). The brain is segregated from the systemic circulation by the blood –brain barrier (BBB), and brain cells can accordingly not utilize the extracellular cholesterol transport system by plasma lipoprotein. Therefore, cholesterol homeostasis in the brain is thought to depend largely on endogenous biosynthesis within the brain (Dietschy and Turley, 2001). Intercellular transport of cholesterol in the brain also depends on its own lipoprotein system. HDL is the only lipoprotein identified in cerebrospinal fluid (CSF) for this function, and it contains apolipoproteins E, A-I, D and J (Roheim et al., 1979; Pitas et al., 1987a,b; Borghini et al., 1995; Koch et al., 2001). ApoE and J are synthesized in astrocytes and microglia and are secreted as HDL (Boyles et al., 1985; Pitas et al., 1987a,b; Poirier et al., 1991; Krul and Tang, 1992; LaDu et al., 1998; DeMattos et al., 2001). The source of apoA-I is not certain, but it is also found in HDL in the brain (Weiler-Guttler et al., 1990; Mockel et al., 1994). Clearance of cholesterol from the CNS is not well understood. A part of cholesterol in the brain appears to be catabolized to 24S-hydroxycholesterol by cholesterol 24hydroxylase localized in microsomes, at least in the neurons (Bjorkhem et al., 1997, 1998; Lund et al., 1999), but it is unclear how much brain cholesterol is catabolized by this pathway. It is also not known how this cholesterol metabolite is transported out from the CNS. A very small portion of cholesterol can be used for the synthesis of steroid hormones, but this amount is negligible compared to the turnover rate of cholesterol in brain. Thus, the major pathway for cholesterol clearance from CNS may be transport by HDL that carry cholesterol from CSF to the systemic circulation. The turnover of cholesterol in human brain is estimated to be 0.02% per day, much slower than that of 0.4% per day in the rat (Dietschy and Turley, 2001). It has been estimated that 2/3 of the brain cholesterol are catabolized to 24S-hydroxycholesterol in the rat brain (Bjorkhem et al., 1998).

3. Lipoproteins and their receptors in brain The delivery and removal of cholesterol to and from cells in the periphery are mainly mediated by lipoproteins in the systemic circulation. However, it is very unlikely that CNS cells could directly exchange cholesterol with plasma lipoproteins, since brain cells are isolated by the blood – brain barrier from the macromolecular components of plasma. Therefore, there must be an autonomous, CNS-specific mechanism for intercellular

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cholesterol transport and regulation of cholesterol homeostasis among neural cells. The main apolipoproteins in CSF are apoE and apoA-I, which are present in CSF as HDL-like particles, suggesting that these two apolipoproteins play central roles in mediating intercellular cholesterol transport in the brain. There have been reports that plasma LDL undergoes transcytosis in capillary endothelial cells and thereby crosses the BBB to enter the brain, and that astrocytes regulate the transcytosis of LDL by secreting some trophic factor (Dehouck et al., 1994, 1997). However, neither LDL nor apoB have been detected in CSF (Koch et al., 2001). Therefore, even though LDL may be able to cross the BBB, it may immediately be incorporated and processed by astrocytes that surround endothelial cells at the BBB. Nevertheless, patients suffering from abetalipoproteinemia (apoBlipoprotein assembly deficiency due to a genetic defect of microsomal triglyceride transfer protein) show severe congenital CNS symptoms (Kane and Havel, 2001), suggesting that apoB-lipoproteins are required for CNS development before the BBB is established, perhaps for cholesterol supply. Although there is one report that apoA-I in CNS is produced by the brain capillary endothelial cells (Mockel et al., 1994), it is unclear whether this production is significant and whether apoA-I is secreted by the endothelial cells directly into brain parenchyma. On the other hand, some authors maintain that the apoA-I synthesized and secreted by the liver or the intestine is transported into the brain through the BBB (Ritas, 1997). In contrast to the ambiguity of the source of apoA-I in CSF, apoE has clearly been shown to be produced in the brain, mainly by astrocytes (Boyles et al., 1985; Pitas et al., 1987a,b; Poirier et al., 1991; Krul and Tang, 1992; Patel et al., 1995; LaDu et al., 1998; Fagan et al., 1999; Fujita et al., 1999; Ito et al., 1999; Xu et al., 2000; DeMattos et al., 2001, Ueno et al., 2002) and also by microglia (Nakai et al., 1996; Stone et al., 1997; Xu et al., 2000). Accordingly, the phenotype of human brain apoE does not change after liver transplantation, proving that brain apoE is indeed produced in the CNS and functions as an intercellular cholesterol transporter in the CNS (Linton et al., 1991). Astrocytes synthesize and secrete apoE along with the cellular cholesterol and phospholipid as cholesterol-rich HDLs. On the other hand, the cells also depend upon extracellular apoAI to generate HDLs (Ito et al., 1999), but these HDLs have a much lower content of cholesterol (Fig. 1, Table 1). Thus, apoE –HDL and apoA-I –HDL in the brain may be produced by slightly different mechanisms and carry out different functions in intercellular lipid transport in CNS. Interactions of apoA-I/HDL with cells are mediated by cellular proteins. Scavenger receptor type-B1 (SR-B1) binds apoA-I/HDL particles and mediates selective uptake of its cholesteryl ester (Acton et al., 1996). Cubilin has been shown to be the HDL binding protein necessary for the uptake of HDL particles or of free apoA-I (Hammad et al., 1999; Kozyraki et al., 1999). The gene encoding ATP-binding cassette transporter protein A-1 (ABCA1) has been identified as a causative gene for HDL deficiency (Bodzioch et al., 1999; Brooks-Wilson et al., 1999; Rust et al., 1999). Its interaction with apolipoprotein for generation of HDL from cellular lipid is under intense investigation by many laboratories (Oram et al., 2000; Chambenoit et al., 2001; Fitzgerald et al., 2002) including our own (Abe-Dohmae et al., 2000; Arakawa and Yokoyama, 2002). SR-B1 (Posse De Chaves et al., 2000) and ABCA1 (Fukumoto et al., 2003) seem to be present in the brain, but there are no data for cubilin, and no

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Fig. 1. Density gradient ultracentrifugation analysis of the culture medium of astrocytes after incubation with apoA-I. The cells in two 3 cm plates (150 ^ 17 mg protein per plate) were incubated with and without apoA-I, 5 mg/1 mL medium, and the medium was analyzed by sucrose density gradient ultracentrifugation. Preloaded radioactivity in cholesterol and phosphatidylcholine was determined for each of twelve fractions from the bottom to the top, and mass of each lipid was calculated from the specific radioactivity of the respective lipid in the cells compared to that in the precursor. Open circles represent phosphatidylcholine and open squares represent cholesterol in each fraction. The thin solid lines indicate density of each fraction. Taken from Ito et al. (1999), with permission.

brain-specific HDL-binding protein has so far been identified. On the other hand, four types of apoE-binding proteins have been identified in the brain: (i) LDL receptor; (ii) very low density lipoprotein (VLDL) receptor; (iii) LDL receptor-related protein (LRP); and (iv) apoE receptor-2 (Pitas et al., 1987a,b; Bu et al., 1994; Oka et al., 1994; Kim et al., 1996). All of these apoE-binding proteins are found in neurons. LDL receptor, VLDL receptor and LRP are found also in astrocytes, but expression of apoE receptor-2 has not been confirmed in astrocytes (Nimpf and Schneider, 2000; Herz, 2001; Herz and

Table 1 Lipid composition of prebeta-HDL-like particles generated by astrocytes. From Ito et al. (1999), with permission Exogenous apolipoprotein Cholesterol (pmol/3 mL) Phosphatidylcholine (pmol/3 mL) Chol/PCa (mol/mol) None ApoA-I Increment

1226 1339 113

823 1235 412

1.49 (1.08) 0.27

The cells in two 3 cm plates (150 mg protein per plate) were incubated with and without apoA-I, 5 mg/mL medium, and the medium was analyzed for its lipid composition. Preloaded radioactivity in cholesterol and phosphatidylcholine was determined and mass of each lipid was calculated from the specific radioactivity of the respective lipid in the cells compared to that in the precursor. ‘Increment’ is the difference between the control (without apoA-I) and the sample with apoA-I. a Molar ratio of cholesterol/phosphatidylcholine.

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Strickland, 2001; Riddell et al., 2001). Recently, Trommsdorff et al. (1999) reported that double deficiency of VLDL receptor and apoE receptor-2 results in reeler/disabled-like disruption of neuronal migration, suggesting that these receptors may also play a role in intracellular signaling.

4. ApoE synthesis and secretion by astrocytes Human apoE is a glycoprotein composed of 299 amino acids with a molecular weight of 37 kDa. While the main source of plasma apoE seems to be the liver, it is also produced by various types of cells such as macrophages and steroidogenic cells. In CNS, apoE is mainly produced and secreted by astrocytes (Boyles et al., 1985; Pitas et al., 1987a,b; Poirier et al., 1991; Krul and Tang, 1992; Patel et al., 1995; LaDu et al., 1998; Fagan et al., 1999; Fujita et al., 1999; Ito et al., 1999; Xu et al., 2000; DeMattos et al., 2001; Ueno et al., 2002), but microglial cells are also capable of doing so (Nakai et al., 1996; Stone et al., 1997; Xu et al., 2000). When apoE is secreted from astrocytes, most of it forms HDL-like particles with diameters of 10– 17 nm with cholesterol and phospholipid (Pitas et al., 1987a,b; Borghini et al., 1995; Shanmugaratnam et al., 1997; LaDu et al., 1998; Ito et al., 1999; Ueno et al., 2002). Astrocytes also secrete apoJ as HDL but in a slightly different form with smaller diameters of 7.5 –12 nm (Fagan et al., 1999). ApoE synthesis and secretion, presumably as HDL, increase as the cells differentiate (Zhang et al., 2000). They also increase during brain development and in the brain recovering from injury, whether acute or chronic (Dawson et al., 1986; Ignatius et al., 1986; Snipes et al., 1986; Mahley, 1988; Boyles et al., 1989; Harel et al., 1989; Popko et al., 1993; Goodrum et al., 1995). It is therefore believed that apoE plays an important role either in the recovery of cholesterol from damaged cell debris or in the supply of cholesterol to regenerating cells, especially neurons. However, the mechanism for upregulation of apoE synthesis and secretion by astrocytes has not been established. We recently reported that apoE secretion by astrocytes is regulated by one or more trophic factor(s) produced in the brain (Ueno et al., 2002). When rat fetal brain astrocytes were grown in primary cultures for 4 weeks followed by 1 week of culturing as secondary cultures, the cells had a lower cholesterol content than corresponding cells prepared by our conventional method of culturing for 1 week as secondary cultures after only 1 week in primary cultures. The older cells synthesized and secreted apoE and cholesterol as HDL more actively, and their HMG-CoA reductase activity was upregulated. Conditioned medium from the cells which had been grown for 4 weeks in primary cultures stimulated astrocytes prepared by our conventional method to increase their synthesis and secretion of apoE and cholesterol. This effect could be reproduced by fibroblast growth factor-1 (FGF-1) (acidic FGF), but not by any other cytokine examined, including FGF-2, and anti-FGF-1 antibody canceled the stimulatory activity of the conditioned medium (Fig. 2). The fetal rat brain astrocytes expressed large amounts of FGF-1 mRNA after 3 weeks in primary culture. These experimental results showed that the FGF-1-like factor is secreted by rat fetal brain cells during long-term primary culture and stimulates production of HDL in astrocytes.

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Fig. 2. Enhancement of cholesterol release (a, b) and apoE secretion (c) from conventionally prepared rat astrocytes by conditioned medium from one-month-old primary culture (M-CM) and inhibition of the enhancement by an anti-FGF-1 antibody, but not by an Anti-apoE antibody. For the antibody experiments the MCM was treated with a goat anti-human aFGF antibody (Santa Cruz Biotech. Inc.) or a rabbit anti-rat apoE antibody conjugated on protein G-Sepharose (Amersham Pharmacia Biotech.) for 4 h at room temperature, and the gels were removed by centrifugation. W/W cells were incubated for 5 days with 0.5 mL/mL of M-CM, which had been pretreated with either antibody or 0.02% BSA/F-10 as a control. Before the experiment the cells had been labeled with 30 mCi/mL of (3H)-acetate for 12 h, washed 3 times with DPBS and incubated in 0.02% BSA/F-10 containing 1 mM acetate for a further 12 h period. The release of newly synthesized cholesterol (from (3H)-acetate) into the medium was determined by counting of the radioactivity in cholesterol. The results were expressed as percentage of labeled cholesterol in total cellular cholesterol in the well (a) and as the radioactivity per mg cell protein (b). Each value represents the average and standard error of triplicate experiments, and ** and *** indicate p , 0:05 and 0.01 from control in the same panel. (c) Stimulation of apoE secretion from rat astrocytes by M-CM, and its inhibition by an anti-aFGF antibody. Rat astrocytes were incubated for 5 days in the fresh 0.02% BSA/F-10 medium containing the indicated conditioned medium. The cells were washed and incubated for further 24 h, and the conditioned media were analyzed by immunoblotting. The gels represent one of a total of three independent experiments. Taken from Ueno et al. (2002), with permission.

The primary cultures that highly express FGF-1 mRNA after 3 weeks in culture, consisted almost exclusively of astrocytes and neurons could hardly be identified. This finding indicates that FGF-1 is produced by astrocytes and stimulates astrocytes in an autocrine manner. Epidermal growth factor is also known to stimulate apoE secretion (Baskin et al., 1997). It reportedly increases apoA-I expression through the Ras-MAP kinase cascade and Sp1 (Zheng et al., 2001). As FGF-1 is known to induce signaling through P21ras/Erk cascade in astrocytes (Asada et al., 1999), it should be investigated if the mechanism for its upregulation of apoE – HDL production involves stimulation of this intracellular signaling pathway. FGF-1 has no signal peptide, so the pathway for its secretion is unknown at present. FGF-1-transfected cells release FGF-1 into the culture medium only during heat-shock conditions (Jackson et al., 1992). FGF-9 produced in the brain and kidney is also known to be secreted, although it lacks a signal peptide, and it is assumed to be secreted via the Golgi apparatus, as it is N-glycosylated (Miyamoto et al., 1993). Thus, specific mechanism(s) may exist for such cytokines to be secreted without any signal peptides.

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5. Production of HDL by astrocytes Astrocytes produce cholesterol-rich HDL with endogenous apoE and generate cholesterol-poor HDL with exogenous apolipoproteins such as apoA-I (Ito et al., 1999) (Fig. 1, Table 1). It is unknown whether these two types of HDL have different functions. Cholesterol-poor HDL or small HDL with apoJ may have a higher capacity to accumulate additional cholesterol and may therefore rather accept cholesterol from other cells, while cholesterol-rich HDL can deliver cholesterol to target cells via apoE-recognizing receptors. It would be of importance to investigate the cholesterol transport network by various HDLs among the CNS cells in normal cholesterol homeostasis and during pathophysiological conditions. Exogenous apoA-I removes less cholesterol and produces cholesterol-poor HDL. However, the cholesterol content in apoA-I – HDL in cultured astrocytes can be increased by digestion of cellular sphingomyelin by extracellular sphingomyelinase (SMase) (Fig. 3), indicating that the mobility of the cholesterol molecule in the cells is restricted by sphingomyelin. This observation suggests that sphingomyelin/cholesterol-rich domains of the cell surface are likely to be used as a source of cholesterol for HDL assembly

Fig. 3. Effect of sphingomyelinase (SMase) treatment on the cholesterol release from the rat astrocytes mediated by apolipoprotein. The astrocytes were cultured in the 3 cm culture plates. LDL labeled with [3H]cholesteryl oleate was added to the cells in fresh 0.1% BSA/F-10 tissue culture medium, followed by incubation at 37 8C for 24 h. After washing with cell buffer and replacement with 0.1% BSA/F-10 medium, the cells were treated with 100 mU SMase (SMase (100 mU)) or left untreated (SMase (2)) for 1 h, and incubated in the presence or absence of apoA-I or apoE at the indicated concentration for another 8 h. Lipid was extracted with organic solvent from the cells and the conditioned medium and analyzed after separation by TLC. Each value represents the average and standard error (if larger than the symbol) of triplicate measurements. In panel A, the single asterisk indicates a significant difference from the respective control in the absence of any apolipoprotein ðp , 0:01Þ; and the double asterisk indicates a difference from the respective SMase (2) ðp , 0:01Þ: Modified from Ito et al. (2000).

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(Ito et al., 2000, 2002, 2003). Although apoA-I does not remove much cholesterol (Table 1), it induces translocation of newly synthesized cholesterol, phospholipid and caveolin-1 to the cytosol in rat astrocytes prior to the appearance of these lipids in HDL in the medium (Ito et al., 2002, 2003). The lipids and caveolin-1 translocated to the cytosol can be recovered as lipid – protein complex particles along with cyclophilin A and are found within the typical density range of HDL. Cyclosporin A, a cyclophilin A-specific binding agent, inhibited both the translocation and the apoA-I-mediated cholesterol release. Thus, at least in astrocytes, cyclophilin A may contribute to the intracellular response to apoA-I that occurs in association with the apoA-I-mediated cholesterol release. This finding is consistent with our previous observation that down-regulation of caveolin-1 resulted in the decrease in cholesterol content in the HDL generated by apoA-I in THP-1 cells (Arakawa et al., 2000). Caveolin-1 is generally thought to play an important role for the intracellular cholesterol trafficking (Fielding and Fielding, 1997). Our findings indicated that caveolin-1 participates in the intracellular cholesterol trafficking linked to the apolipoprotein-mediated generation of HDL in astrocytes. The mechanism(s) triggering such a cholesterol trafficking system is unknown, but the involvement of protein kinase C has been implicated (Li and Yokoyama, 1995; Li et al., 1997). After removal of sphingomyelin from the cells by SMase it is rapidly replenished by transfer of phosphorylcholine from phorphatidylcholine to ceramide, a process, which generates diacylglyceride as a potential signal initiator (Ito et al., 2002, 2003). ABCA1 has been shown to play a key role in the cell – apolipoprotein interaction and subsequent generation of HDL. Mutations of ABCA1 have been shown to result in extremely low plasma HDL levels and lack of apolipoprotein-mediated HDL production in familial HDL deficiency patients, such as Tangier disease (Bodzioch et al., 1999; Brooks-Wilson et al., 1999; Rust et al., 1999) and in ABCA1 knock-out mice (Christiansen-Weber et al., 2000), indicating that the apolipoprotein – cell interaction is the major source of the production of plasma HDL. It is unclear whether ABCA1 mediates production of HDL in CNS cells or whether there is any back-up system for ABCA1 in the CNS (Dean et al., 2001). Despite the extremely low plasma HDL level in the homozygotes with Tangier disease, their risk for atherosclerotic disease is not increased as much as the homozygotes of familial hypercholesterolemia, who have three to five times increased plasma LDL level and almost 100% risk for coronary heart disease by their second decade of life. Also, there is no clear indication that patients with Tangier disease exhibit any CNS symptoms though some patients exhibit peripheral neuropathy (Assmann et al., 2001). It is necessary to analyze the function of ABCA1 and other proteins that may function in generation of HDL in the brain.

6. Brain injury and apoE production There are many reports that the production and secretion of apoE are stimulated in CNS and peripheral nerves after injury. Twenty years ago Skene and Shooter (1983) observed an increase of the production of a 37 kDa protein after injury of the sciatic nerve. The protein was identified as apoE in 1986 (Ignatius et al., 1986; Snipes et al., 1986). Increase of the LDL receptor was also observed in the cells around the injury of the sciatic nerve.

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Since then, numerous papers have been published about the relation between apoE production and nerve injury, including acute and chronic brain damage, such as cerebrovascular infarction and degenerative brain diseases. They indicate a role of apoE in intercellular cholesterol transport in nerve reconstruction and remyelination after the damage (Dawson et al., 1986; Ignatius et al., 1986; Snipes et al., 1986; Mahley, 1988; Boyles et al., 1989; Harel et al., 1989; Popko et al., 1993; Goodrum et al., 1995). As discussed above, the mechanism for upregulation of apoE synthesis in astrocytes is not clear. There are several reports indicating an increase in transcription and translation of FGF superfamily members after injury in the nervous system (Barotte et al., 1989; Tooyama et al., 1991; Eckenstein et al., 1994; Bugra and Hicks, 1997; Yoshimura et al., 2001). Thus hippocampal administration of kainic acid to adult mice induces production of FGF-2 (basic FGF) and increases the density of BrdUrd/NeuN-positive cells (dividing progenitor cells differentiating into neurons) (Yoshimura et al., 2001). This response is attenuated in the FGF-2-deficient mouse, but it can be restored by vector-mediated delivery of FGF-2 to the hippocampus. The choline acetyltransferase activity after brain injury is also enhanced by the microinjection of FGF-2 (Barotte et al., 1989). Our in vitro observation that FGF-1 stimulates apoE –HDL production by a presumably autocrine mechanism (Ueno et al., 2002) suggests the possibility that this growth factor also plays an important role in the recovery of brain injury and therefore suggests a function of FGF-1 as a post-injury survival factor. There are three major isoproteins of human apoE: apoE2, E3 and E4, corresponding to gene alleles of e2, e3 and e4. It is a well established observation that e4 is a strong risk factor for Alzheimer’s disease (Corder et al., 1993; Poirier et al., 1993; Strittmatter et al., 1993a,b; Nathan et al., 1994; Roses et al., 1994), and for the deposition of amyloid protein after head injury (Nicoll et al., 1995; Jordan et al., 1997) or intracranial hemorrhage (Alberts et al., 1995). Since this relationship was discovered, the relationship between apoE and the pathogenesis of Alzheimer’s disease has been extensively studied from various viewpoints. Many approaches were made to characterize isoform specific apoE functions, such as different affinity for amyloid peptides (Strittmatter et al., 1993a,b; LaDu et al., 1994), anti-oxidative activity (Miyata and Smith, 1996), and neurite outgrowthstimulating activity (Fagan et al., 1996; DeMattos et al., 1998). In addition, changes of cellular cholesterol level appear to influence the production of amyloid 1– 40 and amyloid 1– 42 (Chochina et al., 2001; Fassbender et al., 2001).

7. Concluding remarks: glia and cholesterol homeostasis in the brain There is no doubt that astrocytes play a key role in cholesterol metabolism in the brain. This function can be attributed to their ability to synthesize apoE and perhaps apoJ, and thereby generate HDL with their own lipid. HDL produced in this manner is an intercellular carrier of cholesterol in the brain, and seems to function as the major cholesterol supplier for neurons. Efficient neurite growth and synapse formation require external supply of cholesterol. Before the BBB is established, plasma lipoproteins, perhaps mainly LDL, may supply neural cells with cholesterol (Kane and Havel, 2001). However, after the formation of the BBB, glial synthesis of HDL takes over. It is rational to postulate

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that HDL production by astrocytes is stimulated by increased needs of cholesterol, such as after acute and chronic neuronal damage. Our results indicate that apoE and cholesterol synthesis are upregulated by FGF-1, which is released from brain cells (Ueno et al., 2002). Astrocytes or related cells may sense one or more microenvironmental signal(s) to release FGF-1 in order to stimulate apoE – HDL production. It is also of interest that HDL produced by extracellular, rather than endogenous, apolipoprotein is relatively cholesterol-poor (Ito et al., 1999, 2000). Since such HDL particles are capable of accepting more cholesterol released from cells by diffusion, this HDL may function as an acceptor of cholesterol from neurons or cell debris after the damage. HDL is the only carrier of cholesterol to export it from the CNS via the flow of CSF to the systemic circulation. A remaining question is to what extent cholesterol catabolism contributes to 24S-hydroxycholesterol in human brain. Two issues must be addressed: (i) what portion of brain cholesterol is metabolized by this pathway; and (ii) how is 24Shydroxycholesterol transported out of CNS. Regarding the second question, 24Shydroxycholesterol has been reported to appear in HDL, when it is released from the cells (Babiker and Diczfalusy, 1998), but a recent report also indicated that it may be directly exported to the systemic circulation through endothelial cells (Panzenboeck et al., 2002). It is important to investigate regulation of HDL production in astrocytes in order to understand cholesterol homeostasis in the brain. This will lead to further understanding of the regulation of the function of the brain and its individual cell types, and of repair mechanisms following acute and chronic brain damage, and eventually assist in the development of technology to enhance regeneration.

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Non-neuronal cells in the nervous system: sources and targets of neuroactive steroids Roberto C. Melcangi,a In˜igo Azcoitia,b Mariarita Galbiati,a Valerio Magnaghi,a Daniel Garcia-Ovejeroc and Luis M. Garcia-Segurac,* a

Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy b Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, E-28040 Madrid, Spain c,p Instituto Cajal, CSIC, E-28002 Madrid, Spain E-mail: [email protected]

Contents 1. 2.

3.

4.

Introduction Steroid hormone synthesis and metabolism 2.1. Steroidogenesis by non-neuronal cells 2.2. Role of 5a-reductase and the 3a-hydroxysteroid dehydrogenase in glia steroidogenesis 2.3. Aromatase and the production of estradiol by astrocytes 2.4. Functional implications of glial steroidogenesis Non-neuronal cells as targets for steroids 3.1. Steroid hormone receptors in non-neuronal cells 3.2. Effects of steroids on brain endothelial cells 3.3. Effects of steroids on astroglia 3.4. Effects of steroids on microglia 3.5. Effects of steroids on oligodendrocytes and Schwann cells Concluding remarks

Non-neuronal cells synthethize and metabolize steroid hormones and produce local neuroactive steroids that exert neuromodulatory and neurotrophic actions under physiological and pathological conditions. In the central nervous system, the steroids produced by non-neuronal cells, such as pregnenolone, dehydroepiandrosterone (DHEA), testosterone, estradiol, progesterone and other steroid metabolites, regulate synaptic function, affect anxiety, cognition, sleep and behavior and exert neuroprotective and reparative roles. In the peripheral nervous system, progesterone and progesterone Advances in Molecular and Cell Biology, Vol. 31, pages 535–559 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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derivatives produced by Schwann cells, promote myelin formation and the remyelination and regeneration of injured nerves. Non-neuronal cells are also targets for steroids and mediate or participate in many of the actions of these substances in the nervous system. These include: (i) the regulation of blood – brain barrier and cerebrovascular permeability by actions of steroids on endothelial cells; (ii) the regulation of synaptogenesis, synaptic plasticity, neuritic growth and neuroendocrine secretion by actions of steroids on astroglia and (iii) the regulation of neuronal survival and regeneration by actions of steroids on microglia, astroglia and Schwann cells. 1. Introduction The concept that the brain is a target for steroid hormones has abundant experimental support. Steroid hormones regulate the development and function of the nervous system and affect mood, behavior and cognition. For many years, it was considered that actions of steroid hormones in the brain were restricted to specific areas involved in neuroendocrine regulation and the control of behavior. Today it has become evident that steroid hormones exert a broad spectrum of actions both in the central (CNS) and the peripheral nervous systems (PNS). Furthermore, it has been discovered that the nervous system is able to synthethize and metabolize steroid hormones and produce local steroids that affect neural function. Thus, two new concepts have emerged. One is defined by the term neurosteroids, introduced by the laboratory of Baulieu (1998). This term describes those steroids synthesized in the brain directly from cholesterol (which itself is synthesized from glucose within the brain (Morell and Jurevics, 1996)), and gives a name to the concept that nervous tissue is steroidogenic. The other useful concept is defined by the term neuroactive steroids, to comprise those steroids that are able to regulate neural function. It is obvious that these terms are partially overlapping, since several steroids produced by the brain are neuroactive. Neurosteroids and neuroactive steroids include sex steroids and sex steroid metabolites. Some of these steroids act as neuromodulators, regulating the function of ion channels and neurotransmitter receptors and affecting mood, behavior and cognition (Baulieu, 1998; Compagnone and Mellon, 2000; Melcangi and Panzica, 2001). Nonneuronal cells play a central role in the synthesis, metabolism and action of steroids in the nervous system. Steroids, acting on non-neuronal cells and/or produced by them, regulate neural development, neural function and the response of nervous tissue to injury. Therefore, steroids may be considered a new class of molecular signals, with neuromodulatory and neurotrophic actions, involved in the communication of nonneuronal and neuronal cells in the nervous system. An overview of the most recent advances in this recent and fascinating emerging field is provided in this chapter. 2. Steroid hormone synthesis and metabolism 2.1. Steroidogenesis by non-neuronal cells The capability to synthesize steroid hormones is not only a peculiarity of the classical steroidogenic tissues, like for instance the gonads and adrenal gland, but may be also

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Fig. 1. Biosynthesis of steroids in the nervous system. Cholesterol is converted to pregnenolone, catalyzed by the cytochrome P450 side-chain cleavage [P450 scc], and pregnenolone is converted to progesterone, catalyzed by the 3b-hydroxy-steroid dehydrogenase [3b-HSD]. Pregnenolone and progesterone can be further converted, via 17-hydroxy-pregnenolone and 17-hydroxy-progesterone, respectively, to dehydroepiandrosterone and androstenedione, catalyzed by the 17a-hydroxylase/C17-20-lyase [P450 c17], and from there to androstenediol and testosterone, respectively, catalyzed by17b-hydroxylase/C17-20-lyase [17b-HSD]). Androstenedione and testosterone, can be converted to estrone and estradiol, respectively, catalyzed by aromatase (P450 aro). In addition to these pathways, producing androgens and estrogens, progesterone and its derivative 17-hydroxyprogesterone can be converted to the glucocorticoids corticosterone and cortisol, respectively. Cortisol can be further converted to the glucocorticoid cortisone, catalyzed by 11b-hydroxysteroid dehydrogenase (11b-HSD), and corticosterone via 18-hydroxy-corticosterone to the mineralocorticoid aldosterone.

ascribed to the nervous system. Steroidogenesis (Fig. 1) seems to take place prevalently in the non-neuronal compartment. In fact, it has been demonstrated that in the CNS, astrocytes and oligodendrocytes express several enzymes involved in the steroidogenic process (e.g., the cytochrome P450 side-chain cleavage [P450 scc], the 17a-hydroxylase/ C17-20-lyase [P450 c17], the 3b-hydroxy-steroid dehydrogenase [3b-HSD], the 17b-

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hydroxylase/C17-20-lyase [17b-HSD]), and consequently are able to produce different kinds of steroids (Hu et al., 1987; Akwa et al., 1993; Kimoto et al., 1997; Mensah-Nyagan et al., 1999, 2001; Zwain and Yen, 1999a,b; Mellon et al., 2001). In particular, as shown by Zwain and Yen (1999a,b), astrocytes appear to be the most active steroidogenic cells in the brain, since cultures of these cells produce pregnenolone, progesterone, DHEA, androstenedione, testosterone and estradiol. On the contrary, oligodendrocytes seem to be able to form only pregnenolone and androstenedione. The steroidogenic activity of microglia has not been assessed. However, systemic macrophages that share many properties and a common origin with microglia, express aromatase and produce androgenic and estrogenic derivatives (Schmidt et al., 2000). The capability to synthesize steroids seems not to be a peculiarity of the CNS, since also the PNS is able to convert pregnenolone into progesterone; also in this case, this transformation occurs in the glial component, namely in the Schwann cells (Koenig et al., 1995; Guennoun et al., 1997; Schumacher et al., 2001). It is interesting to note that in Schwann cells the formation of progesterone, by 3b HSD activity, is neuronal dependent. As recently shown by Robert and co-workers (2001), the expression and activity of the 3b HSD present in Schwann cells cultured alone is very low; however, when these cells are cultured in contact with sensory neurons, both expression and activity of this steroidogenic enzyme is induced. 2.2. Role of 5a-reductase and the 3a-hydroxysteroid dehydrogenase in glia steroidogenesis As mentioned above, glial cells of the CNS and of the PNS possess several enzymes able to convert steroids into neuroactive steroids. In particular, glial cells are able to metabolize native steroid hormones into their 5a- and 3a-hydroxy-5a reduced derivatives via the enzymatic complex formed by the 5a-reductase (5a-R) and the 3a-hydroxysteroid dehydrogenase (3a-HSD) (Fig. 2) (for review, see Melcangi et al., 1999a, 2001b,c). This enzymatic complex is very versatile, since every steroid possessing the delta 4-3keto configuration may be first 5a-reduced and subsequently 3a-hydroxylated. In particular, testosterone can be converted into dihydrotestosterone and then into 5a-androstane-3a, 17b-diol (3a-diol), progesterone into dihydroprogesterone and subsequently into tetrahydroprogesterone, corticosterone into dihydrocorticosterone, and 11-deoxycorticosterone into dihydrodeoxycorticosterone and then into tetrahydrodeoxycorticosterone (Melcangi et al., 1999a, 2001c). The distribution of the 5a-R activity in the different cell types of the rat brain has been analyzed utilizing either testosterone or progesterone as substrates. In particular, the ability to metabolize testosterone was first studied in freshly isolated cell preparations (Melcangi et al., 1990); subsequent studies were performed in cultures of neurons, of the so-called type-1 astrocytes (the conventional astrocyte culture), of the so-called type-2 astrocytes, and of oligodendrocytes (Melcangi et al., 1993, 1994a). The two different groups of experiments have provided similar data. In particular, utilizing testosterone as substrate, it has been observed that fetal rat neurons possess significantly higher 5a-R activity than neonatal oligodendrocytes and astrocytes (Melcangi et al., 1993). Among glial cells, type 2 astrocytes possess a considerable 5a-R activity, while

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Fig. 2. Metabolism of testosterone and progesterone by the action of the enzymes 5a-reductase and 3a-hydroxysteroid-dehydrogenase (3a-HSD). In a similar manner the 5a-reductase converts corticosterone to dihydrocorticosterone (DHC) and 11-deoxycorticosterone to dihydrodeoxycorticosterone (DHDOC), and the 3a-HSD further converts DHDOC to tetrahydrodeoxycorticosterone (THDOC).

conventional astrocytes are almost devoid of such an activity. On the contrary, the enzyme 3a-HSD appears to be mainly localized in conventional astrocytes (Melcangi et al., 1993). Analogously, also when progesterone was used as substrate, neurons were shown to possess a significantly higher 5a-R activity than any glial cell analyzed. However, it is important to point out that, due to the higher affinity of the 5a-R for progesterone than for testosterone (Melcangi et al., 1999a, 2001c), the formation rate of dihydroprogesterone was about 2 times higher than that of dihydrotestosterone. Also in this case the 3a-HSD activity appeared predominantly concentrated in conventional astrocytes (Melcangi et al., 1994a). However, a consistent formation of tetrahydroprogesterone was also present in oligodendrocytes; the amounts of tetrahydroprogesterone measured in the cultures of these cells were significantly higher than those formed in cultures of type-2 astrocytes and neurons, but significantly lower than those found in conventional astrocytes (Melcangi et al., 1994a). In this context it is important to underline that Gago et al. (2001) have demonstrated that the formation of dihydroprogesterone in fully differentiated oligodendrocytes is 5-fold higher than in oligodendrocyte pre-progenitors and in oligodendrocyte progenitors. On the contrary, the formation of tetrahydroprogesterone is higher in oligodendrocyte pre-progenitors and decreases with oligodendrocyte differentiation (Gago et al., 2001). These findings underline that not only differentiated

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CNS cells possess the 5a-R/3a-HSD system, but that considerable enzymatic activities for conversion of steroid hormones are also present in undifferentiated cells, as shown for the first time on stem cells originating from the mouse striatum (Melcangi et al., 1996a). Interestingly, both the 5a-R activity and the 3a-HSD activity present in conventional astrocytes are stimulated by the simultaneous presence of neurons (Melcangi et al., 1994b), indicating a possible interaction of the two populations of cells in the metabolism of steroid hormones. The ability to convert steroid hormones into 5a- and 3a-hydroxy-5a-reduced derivatives is also present in glial cells of the PNS. Schwann cells possess both 5a-R and 3a-HSD activity (Melcangi et al., 1998, 1999b; Yokoi et al., 1998). In particular, the 5a-R activity present in these cells is at least four times higher than in oligodendrocytes, while the 3a-HSD activity is lower than in oligodendrocytes (Melcangi et al., 1998). 2.3. Aromatase and the production of estradiol by astrocytes The enzyme aromatase (P450 aro), that is able to convert androgens into estrogens, is not present in glial cells of the CNS of mammals under normal circumstances, since only neurons possess such an activity (Negri-Cesi et al., 1992). However, it has been recently demonstrated that rodent astrocytes isolated from the cerebral cortex of neonatal rats are able to produce estradiol and estrone (Zwain et al., 1997; Zwain and Yen, 1999a). It is possible that specific culture conditions may induce aromatase expression in astrocytes. Indeed, recent results (Azcoitia, unpublished) indicate that stressful conditions, such as serum deprivation, induce aromatase expression in cultured astrocytes. Furthermore, aromatase is expressed by astrocytes in the brain of birds and mammals after brain injury (Garcia-Segura et al., 1999b; Peterson et al., 2001), indicating that the enzyme may be induced de novo in these cells under specific circumstances. In contrast to mammals, aromatase is expressed in radial glia in the brain of teleost fish under normal conditions (Forlano et al., 2001). 2.4. Functional implications of glial steroidogenesis The steroids produced by glia may serve a paracrine role regulating synaptic function. These steroids modulate anxiety, cognition, sleep, ingestion, aggression, and reinforcement. Some of them are positive modulators of N-methyl-D -aspartate receptors and enhance cognitive performance. Other steroids produced by glia, such as tetrahydroprogesterone and terahydrocorticosterone, are highly selective and extremely potent modulators of the GABAA receptor, and they elicit marked anxiolytic and anti-stress effects and increase feeding and sleeping (Barbaccia et al., 2001; Engel and Grant, 2001; Lambert et al., 2001; Vallee et al., 2001). Steroids produced by glia may also exert a neuroprotective or reparative role. For instance, steroids produced by Schwann cells (progesterone and its derivatives) regulate the expression of myelin proteins and promote axonal regeneration (Koenig et al., 1995; Magnaghi et al., 2001) (see Section 3.5). Steroids produced by central glia, such as DHEA (Kimonides et al., 1998; Bastianetto et al., 1999; Cardounel et al., 1999) and

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estradiol (Green and Simpkins, 2000; Wise et al., 2000; Garcia-Segura et al., 2001) are neuroprotective. In this regard, it is particularly interesting that P450 aro is expressed by reactive astroglia. Aromatase-expressing astrocytes are observed in all injured brain areas, including the cortex, corpus callosum, striatum, hippocampus, thalamus and hypothalamus (Garcia-Segura et al., 1999b). This indicates that astrocytes from most brain areas have the potential for expressing P450 aro, and therefore to produce estradiol, in response to injury. Estrogen formed by astrocytes may be released as a trophic factor for damaged neurons and may be involved in the compensatory restructuring of injured brain tissue. Estrogen released by astroglia may affect synaptic function, selective regeneration of neuronal processes and local cerebral blood flow, contributing to facilitation of neuronal recovery and reduction of neuronal death. Recent studies indicate that aromatase knock out mice are more sensitive to excitotoxic neurodegenerative injury than wild type mice (Azcoitia et al., 2001b). Furthermore, the susceptibility to kainic acid excitotoxic injury in male rats is greatly enhanced after the intracerebral infusion of the P450 aro inhibitor fadrozole (Azcoitia et al., 2001b). This finding indicates that local cerebral P450 aro activity is involved in neuroprotection. Therefore, the induction of P450 aro in astroglia after brain injury and the local formation of estradiol by these cells may represent a response of the injured neural tissue to limit neurodegenerative damage.

3. Non-neuronal cells as targets for steroids 3.1. Steroid hormone receptors in non-neuronal cells Steroid hormone receptors are expressed in the CNS, both in neurons and in nonneuronal cells. Glucocorticoid and mineralocorticoid receptors are expressed by microglia, astroglia, oligodendroglia and Schwann cells (Neuberger et al., 1994; Garcia-Segura et al., 1996; Tanaka et al., 1997). Estrogen receptors (ERs), androgen receptor (AR) and progesterone receptor (PR) expression has also been described in nonneuronal cells of the CNS, in vitro and in vivo, under normal and/or neurodegenerative conditions, as summarized in Table 1. In addition, Schwann cells (Melcangi et al., 1999b) and conventional astrocytes (see chapter by Hansson and Ro¨nnba¨ck) express the GABAA receptor and consequently may respond to steroids that interact with this neurotransmitter receptor, such as tetrahydroprogesterone and tetrahydrodeoxycorticosterone (for review, see Melcangi et al., 1999a, 2001b).

3.2. Effects of steroids on brain endothelial cells As in the periphery, endothelial cells in the brain are affected by steroids. Several studies have examined the effects of glucocorticoids on brain endothelium. Glucocorticoids exert different effects on brain vascular system, affecting vascular morphogenesis, blood – brain barrier properties, cerebrovascular permeability and brain edema. These effects are in part mediated by the regulation of the expression of endothelin receptors,

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Table 1 Sex steroid receptors in non-neuronal cells Cell type

Condition

References

ERa

Astroglia

In vitro In vivo In neurodegeneration In vivo In vivo In vitro In vitro In vitro In vivo

Jung-Testas, 1992; Santagati et al., 1994; Buchanan et al., 2000 Langub and Watson, 1992; Milner et al., 2001 Azcoitia et al., 2001a; Blurton-Jones and Tuszynski, 2001; Garcia-Ovejero et al., 2002 Langub and Watson, 1992 Gudin˜o-Cabrera and Nieto-Sampedro, 1999 Santagati et al., 1994 Thi et al., 1998 Vegeto et al., 2001; Bruce-Keller et al., 2000 Langub and Watson, 1992

In vitro In vivo In vitro

Buchanan et al., 2000; Ho¨sli et al., 2001 Azcoitia et al., 1999; Cardona-Gomez et al., 2000b Mor et al., 1999; Li et al., 2000; Vegeto et al., 2001

In vitro In vivo In neurodegeneration In vivo In neurodegeneration In neurodegeneration

Jung-Testas et al., 1992; Ho¨sli et al., 2001 Finley and Kritzer, 1999 Puy et al., 1995 Finley and Kritzer, 1999 Puy et al., 1995 Garcia-Ovejero et al., 2002; Puy et al., 1995

In vitro In vitro In vivo

Jung-Testas et al., 1992 Jung-Testas et al., 1996; Thi et al., 1998 Magnaghi et al., 1999, 2001

Ependymoglia Growth promoting gliaa Oligodendroglia Schwann glia Microglia Endothelial cells ERb

Astroglia Microglia

AR

Astroglia

Oligodendroglia Microglia PR

a

Astroglia Schwann glia

Olfactory ensheathing cells, tanycytes, pituicytes, pineal glia, retinal Mu¨ller cells, and cerebellar Bergmann glia.

R.C. Melcangi et al.

Receptor

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histamine receptors or matrix metalloproteinases in vascular endothelium (Stanimirovic et al., 1994; Karlstedt et al., 1999; Harkness et al., 2000). The effect of estradiol on brain endothelium has been studied as well. As shown in Table 1, Langub and Watson (1992) reported ER alpha immunoreactivity in guinea pig brain endothelial cells by electron microscopy. Furthermore, estrogen increases rat brain endothelial nitric oxide synthase via ERs (McNeill et al., 2002). Part of the neuroprotective effects of estrogen may be due to hormonal actions on brain endothelium (Shi et al., 1997; Watanabe et al., 2001; Galea et al., 2002). For instance, estrogen treatment increases the endothelial cell glucose transporter GLUT1 and protects against brain capillary endothelial cell loss, which may in turn reduce focal ischemic brain damage (Shi et al., 1997). Estradiol may also exert protective effects in cerebral ischemia by blockade of leukocyte adhesion in cerebral endothelial cells (Santizo and Pelligrino, 1999), an effect that is a consequence of the hormonal down-regulation of the expression of adhesion molecules in cerebral endothelium (Galea et al., 2002).

3.3. Effects of steroids on astroglia There is an abundant literature showing that steroids affect astroglia cell shape and gene expression (Garcia-Segura et al., 1996, 1999a, Jones et al., 1999; Jordan, 1999; Mong and McCarthy, 1999; Nichols, 1999; Vardimon et al., 1999). Steroid hormone precursors and neurosteroids, such as pregnenolone and DHEA, affect astroglia cell shape and glial fibrillary acidic protein (GFAP) expression in different brain areas (Del Cerro et al., 1996; Legrand and Alonso, 1998; Garcia-Estrada et al., 1999). Sex steroids affect GFAP expression as well. For instance, castration decreases GFAP immunoreactivity in the hypothalamus of male rats (Day et al., 1993), and this phenomenon is counteracted by testosterone or dihydrotestosterone administration, but not by estradiol (Day et al., 1993). The situation is quite different in the hippocampus, since it has been demonstrated that in male rats castration increases the levels of GFAP mRNA and protein in this cerebral structure (Day et al., 1990, 1993). Among the gonadal steroids tested (estradiol, testosterone and dihydrotestosterone), only estradiol proved able to counteract the effect of castration (Day et al., 1993). After a penetrating brain injury, sex steroids (estradiol and progesterone in females, or testosterone in males) are able to decrease the process of gliosis and astrocyte proliferation, resulting in a decrease in the number of reactive astrocytes in the cerebral cortex and in the hippocampus (Garcia-Estrada et al., 1993). It has also been observed that the levels of GFAP mRNA and immunoreactivity show sex differences in the arcuate nucleus of the rat, lower levels being found in females than in males (Chowen et al., 1995). Androgenization of neonatal females increases GFAP mRNA to male levels, while castration of newborn males decreases GFAP mRNA to the levels found in females (Chowen et al., 1995). In the arcuate nucleus and in the hilus of the dentate gyrus of adult female rats, the surface density of GFAP-immunoreactive cells fluctuates throughout the estrous cycle, decreases after ovariectomy and increases after the administration of estradiol (Luquin et al., 1993; Garcia-Segura et al., 1994a,c). More recently, Stone et al. (1998) have analyzed GFAP transcription and the GFAP mRNA levels in the hypothalamus and hippocampus of rats during the estrous cycle. They

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observed that on the afternoon of proestrous, both parameters increase in the arcuate nucleus of the hypothalamus and in the outer molecular layer of the dentate gyrus. On the contrary, neither GFAP transcription nor GFAP mRNA exhibits variations in the hilus of the hippocampus during the estrous cycle. This is particularly interesting since, as mentioned above, the extension of GFAP-immunoreactive astrocytic processes shows striking changes during the estrous cycle in the hilus (Luquin et al., 1993), suggesting that ovarian hormones may affect GFAP redistribution and growth or retraction of astrocytic processes without affecting GFAP synthesis in this brain area. Furthermore, it should be mentioned that although estrogens may act directly on astrocytes, the direction of the transcriptional response is influenced by interactions of astrocytes with neurons. Thus, estradiol, when added to cortical astrocytes in culture, increases GFAP transcription, while the same treatment, performed in astrocytes co-cultured with neurons, induces a decrease of this parameter (Stone et al., 1998). Cell adhesion molecules, such as PSA-NCAM (Garcia-Segura et al., 1995a), and neurotransmitters, such as GABA (Mong et al., 2002), have been shown to be involved in estrogen-induced neuron to astrocyte signaling. Corticosteroids are also able to influence GFAP expression. Treatments with corticosterone and other glucorticoids (e.g., dexamethasone) inhibit GFAP expression in the neonatal and adult rat brain (Nichols et al., 1990a,b; Laping et al., 1991; Tsuneishi et al., 1991). In line with these observations, adrenalectomy, performed in adult rats, has been shown to increase GFAP mRNA and protein, an effect, which is reversed by corticosterone administration (Laping et al., 1994). However, a different pattern of activity is shown by corticosterone in vitro, since exposure of cultured astrocytes to this steroid induces an increase in GFAP mRNA and protein (Rozovsky et al., 1995). Surprisingly, this effect of corticosterone on GFAP is reversed if astrocytes are co-cultured with neurons, a finding which suggests that there is a cross-talk between the two types of cells, and provides a possible explanation for the discrepancies between the results obtained in vivo and in vitro (Rozovsky et al., 1995). Glucocorticoids influence other important functional parameters of astrocytes as well. For instance, methylprednisolone and dexamethasone enhance astrocytic calcium signaling, increasing both resting cytosolic calcium ([Ca2þ]i) levels and the extent and the amplitude of Ca2þ wave propagation (Simard et al., 1999). The glucocorticoidassociated potentiation of Ca2þ signaling may result from up-regulation of the cellular ability to mobilize Ca2þ and to release the purinergic transmitter ATP (see chapter by Hansson and Ro¨nnbck), because both ATP-induced [Ca2þ]i increments and ATP release were proportionally enhanced by glucocorticoids. Moreover, glucocorticoids are also able to influence the metabolism of glutamate in astrocytes, increasing the expression of both glutamine synthetase and glutamate dehydrogenase in these cells (Hardin-Pouzet et al., 1996; Vardimon et al., 1999). Finally, corticosteroids can modulate the expression of growth factors, such as basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor, in astrocytes (Magnaghi et al., 2000). In many cases the effect exerted by steroid hormones on GFAP expression are due to their conversion into active metabolites (i.e., neuroactive steroids). For instance, metabolites of testosterone and progesterone (respectively, dihydrotestosterone and dihydroprogesterone or tetrahydroprogesterone) are able to modulate gene expression of GFAP in cultures of conventional astrocytes (Melcangi et al., 1996b). This seems to be

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the case also for corticosteroid metabolites, since while the mineralocorticoid deoxycorticosterone is ineffective, its 5a-reduced derivative, dihydrodeoxycorticosterone, strongly inhibits GFAP gene expression (Melcangi et al., 1997). 3.3.1. Implications of steroid effects on astroglia for synaptogenesis and synaptic plasticity The effects of sex steroids on astrocytes are linked to the regulation of synaptic connectivity. In the hypothalamic arcuate nucleus there are sex differences in the number of axo-somatic and axo-dendritic synapses that are accompanied by sex differences in the levels of GFAP, the differentiation of astrocytes and the amount of neuronal membrane surface covered by astroglial cell processes (Chowen et al., 1995; Garcia-Segura et al., 1995b; Mong et al., 1996, 1999). These sex differences are induced by the perinatal secretion of testosterone in male rats. This hormone induces stellation of astrocytes, an increased expression of GFAP and an increased coverage of neuronal membrane by astrocytic processes. Coincident with these changes in astrocytic morphology there is a strong reduction in the density of dendritic spines and axo-somatic synapses on arcuate neurons (Garcia-Segura et al., 1995b; Mong et al., 1996, 1999). The effect of testosterone on astrocytes and synapses is probably mediated by its conversion to estradiol. In the arcuate nucleus of adult female rats estradiol has similar effects as those of testosterone during development, with the important difference that estradiol effects in adults are transient and those of testosterone during development are permanent. In the afternoon of proestrus, the surge of estradiol induces a transient growth of astrocyte processes on the surface of arcuate neurons (Garcia-Segura et al., 1994c). As a consequence, there is a transient disconnection of inhibitory GABAergic synapses from arcuate neuronal somata by the interposed glial processes (Garcia-Segura et al., 1994b). These changes are also elicited by the administration of estradiol to adult ovariectomized rats (Garcia-Segura et al., 1994a,b). Estradiol also increases the number of synapses on dendritic spines in the arcuate nucleus during the estrous cycle. A similar effect in the hippocampus is accompanied by an increased expression of tyrosine kinase A (TrkA) receptors in astrocytes (McCarthy et al., 2002). Since nerve growth factor, the ligand for TrkA, stimulates astrocytes to function as substrates for axon growth (Kawaja and Gage, 1991), McCarthy et al. (2002) have proposed that estradiol may regulate axonal growth and synaptic plasticity in the hippocampus by the induction of TrkA receptors in astrocytes. In addition, soluble factors released by astrocytes from target brain areas have been shown to influence the neuritogenic effect of estradiol on cultured hypothalamic neurons (Cambiasso et al., 2000). Finally, astrocytes secrete laminin in response to estrogen, facilitating neurite extension when co-cultured with neurons (Rozovsky et al., 2002). 3.3.2. Implications of steroid effects on astroglia for neuroendocrine regulation It has recently been observed that astrocytes modulate the function of the hypothalamic neurons synthesizing and secreting the luteinizing hormone releasing hormone (LHRH). The most important principles, released from glial cells and apparently responsible for these effects, are transforming growth factor a (TGFa), transforming growth factors b1

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(TGFb1), transforming growth factor b2 (TGFb2), bFGF and insulin-like growth factor-I (IGF-I). The astrocytic involvement provides an additional and new mode of control of LHRH secretion, which is also regulated by neuronal inputs, as well as by steroid hormones acting via positive or negative feedback signals (see also chapter by Pre´vot et al.). The possibility of a functional co-operation between growth factors and steroid hormones at the level of astrocytes and tanycytes in the control of the LHRHsecreting neurons has recently been taken in consideration (for review, see Melcangi et al., 2002). For instance, it has been demonstrated that estradiol is able to induce an increase in TGFa and bFGF mRNA levels in cultures of hypothalamic astrocytes (Ma et al., 1994, Galbiati et al., 2002). It is interesting to note that in hypothalamic astrocytes, estradiol is also able to facilitate the effect of prostaglandin PGE2, one of the humoral factors intervening in the control of the secretion of LHRH (Rage et al., 1997). In particular, it has been demonstrated that hypothalamic astrocytes treated with estradiol produce a conditioned medium that after application to a cell line of LHRH-secreting neurons (i.e., GT1 cells), induces a selective up-regulation of two of the four known members of the PGE2 receptor family (i.e., EP1-R and EP3g-R) (Ojeda and Ma, 1999). Estrogens and DHEA are able to influence the release of TGFb1 from cultures of hypothalamic astrocytes (Buchanan et al., 2000; Zwain et al., 2002). Moreover, in this case, an effect of progesterone is also evident. In particular, it has been observed that treatment of cultured hypothalamic astrocytes for 6 h with either progesterone or one of its derivatives (dihydroprogesterone and tetrahydroprogesterone) results in a stimulation of TGFb1 gene expression (Melcangi et al., 2001a). However, with longer treatments (24 h), only dihydroprogesterone and tetrahydroprogesterone are able to increase the mRNA levels of TGFb1. On the basis of this time-dependent effect, it has been hypothesized that the effect of progesterone might be due to its conversion into dihydroprogesterone and tetrahydroprogesterone, since, as mentioned above, 5areductase and 3a-hydroxysteroid dehydrogenase activities are present in conventional astrocytes (Melcangi et al., 1993, 1994a). In contrast, dihydrotestosterone, the 5a-reduced metabolite of testosterone, reduces the expression of bFGF in astrocytes (Melcangi et al., 2001a). This effect may be due to an interaction with the AR, which is expressed in the astrocytes in vitro (Melcangi et al., 2001b). However, testosterone, which is also able to bind to this receptor, albeit with a lower affinity than dihydrotestosterone, does not affect bFGF expression (Melcangi et al., 2001a). Another factor involved in the regulation of LHRH neurons is IGF-I. Tanycytes in the hypothalamus and the median eminence accumulate IGF-I from cerebrospinal fluid and plasma and may therefore contribute to LHRH regulation by modulating local IGF-I levels (Garcia-Segura et al., 1999a). Gonadal steroids regulate the expression and distribution of IGF-I receptors and IGF binding proteins in tanycytes (Cardona-Gomez et al., 2000a) and this, in turn, affects the uptake of IGF-I originating from blood or cerebrospinal fluid (Garcia-Segura et al., 1999a). Therefore, tanycytes integrate hormonal signaling from the reproductive axis (gonadal steroids) and the growth hormone axis (IGF-I) to regulate LHRH release. In summary, sex steroids affect the synthesis, accumulation and release by hypothalamic astrocytes and tanycytes of different growth factors that regulate LHRH

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neurons. Therefore, astrocytes and tanycytes may play an important role as mediators of the action of peripheral steroid hormones in the control of neuroendocrine regulation.

3.3.3. Implications of steroid effects on astroglia for neuroprotection The effects of steroids on astroglia have implications for neuroprotection and brain repair as well. Sex steroids and neuroactive steroids may affect brain responses to pathological conditions by regulating reactive gliosis (Del Cerro et al., 1996; Garcia-Estrada et al. 1993, 1999) and the expression of molecules in reactive astroglia that are part of the response of astrocytes to injury. For instance, DHEA inhibits production of tumor necrosis factor alpha and interleukin-6 in astrocytes (Kipper-Galperin et al., 1999). Estrogen down-regulates the expression of bFGF in astrocytes (Flores et al., 1999) and increases the expression of apolipoprotein E (ApoE), a molecule involved in neuroregulation after injury, in astrocytes and microglia (Stone et al., 1997). A very interesting finding with implications for reactive astroglia is that estradiol in cultured astrocytes reduces activation of NF-kappaB induced by amyloid A beta (1-40) and lipopolysacccahride (LPS) (Dodel et al., 1999). Since NF-kappaB is a potent immediateearly transcriptional regulator of numerous pro-inflammatory genes, the hormonal regulation of this molecule in astrocytes may play a crucial role in the neuroprotective effects of estrogen. Effects of steroids on astrogliosis may be a contributing factor to neural regeneration. This is suggested by studies of the effects of the estradiol precursor testosterone on the regulation of the central astrocytic response to peripheral nerve injury. In adult male hamsters, testosterone propionate administration reduces the increase in GFAP mRNA in the facial nucleus after facial nerve axotomy (Jones et al., 1997b, 1999), attenuates glial-mediated synaptic stripping of axotomized motoneurons (Jones et al., 1997a, 1999) and increases facial nerve regeneration (Kujawa et al., 1991). The relationship between reduced astrogliosis in the facial motor nucleus and increased axonal regeneration is still unknown. However, the results strongly suggest that hormonal regulation of astrogliosis may contribute to the regenerative mechanisms of testosterone and estradiol on facial motoneurons. These effects may also explain why estradiol decreases GFAP expression and promotes neurite outgrowth in the hippocampus after deafferenting lesion of the entorhinal cortex (Rozovsky et al., 2002), rather than causing the increase in GFAP seen in unlesioned animals (see above). Other effects of steroids on astrocytes may also be relevant under neuropathological conditions. For instance, glucocorticoids may be detrimental to reactive astrocytes, by mechanisms involving depletion of intracellular ATP levels and deterioration of mitochondrial transmembrane potentials (Shin et al., 2001). In contrast, estradiol increases the expression of heat shock proteins in astrocytes (Mydlarski et al., 1995), an effect that has also been observed in striatal astrocytes after global ischemia in gerbils (Lu et al., 2002), and may be related to the protective effects of the hormone in animal models of brain ischemia. Furthermore, estradiol increases glutamate uptake in astrocytes derived from Alzheimer’s patients (Liang et al., 2002), which may contribute to the potential

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protective hormonal effect against this neurodegenerative disease, where the extracellular glutamate concentration appears to be increased (see chapter by Barger). 3.4. Effects of steroids on microglia The well known anti-inflammatory effects of steroids in peripheral tissues have been also observed in the brain. Steroids exert their anti-inflammatory actions in the brain acting on non-neuronal cells, mainly on microglia. Microglia are, as macrophages, target of steroids and both cell types show similar anti-inflammatory responses to glucocorticoids. For instance, cortisol represses LPS induction of nitric oxide production in primary cultured microglia and transformed N9 microglial cells (Drew and Chavis, 2000a). Therefore, although cortisol may be directly toxic to neurons, it may indirectly protect neurons by blocking the production of cytotoxic molecules by microglia (Drew and Chavis, 2000a). Other steroids, such as DHEA and progesterone, also inhibit nitric oxide production in primary cultures of microglia in response to LPS (Barger et al., 2000; Drew and Chavis, 2000b). The effect of DHEA may be related to the neuroprotective effects of this steroid. Furthermore, Drew and Chavis (2000b) have proposed that the progesteronemediated inhibition of microglial cell activation may contribute to the decreased severity of multiple sclerosis symptoms commonly observed during with pregnancy. Several studies have analyzed the effect of estradiol on microglia, in search for a basis for the neuroprotective effects of the hormone. Stone et al. (1997) showed that estradiol enhances ApoE secretion by microglia in vivo. Vegeto et al. (1999) reported that estradiol inhibits apoptosis in microglia cultures by a receptor-mediated enhancement of Nip2 protein production. Subsequent studies in microglia cultures have shown that estradiol inhibits the induction of inducible nitric oxide synthase, and the consequent production of nitric oxide, in response to LPS and to the pro-inflammatory cytokines interferon-g and TNF-a (Bruce-Keller et al., 2000; Drew and Chavis, 2000b; Vegeto et al., 2001). Furthermore, Vegeto et al. (2001) have found that estradiol also reduces LPS-induced production in microglia of other inflammatory mediators, such as PGE2, and metalloproteinase-9. Estradiol is also able to enhance uptake of amyloid beta-protein (Ab) by microglia derived from the human cortex (Li et al., 2000), an effect that may be relevant for the protective effect of this hormone against Alzheimer’s disease. Bruce-Keller et al. (2000, 2001) have studied the mechanism involved in the antiinflammatory actions of estradiol on microglia. These authors have found that estrogen receptor-dependent activation of MAP kinase is involved in the hormonal action. This opens the possibility for a co-ordinated regulation of microglia by estradiol and growth factors via activation of the MAP kinase signaling pathway. 3.5. Effects of steroids on oligodendrocytes and Schwann cells The effects of steroid hormones on central myelin and oligodendroglia have not been sufficiently explored. Very little is known about effects of gonadal hormones on oligodendroglia. Glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis during neonatal development (see Garcia-Segura et al., 1996, for review).

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However, these hormones inhibit the proliferation of oligodendrocyte precursors located throughout the white and gray matter regions of the adult rat brain. Since the proliferation of oligodendrocyte precursors plays a major role during remyelination, these data raise the question of possible detrimental effects of therapeutic treatments of CNS trauma in adult individuals by administration of glucocorticoids (Alonso, 2000). Furthermore, dihydrocorticosterone decreases the expression of myelin basic protein in oligodendrocytes (Melcangi et al., 1997), and it has been reported that prenatal corticosteroid administration delays myelination of the corpus callosum in fetal sheep (Huang et al., 2001). During the last few years, several studies have been performed to evaluate possible effects of steroid hormones on physiological parameters of Schwann cells. For instance, it has been shown that dexamethasone, a synthetic glucocorticoid, is able to strongly enhance the mitogenic activity exerted by an axolemma-enriched fraction in Schwann cell cultures (Neuberger et al., 1994). In the same experimental model, no co-mitogenic action was exerted by progesterone, testosterone, estradiol, or aldosterone (Neuberger et al., 1994). In partial disagreement with these observations, it has been shown that estrogens are able to promote Schwann cell proliferation in culture in the presence of agents elevating intracellular cAMP (Jung-Testas et al., 1993). The effects of estrogens and progesterone on the proliferation of Schwann cells have also been studied in cultures of segments of the rat sciatic nerve obtained from adult or newborn male and female rats (Svenningsen and Kanje, 1999). In this experimental model, these two sex steroids are able to enhance [3H] thymidine incorporation into Schwann cells, an effect that was blocked by their respective receptor antagonists. However, it is important to underline that this effect depends on the sex of the animals. Thus, estrogens are effective on Schwann cell proliferation only in segments from male rats, while progesterone increases Schwann cell proliferation only in segments obtained from females. An effect of sex steroids is also evident on Schwann cells that cover motoneuron terminals. As shown by Lubischer and Bebinger (1999), the number of this particular type of non-myelinating Schwann cells present in an androgen-sensitive muscle of the rat (i.e., the levator ani) decrease after castration. The decline is reversed by testosterone treatment. This steroid effect seems to be indirect, since Schwann cells do not express AR (Magnaghi et al., 1999), and the involvement of motoneurons and/or muscle fibers expressing AR might be hypothesized (Jordan et al., 1997; Jordan and Williams, 2001). One of the major products of Schwann cells is myelin. Consequently, several studies have been performed both in vivo (i.e., in the whole sciatic nerve of rat) and in vitro (i.e., in cultures of rat Schwann cells) to evaluate whether steroid hormones and/or neuroactive steroids might influence this cellular component. In particular, the attention of different laboratories has been directed towards the proteins proper of the peripheral myelin, like the glycoprotein Po (Po) and peripheral myelin protein 22 (PMP22) (for review, see Melcangi et al., 2000b; Magnaghi et al., 2001). Data obtained so far strongly suggest an important role of steroid hormones in modulating the expression of these two crucial myelin proteins. In particular, progesterone and its physiological 5a- and 3a-5a-reduced derivatives, dihydroprogesterone and tetrahydroprogesterone, stimulate the expression of Po in intact or transected sciatic nerve of male rats. These effects are also evident in rat Schwann cell cultures, indicating a direct effects of these steroids on the cells producing Po (De´sarnaud et al., 1998; Notterpek et al., 1999; Melcangi et al., 2000a,b;

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Magnaghi et al., 2001). In the same models, the gene expression of PMP22 is influenced only by tetrahydroprogesterone (Melcangi et al., 1999b). However, at variance with these findings, other observations indicate that the expression of PMP22 may also be stimulated by progesterone itself. In fact, utilizing Schwann cells transiently transfected with a reporter construct in which the expression of the luciferase is controlled by the promoter region of the PMP22 gene, De´sarnaud and co-workers (1998) showed that progesterone stimulates the gene expression of this myelin protein, acting on promoter 1, but not on promoter 2 of the PMP22 gene. It has been proposed that the effects of progesterone, dihydroprogesterone and tetrahydroprogesterone (after retro-conversion into dihydroprogesterone) on gene expression of protein Po are linked to an interaction with the PR, which is present in Schwann cells (Magnaghi et al., 1999), while effects of tetrahydroprogesterone on protein PMP22 appear to be due to an interaction of this steroid with the GABAA receptor, whose subunits have been found in Schwann cells (Melcangi et al., 1999b). This hypothesis is based on the findings that, in primary cultures of Schwann cells isolated from neonatal rat sciatic nerves: (i) mifepristone (RU38486), an antagonist at the PR, is able to block the effects of progesterone, dihydroprogesterone and tetrahydroprogesterone, on the expression of protein Po; and (ii) the effect of tetrahydroprogesterone on PMP22 is blocked by bicuculline, a classical antagonist of the GABAA receptor (Magnaghi et al., 2001). The conclusion that the expression of Po seems to be under the control of the PR, while that of PMP22 is under GABAA receptor influence is further supported by the findings that the GABAA receptor agonist muscimol does not increase the mRNA levels of protein Po, while it increases the expression of protein PMP22 (for review, see Magnaghi et al., 2001). An effect of progesterone and its derivatives on Po gene expression through the PR might suggest a genomic mechanism, in which the complex ligand – receptor interacts with steroid responsive elements located in the promoter region of Po. In agreement with this hypothesis, a computer analysis has demonstrated that putative progesterone responsive elements are present on the Po promoter (Magnaghi et al., 1999). It is also interesting to recall that progesterone stimulates gene expression of the transcription factor Krox-20, which plays an important role in myelination of peripheral nerves, as well as of other transcription factors present in Schwann cells (for review, see Schumacher et al., 2001). In particular, it has been observed that progesterone induces a rapid increase in the gene expression of Krox-20, Krox-24, Egr-3 and Fos B (Guennoun et al., 2001; Mercier et al., 2001), suggesting that this steroid hormone might also co-ordinate the signaling pathways involved in the initiation of myelination. The possible involvement of the GABAA receptor in the control of the expression of PMP22 is also evident in experiments, in which rat Schwann cells were exposed to testosterone, dihydrotestosterone and 3a-diol (see Fig. 2). In fact, in this case only 3a-diol was able to influence PMP22 expression (Melcangi et al., 2000a). It is interesting to note that this steroid, which does not interact with the AR, is able to interact with the GABAA receptor (Frye et al., 1996a,b). On the other hand, it is important to mention that androgen molecules themselves are not completely ineffective on Po expression. Removal of circulating androgens by castration decreases mRNA levels of Po in the sciatic nerve, a phenomenon, which is counteracted by subsequent treatment with dihydrotestosterone

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(Magnaghi et al., 1999). However, since Schwann cells as previously mentioned do not express the AR (Magnaghi et al., 1999), it has been hypothesized that the gene expression of Po might be stimulated by androgen-dependent mechanisms acting on Schwann cells through the adjacent neuronal component, which seems to express AR (Magnaghi et al., 1999). Finally, also glucocorticoids seem to be able to stimulate the transcription from Po and PMP22 promoters in Schwann cells. As shown by De´sarnaud et al. (2000), dexamethasone and corticosterone are able to stimulate Po expression and both promoters of the PMP22 gene. Since Po and PMP22 play an important physiological role in the maintenance of the multilamellar structure of peripheral myelin (for review, see Melcangi et al., 2000a; Magnaghi et al., 2001), these observations might suggest the possible utilization of neuroactive steroids after peripheral injury, during aging or in particular in demyelinating diseases (e.g., Charcot-Marie-Tooth type 1a and 1b, De´je´rine-Sottas syndrome), in which rebuilding of myelin is needed.

4. Concluding remarks This chapter has reviewed recent information indicating that non-neuronal cells of the nervous system synthesize and metabolize steroids and are targets for hormonal and neuroactive steroids. We have noted that glia affect neuronal function by producing steroids and metabolizing hormonal steroids into neuroactive steroids. On the other hand, endothelial cells and glia are targets for hormonal and neuroactive steroids and mediate some of the effects of these molecules in the brain. However, there is still an important gap in our knowledge of all the implications and mechanisms involved in the communication between non-neuronal and neuronal cells via steroids. In some cases, there is evidence that steroids produced by glia exert specific functions on adjacent neuronal constituents. The best example is the production of progesterone and its derivatives by Schwann cells and their involvement in peripheral nerve remyelination and regeneration. This is also the case of the neuroprotective actions of estradiol produced by reactive astroglia. There is also considerable evidence for specific consequences of steroid actions on glia. For instance, steroids modulate synaptic plasticity and neuroendocrine secretion by an action on astroglia, and they regulate the response of nerve tissue to injury by an action on microglia. However, there are still many unsolved questions and unexplored areas in relation to steroids and non-neuronal cells. For instance, further studies are necessary to determine to what extent steroids produced by glia affect brain development and CNS myelination, whether physiological or pharmacological modifications of glial steroidogenesis affect mood, cognitive function and behavior, and whether the production of neuroactive steroids by glia is a general mechanism for regulation of synaptic function. The resolution of these unsolved questions may broaden our perception of the manners in which integration of neuronal and non-neuronal mechanisms regulate nervous system function. In addition, understanding of steroid-mediated communication between neurons and non-neuronal cells in the nervous system has obvious implications for the treatment of neurodegenerative diseases and psychiatric disorders.

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Acknowledgements We want to acknowledge financial support from the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6-CT-2000-00179.

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Expression of neurotrophic factors and cytokines and their receptors on astrocytes in vivo Takao Nakagawa1 and Joan P. Schwartzp Neurotrophic Factors Section, NINDS, National Institutes of Health, Building 1, Room 135, Bethesda, MD 20892-0151, USA p Correspondence address: Tel.: þ 1-301-496-1248; fax: þ 1-301-402-0027. E-mail: [email protected](J.P.S.)

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Introduction Neurotrophins TGF-b superfamily Insulin-like growth factor family Fibroblast growth factors Vascular endothelial growth factor Neuropoietic cytokines Pro-inflammatory cytokines Anti-inflammatory cytokines Chemokines Concluding remarks

The functions of astrocytes following injury are mediated in part by synthesis of neurotrophic factors as well as cytokines. For this chapter we have chosen to review data on the in vivo synthesis and expression of various neurotrophic factors and cytokines/chemokines, as well as their receptors, in astrocytes from control and injured brain. We present data only for the central nervous system and discuss a variety of different models of injury, as well as results from human neurological diseases. These data from the literature are compared with our cDNA microarray results for astrocytes isolated in vivo from normal or 6-hydroxy-dopamine-lesioned rat striatum. 1. Introduction The central nervous system (CNS) is composed of several cell populations, primarily neurons, macroglia and microglia, with astrocytes and oligodendrocytes the principal 1

Present address: Department of Neurosurgery, Fukui Medical University, Fukui, Japan.

Advances in Molecular and Cell Biology, Vol. 31, pages 561–573 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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macroglial cell types. Astrocytes constitute nearly 40% of the total CNS cell population (Rutka et al., 1997), yet our knowledge of the function of astrocytes is still incomplete. Astrocytes are stellate cells with multiple fine processes, some of which contact cells of mesodermal origin (most frequently capillary endothelial cells) and some of which are intertwined within the neuropil and ensheath synaptic contacts (Rohlmann and Wolff, 1996). These quantitative and morphological findings support the idea that astrocytes have important functions in maintaining or modulating CNS function. Among these are metabolic support for neurons and uptake of neurotransmitters (see chapter by Schousboe and Waagepetersen), ion homeostasis (see chapter by Walz), guidance for neuronal cell migration and axon outgrowth during development (see chapter by Gomes and Rehen), and preservation of host tissue integrity following injury, a property of activated, or reactive, astrocytes. Reactive gliosis, which occurs in the CNS in response to virtually all forms of brain injury, has been defined as an increase in the size of the astrocyte cell body and its processes, with an increase of glial fibrillary acidic protein (GFAP) being the prototypical biochemical change (see chapter by Kalman). Prominent reactive astrocytosis is seen in acute traumatic brain injury (Faden, 1993), neurodegenerative diseases such as Alzheimer’s and Parkinson’s (Delacourte, 1990; Pike et al., 1995; Knott et al., 2002), and following exposure to toxins such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, 6-hydroxydopamine (6-OHDA) and kainic acid (Forno et al., 1992; O’Callaghan and Miller, 1993). A distinguishing characteristic among these different types of injuries is whether inflammation occurs, thus involving immune cells and/or cytokines. Cytokines and chemokines, important mediators of the host defense system and the inflammatory response, are elevated in a variety of neurological diseases (Eng and Ghirnikar, 1994; Zhao and Schwartz, 1998). Both astrocytes and microglia can produce and respond to cytokines (Giulian et al., 1994a,b). Thus, cytokines or chemokines elevated after brain injury may act as signals among microglia, neurons, and astrocytes but may also be functioning in trophic roles. The functions of astrocytes during development and following injury are mediated in part by astrocyte synthesis of neurotrophic factors as well as cytokines (Eddleston and Mucke, 1993; McMillian et al., 1994; Ridet et al., 1997). Furthermore, the presence of growth factor and cytokine receptors on astrocytes suggests that these factors modulate astrocyte functions in autocrine or paracrine ways (Ridet et al., 1997). Some of the neurotrophic factors and cytokines have been detected in embryonic and neonatal brain in vivo. Few are present in the normal adult brain, but synthesis of both neurotrophic factors and cytokines/chemokines is turned on following brain injury. Of great interest has been the specific expression of these factors in astrocytes, both developmentally and following injury, since some studies have suggested that reactive astrocytes recapitulate the properties of neonatal astrocytes. Furthermore, both neurotrophic factors and cytokines have been shown to rescue damaged neurons in various injury models. What factors do astrocytes synthesize in the developing or injured brain? For this chapter we have chosen to review data on the in vivo synthesis and expression of various neurotrophic factors and cytokines/chemokines in astrocytes. We will present data only for the CNS and will compare a variety of different models of injury, as well as results from human neurological diseases. Summarized in Table 1 are the various families of

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Table 1 Neurotrophic factors, cytokines, chemokines and their receptors expressed in astrocytes Family Neurotrophic factors Neurotrophins

TGF-b superfamily

Insulin-like growth factors

Fibroblast growth factors (23 family members, 10 in CNS) Vascular endothelial growth factor Cytokines Neuropoietic cytokines

Pro-inflammatory cytokines Anti-inflammatory cytokines

Chemokines CC family

CXC family

CX3C family a

Nerve growth factor. Brain-derived neurotrophic factor. c Glial cell derived neurotrophic factor. d Ciliary neurotrophic factor. e Leukemia inhibitory factor. f Macrophage inflammatory protein. g Monocyte chemoattractant protein. h Interferon-g-inducible protein. i Macrophage interferon-g-inducible protein. b

Factor

Receptor(s)

NGFa BDNFb Neurotrophin-3 (NT-3) GDNFc Neurturin Artemin Persephin IGF-1 IGF-2 FGF-1 (acidic) FGF-2 (basic) VEGF VEGF-B to VEGF-E

trkA, p75 trkB, p75 trkC, p75 Ret þ GFRa1 Ret þ GFRa2 Ret þ GFRa3 Ret þ GFRa4 IGF1R IGF2R IGFBP-1 to IGFBP-6 FGFR-1 to FGFR-4 FGFR-1 to FGFR-4 Flt-1 (VEGFR-1) Flk-1 (VEGFR-2)

CNTFd LIFe IL-6 Oncostatin-M Cardiotropin IL-1b TNFa TGF-b IL-1ra IL-4 IL-10

gp130 þ CNTFRa gp130 þ LIFR gp130 þ IL-6Ra gp130 þ LIFR þ OsMR gp130 þ LIFR þ CT1Ra IL-1R1, IL-1R2 TNF-R1, TNF-R2 TGF-bR1 IL-1R1 IL-4R IL-10R

MIP-1af MIP-1b MCP-1g MIP-3a MIP-3b RANTES Eotaxin IP-10h Migi MIP-2 IL-8 Fractalkine

CCR1, CCR5 CCR1, CCR5 CCR1, CCR2 CCR6 CCR7 CCR1, CCR3, CCR5 CCR3, CCR5 CXCR3 CXCR3 CXCR2 CXCR1, CXCR2 CX3CR

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neurotrophic factors (growth factors acting on neurons and/or glial cells), cytokines (factors regulating development and function of hematopoietic cells and cells of the immune system), and chemokines (factors regulating motility of immune cells) to be discussed, with their associated receptors. Corresponding factors and receptors that have been found on astrocytes in culture but presently have not been demonstrated on astrocytes in vivo (e.g., the epithelial growth factor family) are not included. These data from the literature will be compared with our cDNA microarray results (manuscript in preparation). The microarray methodology has become a popular way to examine expression of a variety of genes simultaneously (reviewed in Luo and Geschwind, 2001). We have specifically taken advantage of microarrays to determine striatal astrocyte genes that are affected following substantia nigra lesion with 6-OHDA. Reactive astrocytes are induced by the 6-OHDA lesion, with the peak number of cells and intensity of GFAP increase occurring seven days after the lesion (Sheng et al., 1993). Combining this animal model of Parkinson’s disease (PD) with the new methodologies for isolating acutely dissociated astrocytes allowed us to pick up individual cells from normal adult rat brain striatum and from the striatum on the lesioned side of the 6-OHDA-lesioned brain. RNA was prepared and the identity of astrocytes confirmed by single cell RT-PCR for GFAP mRNA (Zhou et al., 2000). Amplification of the cDNA allowed preparation of sufficient material to carry out microarray analysis using Clontech nylon arrays (Eberwine et al., 1992, 2001; Spirin et al., 1999; Wang et al., 2000). The results from the literature will be compared with these results, and we will refer to the astrocytes isolated in vivo as ‘in vivo reactive astrocytes’.

2. Neurotrophins The family of neurotrophins, of which nerve growth factor (NGF) was the first to be isolated and studied, was originally localized to target neurons. More recent data, using immunohistochemistry and in situ hybridization, have shown expression of NGF, brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in astrocytes in normal adult brain (Arendt et al., 1995; Dreyfus et al., 1999; Fiedorowicz et al., 2001; Knott et al., 2002) and spinal cord (Dreyfus et al., 1999) in vivo. To date, no studies have examined astrocyte expression of neurotrophins developmentally. Expression of both NGF and BDNF is enhanced in reactive astrocytes induced by a variety of lesions in animals, including spinal cord transection (Krenz and Weaver, 2000), kainic acid (Bakhit et al., 1991; Strauss et al., 1994), electrolytic lesion (Oderfeld-Nowak et al., 1992), stab wound (Goss et al., 1998), ischemia (Shozuhara et al., 1992) and insertion of a nitrocellulose filter (McKeon et al., 1997). Elevated levels of NGF in reactive astrocytes are reported following ethanol or trimethyltin toxicity in rats (Arendt et al., 1995; Koczyk and Oderfeld-Nowak, 2000; Fiedorowicz et al., 2001), while the content of BDNF and NT-3 in reactive astrocytes is elevated in PD and multiple sclerosis (MS) (Knott et al., 2002; Stadelmann et al., 2002). Along with this increased synthesis of the neurotrophins comes increased expression of their receptors. The neurotrophin receptors trkA, trkB and trkC (Table 1) have all been identified on astrocytes in normal adult brain (Knott et al., 2002; Stadelmann et al., 2002), and are present on reactive astrocytes

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in PD (Knott et al., 2002; Stadelmann et al., 2002). Experimental autoimmune encephalitis (EAE) upregulates trkA expression on reactive astrocytes in the spinal cord (Oderfeld-Nowak et al., 2001), while transection or mechanical trauma increases both trkA and trkB (Frise´n et al., 1993; Foschini et al., 1994; Dougherty et al., 2000), and implantation of a nitrocellulose filter elevates expression of trkB (McKeon et al., 1997). In the brain, trkB expression is induced on reactive astrocytes by trimethyltin toxicity (Koczyk and Oderfeld-Nowak, 2000), and by nitrocellulose filter implants (McKeon et al., 1997). These findings suggest autocrine or paracrine effects of NGF and the other neurotrophins on astrocytes. Our analyses of in vivo reactive astrocytes found increased content of trkB and NGF.

3. TGF-b superfamily Glial cell derived neurotrophic factor (GDNF) was first cloned from a glial cell line (and named ‘glial cell-line derived neurotrophic factor’), but like the neurotrophins, it and the other family members, neurturin, artemin and persephin, have been localized primarily to neurons in vivo (Stro¨mberg et al., 1993; Poulsen et al., 1994). However, there is one report of GDNF being expressed in adult astrocytes, measured by in situ hybridization (Ho et al., 1995), and both kainic acid lesions (Bresjanac and Antauer, 2000; Marco et al., 2002) and ischemia (Miyazaki et al., 2001) induce its expression in reactive astrocytes. Kainate also induces expression of the GDNF receptor a1 (GFRa1) on reactive astrocytes (Bresjanac and Antauer, 2000; Marco et al., 2002). Our results for in vivo reactive astrocytes also show increased expression of GDNF mRNA.

4. Insulin-like growth factor family Insulin-like growth factor-1 (IGF-1) has not been detected in astrocytes under control conditions although the IGF-Binding Protein-2 (IGFBP-2) has (Lee et al., 1993). Cuprizone exposure turns on IGF-1 expression in reactive astrocytes (Mason et al., 2001), while either ischemia or high potassium enhances expression of IGFBP-2 (Lee et al., 1997; Holmin et al., 2001). In contrast, we find that IGF-1 is unchanged in in vivo reactive astrocytes, IGFBP-2 expression is decreased, and IGFBP-1 and IGFBP-3 are increased.

5. Fibroblast growth factors Astrocyte expression of fibroblast growth factor 2 (basic FGF) and of its receptor FGFR1 has been shown for both neonatal astrocytes (Smith et al., 2001; Ganat et al., 2002) and for adult astrocytes (Clarke et al., 2001) in vivo. Furthermore, it has been well established that FGF-1 and FGF-2 are increased in reactive astrocytes following a variety of insults, including electrolytic lesions (Buytaert et al., 2001), ischemia (Ganat et al., 2002), stab wounds (Smith et al., 2001; Ganat et al., 2002), and spinal cord transection

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(Leme and Chadi, 2001). FGF-2 stimulates astrocytic proliferation and differentiation into a reactive phenotype in vivo (Eclancher et al., 1996) and also induces astrocyte migration in vivo (Holland and Varmus, 1998). Thus, FGF-2 may also have autocrine or paracrine effects on astrocytes. 6. Vascular endothelial growth factor Vascular endothelial growth factor (VEGF) has neurotrophic effects in addition to its better-known angiogenic effects (Sondell et al., 2000; Wick et al., 2002). Moreover, VEGF stimulates proliferation of astrocytes in vivo (Krum et al., 2002). It is expressed only in neurons in control adult brain (Papavassiliou et al., 1997; Krum and Rosenstein, 1998) but expression is turned on in reactive astrocytes following ischemia (Beck et al., 1993; Salhia et al., 2000), stab wound (Papavassiliou et al., 1997; Krum and Rosenstein, 1998; Salhia et al., 2000; Sko¨ld et al., 2000), freeze injury (Papavassiliou et al., 1997) or implantation of grafts (Krum and Rosenstein, 1998). In addition, VEGF expression is elevated in the senile plaques of Alzheimer’s disease (Salhia et al., 2000). Two receptors have been identified, Flt-1 or VEGFR-1, and Flk-1 or VEGFR-2. Stab wounds and graft implantation (Krum and Rosenstein, 1998), as well as infusion trauma (Krum et al., 2002), induce expression of Flt-1 on reactive astrocytes, while Sko¨ld et al. (2000) showed the presence of Flk-1 on reactive astrocytes in the spinal cord following transection.

7. Neuropoietic cytokines The family of neuropoietic cytokines (Allan and Rothwell, 2001), that includes ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), IL-6, oncostatinM and cardiotropin, has a variety of effects in the CNS. The family shares one common receptor component, gp130, with different additional components required by each of the various family members (Table 1). CNTF itself has been implicated in induction of reactive gliosis (Winter et al., 1995; Clatterbuck et al., 1996; Levison et al., 1996; Albrecht et al., 2002), although other work has not supported that conclusion (Emerich et al., 1996; Lisovoski et al., 1997). Both CNTF and IL-6 have been demonstrated in neonatal astrocytes in vivo (Acarin et al., 2000; Widenfalk et al., 2001). CNTF was reported to be present in normal adult astrocytes (Widenfalk et al., 2001; Dallner et al., 2002), although Lee et al. (1997) did not find it. Friedman (2001) could not detect IL-6 in normal adult astrocytes. A variety of injuries are capable of inducing either CNTF, e.g., a stab wound (Dallner et al., 2002) or spinal cord transection (Widenfalk et al., 2001), or IL-6, e.g., NMDA administration (Acarin et al., 2000) or ischemia (Friedman, 2001) in reactive astrocytes. In addition, induction of long term potentiation turns on IL-6 expression in astrocytes, although it remains to be established whether they are reactive (Jankowsky et al., 2000). The CNTF-specific receptor subunit CNTFRa was shown to be expressed on normal adult astrocytes (Dallner et al., 2002) and to be increased following a stab wound (Lee et al., 1997). Other receptor subunits have not been examined. Our results show induction of the IL-6Ra on in vivo reactive astrocytes. LIF was also expressed in in vivo reactive

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astrocytes, in support of results from Banner et al. (1997) using a stab wound model, whereas Lemke et al. (1999) have not detected it in astrocytes after 192IgG-saporin cholinergic lesion.

8. Pro-inflammatory cytokines IL-1b, TNF-a, and IL-6 have often been referred to as the pro-inflammatory cytokines, although it is clear that IL-6 may be protective under certain conditions in the CNS (Merrill and Benveniste, 1996; Allan and Rothwell, 2001). As discussed in Section 7, IL-6 is induced in reactive astrocytes following various forms of injury. Neither IL-1b nor TNF-a has been detected in either neonatal (Acarin et al., 2000) or adult astrocytes (Friedman, 2001). However, NMDA treatment induces not only IL-6 (see above), but also IL-1b and TNF-a in reactive astrocytes (Acarin et al., 2000). Ischemia (Orzylowska et al., 1999; Friedman, 2001) and cuprizone exposure (Mason et al., 2001) both induce expression of IL-1b and IL-6 in reactive astrocytes. The IL-1b receptor subunit IL-1R1, expressed on neurons developmentally, is induced on reactive astrocytes following a stab wound (Friedman, 2001). We find that both TNF-a and its receptor are expressed on in vivo reactive astrocytes.

9. Anti-inflammatory cytokines None of the anti-inflammatory cytokines, TGF-b, IL-1ra (the IL-1R antagonist), IL-4, and IL-10 (Allan and Rothwell, 2001), is expressed in neonatal astrocytes (Acarin et al., 2000) although IL-4 and IL-10 have been identified in adult astrocytes (Hulshof et al., 2002). Acarin et al. (2000) have shown that NMDA treatment induces expression of TGF-b in reactive astrocytes, while both IL-4 and IL-10, as well as their receptors, are turned on in reactive astrocytes in MS plaques (Hulshof et al., 2002). The a subunit of IL-4R has also been reported to be present on reactive astrocytes in the vicinity of epileptic foci and CNS tumors (Liu et al., 2000). No changes were detected in our in vivo reactive astrocytes. 10. Chemokines Chemokines, a large family of proteins that stimulate motility of immune cells, have been classified into four groups, of which three have been found in astrocytes in the brain in vivo, whereas no data are available suggesting any function of the fourth chemokine family member (C) in astrocytes. We will discuss some of the members of the CC family, the CXC family, and the CX3C member fractalkine (Table 1). Corresponding to these families are families of receptors, designated the CC, CXC, and CX3C receptors. The interest in this group of compounds has grown very considerably following the discovery that they play major roles in the neurological complications of HIV (see chapters by Zsembery et al. and by Ghorpade and Gendelman).

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Only one of the CC family chemokines, MIP-1b, has been detected in control adult astrocytes (Sanders et al., 1998). Che et al. (2001) showed that MCP-1 is induced by ischemia in rats. MIP-1b has been identified in reactive astrocytes in brains from patients with HIV-encephalitis (Sanders et al., 1998) and Alzheimer’s disease (Xia et al., 2000). MCP-1 expression occurs in reactive astrocytes in EAE, the animal model of MS (DeGroot and Woodroofe, 2001), as well as in MS (Simpson et al., 2000; DeGroot and Woodroofe, 2001). The other CC chemokines, including MCP-2, MCP-3, MIP-1a, MIP1b, and RANTES (regulated on activation, normal T cell expressed and secreted), are also expressed in reactive astrocytes in MS (DeGroot and Woodroofe, 2001). Toxoplasma encephalitis results in MCP-1, but not RANTES, expression in reactive astrocytes (Strack et al., 2002). Results on the expression of the CC receptors in control adult brain astrocytes is highly variable, with lack of expression of CCR1, CCR3 and CCR5 reported (Ghorpade et al., 1998; Sanders et al., 1998) but positive expression for CCR3 and CCR5 also reported (Rottman et al., 1997; Ghorpade et al., 1998; Westmoreland et al., 1998; Simpson et al., 2000; Otto et al., 2001). CCR2, CCR3 and CCR5 are all present on reactive astrocytes in MS plaques (Simpson et al., 2000) and in both macaque SIV-encephalitis (Westmoreland et al., 1998) and human HIV-encephalitis brain (Ghorpade et al., 1998). MIP-1a was induced in in vivo reactive astrocytes. Three members of the CXC family have been localized to astrocytes in the CNS. Of these, Mig is not present under normal conditions (Asensio et al., 2001), IL-8 has been detected (Sanders et al., 1998), and reports about IP-10 are conflicting (Xia et al., 2000; Asensio et al., 2001). All are elevated in human diseases. IP-10 is present in reactive astrocytes in brains from patients with HIV-encephalitis (Sanders et al., 1998) and in an animal model of HIV encephalopathy (Asensio et al., 2001), in MS (and EAE) (DeGroot and Woodroofe, 2001) and Alzheimer’s disease (Xia et al., 2000), as well as in Toxoplasma encephalitis (Strack et al., 2002). Mig is also found in reactive astrocytes in MS (DeGroot and Woodroofe, 2001) but not following Toxoplasma infection (Strack et al., 2002). IL-8 is induced in reactive astrocytes in HIV-encephalitic brain (Sanders et al., 1998). There are reports of expression of the receptors CXCR2 and CXCR4 on astrocytes in adult brain (Westmoreland et al., 1998; Otto et al., 2001), whereas CXCR3 has been localized to neurons (Xia et al., 2000) and is not found on astrocytes (Asensio et al., 2001). Expression of CXCR2 is enhanced on reactive astrocytes after a stab wound (Otto et al., 2001). CXCR3 is expressed on reactive astrocytes in MS plaques (DeGroot and Woodroofe, 2001) while CXCR4 is present on reactive astrocytes in the HIV-encephalitic brain (Sanders et al., 1998). Fractalkine, the only CX3C ligand, is present in adult astrocytes and increased in reactive astrocytes in a prion model of chronic neurodegeneration and inflammation (Hughes et al., 2002). None were detected in our studies.

11. Concluding remarks This review supports the view that whereas astrocytes produce neurotrophic factors and cytokines in the developing and the injured brain, expression in the normal adult brain is very low. Factors fall into one of three patterns: many are not expressed at all in the normal

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adult brain, but can be induced in reactive astrocytes (IGF-1, IL-1b, TNF-a, most of the chemokines); some are expressed at low levels in normal astrocytes (NTFs, FGFs, CNTF, MIP-1b, IL-4, IL-8 and IL-10) and upregulated in reactive astrocytes; and some are expressed exclusively in neurons, but can be induced in reactive astrocytes in response to injury (GDNF family, VEGF, IL-6). In addition to their functions as neurotrophic and immunological factors, these factors can have other functions such as stimulating the proliferation, differentiation and migration of astrocytes, and enhancing both angiogenesis and gliogenesis. Induction of many of the receptors on reactive astrocytes suggests a set of autocrine/paracrine functions. By comparing these factors in the normal versus the injured brain, versus the developing brain, we will further understand which functions are important in which circumstances. Our microarray analysis of gene expression in acutely isolated astrocytes from the rat 6OHDA model of PD revealed upregulation of a number of the same neurotrophic factor and cytokine genes as are seen in other models of injury. Too few studies have examined brains from PD to provide the data with which to compare ours, but in general the microarray findings support the idea that a specific brain lesion induces a specific increase in a specific set of genes. Understanding which genes those are, and what the functions of their respective products are, may allow us to address treatments for these neurological diseases.

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The nitric oxide/cyclic GMP pathway in CNS glial cells Agustina Garcı´aa,* and Marı´a Antonia Baltronsb a

Instituto de Biotecnologı´a y Biomedicina V. Villar Palası´, Universidad Auto´noma de Barcelona, 08193 Bellaterra (Cerdanyola del Valle´s), Barcelona, Spain * Correspondence address: E-mail: [email protected](A.G.) b Instituto de Biotecnologı´a y Biomedicina V. Villar Palası´ and Departamento de Bioquı´mica y Biologı´a Molecular, Universidad Auto´noma de Barcelona, 08193 Bellaterra (Cerdanyola del Valle´s), Barcelona, Spain

Contents 1. 2.

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5. 6.

Introduction NO formation in glial cells 2.1. Expression and regulation of calcium-dependent NOS isoforms 2.2. Induction and regulation of NOS2 Cyclic GMP synthesis 3.1. Guanylyl cyclases 3.2. NO-sensitive guanylyl cyclase expression in glial cells 3.3. Regulation of NO-sensitive guanylyl cyclase in astroglial cells Cyclic GMP inactivation in astroglial cells 4.1. Cyclic GMP phosphodiesterases 4.2. Cyclic GMP efflux Targets and actions of cGMP in glial cells Concluding remarks

The NO-cGMP signaling cascade participates in essential CNS functions. NO has been also recognized as a neuropathological agent mediating excitotoxic cell death and neuroinflammatory cell damage. All CNS parenchymal cells have the capacity to synthesize NO but only neurons and astroglial cells appear to express all the molecular components required for NO-cGMP-PKG signaling. Recent evidence implicates this pathway in the regulation of important aspects of astroglial physiology—calcium homeostasis, gene expression and survival—that are relevant for neuronal function. 1. Introduction Shortly after the identification of NO as the endothelium-derived relaxing factor (Ignarro et al., 1987; Palmer et al., 1987), it was reported that this free radical gas was also Advances in Molecular and Cell Biology, vol. 31, pages 575–593 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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a signaling molecule in the CNS. The observation that stimulation of cyclic GMP (cGMP) formation by excitatory amino acids in brain cells was mediated by NO (Garthwaite et al., 1988) lead to a plethora of studies that have established the role of NO as an atypical neurotransmitter in both CNS and PNS, as well an important modulator of synaptic plasticity processes underlying memory formation and behavior, brain development, visual and sensory processing, neuroendocrine secretion and cerebral blood flow. Moreover, NO has been recognized as a neuropathological agent responsible for excitotoxic cell death and neuroinflammatory cell damage in conditions such as epilepsy, stroke and neurodegenerative disorders (for reviews see Zhang and Snyder, 1995; Szabo´, 1996; Bredt, 1999; Bolan˜os and Almeida, 1999; Murphy, 2000; Garthwaite, 2000). NO signaling begins with its synthesis by a nitric oxide synthase (NOS). Three NOS isoforms (types 1– 3) encoded by distinct genes and several splice variants have been identified. In their active form NOS are homodimers that catalyze the formation of NO and L -citrulline from L -arginine, using O2 and NADPH as co-substrates, and FMN, FAD, heme and tetrahydrobiopterin as cofactors. NOS1 and NOS3, also known as nNOS and eNOS because they were first identified in neurons and endothelial cells, are constitutively expressed and activated by binding calmodulin in a reversible calcium-dependent manner. In contrast, NOS2 or iNOS, originally identified in macrophages, is regulated by transcriptional induction and does not require calcium increase for activity, since it has tightly bound calmodulin (for reviews see Fo¨rstermann and Kleinert, 1995; Nathan, 1997; Alderton et al., 2001). The three NOS isoforms can be expressed in CNS parenchymal cells. In the normal brain, NOS1, predominantly expressed in discrete populations of neurons, is the main NOS responsible for controlled, transient generation of low concentrations of NO. However, in neuropathological conditions associated with inflammation, glial cells become an important source of sustained and high production of NO by the action of NOS2. As in the cardiovascular system, the major physiological target for NO in the CNS is a NO-sensitive guanylyl cyclase that catalyzes the conversion of GTP into cGMP. This nucleotide mediates most of the low concentration effects of NO through interaction with cGMP-dependent protein kinases (PKGs), cGMP-regulated phosphodiesterases (PDEs) and cGMP-regulated ion channels (Schmidt et al., 1993; Garthwaite, 2000; Lucas et al., 2000). In contrast to the potentially ubiquitous NO synthesis in CNS cells, NO-dependent cGMP formation appears to occur mainly in neurons and astrocytes. The role of NO as a signaling molecule in neuronal cells and as a pathological agent in CNS disorders has been extensively reviewed. Here we review evidence on the expression and regulation of components of the NO-cGMP signaling pathway in CNS glial cells and their implication in the regulation of glial and neuronal activity. 2. NO formation in glial cells 2.1. Expression and regulation of calcium-dependent NOS isoforms Most of the NOS1 constitutively expressed in brain cells is found in discrete populations of interneurons in different brain nuclei (Zhang and Snyder, 1995; Bredt, 1999). Of the three splice variants of NOS1 (a, b and g) expressed in rodent brain,

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NOS1a is the most abundant and the predominant form in neurons. NOS1a contains exon 2 that carries a PDZ domain that is absent in the b and g variants. The PDZ domain interacts with postsynaptic density protein PSD-95 that also binds to glutamate NMDA receptors, thus allowing an efficient stimulation of NOS1 by calcium entering through the NMDA channel (reviewed by Tomita et al., 2001). The presence of the other calcium-dependent isoform NOS3 in populations of neurons has been also reported (Dinerman et al., 1994). Although initially controversial there is now solid evidence that NOS1 and NOS3 are also expressed in populations of astrocytes. In contrast, calciumdependent NOS activities have not been detected in microglia or oligodendrocytes (Agullo´ et al., 1995; Keilhoff et al., 1998). The first indication of the expression of calcium-dependent NOS activities in astrocytes was obtained using primary cultures from rat brain (reviewed in Murphy et al., 1993). In our laboratory, results from a comparative study in cultured forebrain neurons and astrocytes, using a series of agonists known to stimulate cGMP formation in different CNS preparations, showed that while several agonists stimulated NOdependent cGMP formation in astrocytes, only glutamate (acting on NMDA receptors) gave significant responses in neurons (Agullo´ and Garcı´a, 1992). Noradrenaline acting on a1-adrenoceptors was the most effective agonist in astrocytes from different brain regions and the largest response was observed in cerebellar cells (Agullo´ et al., 1995). Since noradrenaline had no effect on neuronal cultures we suggested astrocytes as the site of the a1-adrenoceptor-mediated stimulation of cGMP formation well documented in cerebellar slices (Wood, 1991). We later showed that glutamate acting on AMPA receptors and endothelin acting through ETA receptors also elicited large responses in cerebellar astroglia (Baltrons and Garcı´a, 1997; Saadoun and Garcı´a, 1999). Immunoreactivity towards different anti-NOS1 antibodies as well as the histochemical reaction for NADPH diaphorase was demonstrated in the cerebellar astroglial cultures fixed with low concentration of aldehyde (Arbone´s et al., 1996). Immunohistochemical studies using higher aldehyde concentrations showed weak NOS1 immunoreactivity in astroglial cells in different brain regions of rodents and humans (Schmidt et al., 1992; Wendland et al., 1994; Egberongbe et al., 1994; Lu¨th, 1997). More convincing evidence for the expression of NOS1 in astrocytes in situ was reported by Kugler and Drenckhahn (1996). These authors, using freeze-dried tissue sections to minimize diffusion artifacts and avoid loss of antigenicity due to fixation with aldehydes, demonstrated strong NOS1 immunostaining in rat hippocampal and cerebellar astrocytes and Bergmann glia. The NOS1 expressed in rat cerebellar astroglial cells has the same biochemical characteristics (Km for L -arginine, EC50 for calcium, inhibitory potency of L -arginine analogs) as the isoform expressed in cerebellar granule cells, but the astroglial activity is totally cytosolic, while a significant fraction of the neuronal activity is membrane-bound (Arbone´s et al., 1996). The more labile association of the astroglial enzyme to membranous structures could explain its higher sensitivity to fixation conditions, and it suggests that it may be a variant lacking the PDZ motif. This suggestion is supported by a recent report showing increased expression of the PDZ-lacking NOS1 splice variants b and g in astrocytes of amyotrophic lateral sclerosis (ALS) patients (Catania et al., 2001). This and other studies additionally suggest a differential regulation of the neuronal and

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astroglial NOS1 variants and an implication of the astroglial enzyme in brain pathologies. In ALS patients (Catania et al., 2001) and in a transgenic mice model of ALS, overexpressing a human SOD-1 mutation (Cha et al., 1998), NOS1 is up-regulated in reactive astrocytes in the spinal cord but not in neurons. Intense NOS1 immunopositive reactive astrocytes have also been observed around b-amyloid plaques in hippocampus and entorhinal cortex of brains from Alzheimer disease patients (Simic et al., 2000), and increased NOS1 expression has been documented in astrocytes after cortical spreading depression (Caggiano and Kraig, 1998; Shen and Gundlach, 1999). In this context, NO produced by astroglial NOS1 may be involved in immunomodulatory actions of neurotransmitters, as suggested by reports showing that it inhibits expression of MHC class-II (Heuschling, 1995) and activation of the redox-sensitive transcription factor NFkB (Togashi et al., 1997). In recent studies, NOS3 immunoreactive astrocytes have been detected in different brain regions of rat and primate species (Gabbot and Bacon, 1996; Wiencken and Casagrande, 1999). As for NOS1 (Kugler and Drenckhahn, 1996), astrocyte processes immunopositive for NOS3 were observed in close contact with the external surface of capillary walls, suggesting that NO generated in astroglia in response to neurotransmitters may couple neuronal activity to changes in blood flow. However, a role for astroglial NOS3 in brain pathologies is also suggested by the observation of increased NOS3 expression in rodent brain astrocytes following a neurotropic viral infection and after intraperitoneal injection of cytokines or bacterial endotoxin (lipopolysaccharide; LPS) (Barna et al., 1996; Iwase et al., 2000), as well as in brains of victims of Alzheimer’s disease and other neurodegenerative conditions (Sohn et al., 1999).

2.2. Induction and regulation of NOS2 Since the initial demonstration ten years ago (reviewed by Murphy et al., 1993), that LPS and combinations of proinflammatory cytokines (IL-1b, TNF-a, INF-g) induced NOS2 expression in rodent microglial and astroglial cultures, and that glial-derived NO was toxic to neurons and oligodendrocytes, a large amount of literature has accumulated showing expression of NOS2 in several inflammatory and degenerative diseases of the CNS. The controversial issue of the implication of NO produced by NOS2 in neurotoxicity/neuroprotection continues to stimulate interest in the investigation of the cellular source and factors that regulate NOS2 expression. A large amount of evidence gathered both in vitro and in vivo indicates that in rodent brain microglia and astroglia are the main cellular site of NOS2 expression. Studies in cultured cells have shown that apart from LPS and cytokines other substances that participate in pathogenic processes leading to neurodegeneration, such as b-amyloid peptides (Rossi and Bianchini, 1996; Hu et al., 1998), protein S100b (Hu et al., 1996), HIV coat proteins (Koka et al., 1995; Hori et al., 1999), chromogranin A (Taupenot et al., 1996) and prion protein fragments (Fabrizi et al., 2001) cause transcriptional activation of NOS2. Additionally, astroglial and/or microglial NOS2 induction has been detected in brains of animals with acute and chronic viral infection (Sun et al., 1995; Grzybicki et al., 1997),

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following traumatic brain injury (Grzybicki et al., 1998), global and focal cerebral ischemia (Endoh et al., 1994; Loihl et al., 1999), excitotoxic damage (Calka et al., 1996; Acarin et al., 2002), or experimental allergic encephalitis (Okuda et al., 1997) and in transgenic mouse models of familial ALS (Almer et al., 1999) and Alzheimer’s disease (Lu¨th et al., 2001). In contrast, the predominant site of NOS2 expression in humans appears to be astrocytes rather than microglia. NOS2 induction in response to IL-1b and INF-g or TNF-a is readily observed in fetal and adult human astrocytes (Lee et al., 1993; Hu et al., 1995; Zhao et al., 1998), but only occasionally demonstrated in microglia (Colasanti et al., 1995; Ding et al., 1997). Astroglial NOS2 immunoreactivity has been reported in postmortem brain tissue of patients suffering from Multiple Sclerosis (Bo¨ et al., 1994), Alzheimer’s disease (Wallace et al., 1997), Parkinsonism (Hunot et al., 1996) and Krabbe’s disease (Giri et al., 2002) and in the optic nerve head of human glaucomatous eyes (Liu and Neufeld, 2000). There is increasing evidence from in vitro and in vivo studies that NOS2 can also be expressed in immunostimulated neurons in rodents and humans (review by Heneka and Feinstein, 2001). However, expression of NOS2 in oligodendrocytes has been demonstrated only in cultured rodent cells (Merrill et al., 1997; Molina-Holgado et al., 2001). Expression of glial NOS2 is mainly regulated at the transcriptional level, although changes in mRNA stability in response to some agents have also been described (review in Murphy, 2000; Colasanti and Persichini, 2000; Baltrons and Garcı´a, 2001). As found for other cell types, mitogen-activated protein kinase (MAPK)-mediated signaling appears to play a prominent role in endotoxin and cytokine transcriptional activation of glial NOS2. The promoter regions of NOS2, as well as other proinflammatory molecules, contain consensus binding sites for numerous transcription factors such as NFkB, AP-1, CRE and C/EBP (Eberhardt et al., 1996) that are directly or indirectly phosphorylated by MAPKs. NFkB, whose activation has been described during neurological disease and trauma (Mattson and Camandola, 2001), plays a key role in the transcriptional activation of NOS2 expression. The activation of NFkB involves its release from a complex with IkB followed by translocation to the nucleus. Phosphorylation of IkB by a cytokine-activated kinase marks it for destruction by the 20S proteasome. Several factors prevent NOS2 induction by interfering with NFkB activation or binding to the gene promoter. In astrocytes, as in other cells, exogenous NO has been shown to reduce NOS2 transcriptional activation by both of these mechanisms, suggesting an explanation for the transient expression of NOS2 (review in Colasanti and Persichini, 2000). The heat shock response induced by hyperthermia or treatment with the fungal-derived antibiotics ansamycins blocks LPS and cytokinedependent activation of NFkB and expression of NOS2 in cultured astroglia and in vivo by increasing IkBa (Feinstein et al., 1996; Heneka et al., 2000; Murphy et al., 2002). The same mechanism appears to be responsible for suppression of NFkB activation and NOS2 induction by other anti-inflammatory compounds like glucocorticoids (Quan et al., 2000). Furthermore, the neurotransmitter noradrenaline, that blocks NOS2 expression in astrocytes via the cAMP/PKA pathway, increases IkB expression (Galea and Feinstein, 1999). Conversely, depletion of central noradrenaline levels by lesioning of the locus ceruleus reduces IkB levels and potentiates inflammatory responses to LPS (Gavrilyuk et al., 2002).

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3. Cyclic GMP synthesis 3.1. Guanylyl cyclases Cyclic GMP is formed by an ubiquitous family of guanylyl cyclases (GCs) that comprises peptide-regulated and NO-sensitive isoforms. Peptide-regulated GCs or particulate GCs (pGCs) are homodimers or homotetramers of single membrane-spanning subunits that contain a N-terminal extracellular ligand binding domain and intracellular regulatory and catalytic domains (for reviews see Garbers, 1999; Lucas et al., 2000). The pGCs expressed in the CNS belong to the natriuretic peptide receptor group. Using immunocytochemical techniques, accumulation of cGMP in response to atrial natriuretic peptides has been observed in astrocytes in restricted areas of the adult rat brain, but only in a few neuronal structures (De Vente and Steinbusch, 2000), and not in oligodendrocytes or microglial cells (Tanaka et al., 1997). The NO-sensitive GC, a major target for NO, was until very recently (see below) considered a cytosolic enzyme and usually referred to as soluble GC. For the sake of simplicity we will use the abbreviation sGC for the NO-sensitive GC throughout this article. sGCs are largely heterodimers of a and b subunits containing one protoporphyrin IX type heme that is the binding site for NO. Each subunit contains a N-terminal domain involved in heme binding, a central domain thought to be implicated in dimerization and a C-terminal catalytic domain homologous to pGC and adenylyl cyclase catalytic domains. The a1 and a2 subunits can form functional heterodimers with the b1 subunit that are functionally indistinguishable, and both a1b1 and a2b1 have been found at the protein level (for reviews see Denninger and Marletta, 1999; Russwurm and Koesling, 2002). The b2 subunit, primarily expressed in kidney, presents activity in the absence of other subunits (Koglin et al., 2001). Recent reports question the concept of NO-activated GC as a soluble enzyme. It has been shown that the a2/b1 isoform associates with brain membranes by interaction of the a2 subunit C-terminal amino acids with a PDZ domain of PSD-95 (Russwurm et al., 2001). This interaction will position the enzyme close to the NMDA receptor-activated NOS1 (see Section 2.1) leading to an efficient stimulation of NO-dependent cGMP formation by glutamate. However, NOS1 associated sGC may be a minor portion of brain sGC, since immunocytochemical studies have generally found that co-localization of NOS1 and NOstimulated cGMP accumulation is minor, whereas independent but complementary staining is abundant throughout the brain (reviewed by De Vente and Steinbusch, 2000). The a1b1 dimer was also reported to be partially associated with membranes in different tissues, including brain, in a state sensitized to NO. Activation of the enzyme increases its translocation to the membrane in a manner dependent on increased intracellular calcium (Zabel et al., 2002). This regulatory mechanism contrasts with the reported inhibition of NO-stimulated sGC activity by calcium (Parkinson et al., 1999). 3.2. NO-sensitive guanylyl cyclase expression in glial cells In situ hybridization studies in adult (Matsuoka et al., 1992; Furuyama, 1993) and postnatal (Gibb and Garthwaite, 2001) rat brain demonstrate a widespread expression of

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the sGC b1 subunit and a more limited distribution of the a1 and a2 subunits, indicating that different regions predominantly express either the a1b1 or the a2b1 isoform. A differential expression of a1 and b1 subunits in regions of the human brain was also reported (Zabel et al., 1998). Early immunohistochemical studies with antibodies against purified sGC demonstrated immunoreactivity in rat brain neurons and astrocytes (Ariano et al., 1982; Nakane et al., 1983). Furthermore, differential lesioning of cell types in slice preparations (Garthwaite and Garthwaite, 1987) and measurement of GC activity in acutely dissociated cells from rat cerebellum (Bunn et al., 1986) indicated that astrocytes are a major site of NO-dependent cGMP accumulation. In agreement with this, cGMP immunoreactivity was demonstrated in astrocytes in different regions of adult and immature rat brain slices stimulated with NO donors, more prominently in hippocampal and cerebellar astrocytes and in Bergmann glia (reviewed by De Vente and Steinbusch, 2000). Recently, the b1 subunit of sGC was shown to co-localize with cGMP inmunoreactivity in hippocampal astrocytes (Teunissen et al., 2001). In contrast, cGMP immunoreactivity in oligodendrocytes is only observed in immature but not adult rat brain and microglia does not appear to accumulate cGMP in response to NO (Tanaka et al., 1997). Our studies in primary cultures also demonstrate regional differences in NOdependent cGMP formation in rat astrocytes and the lack of sGC activity in microglia (Agullo´ et al., 1995). In humans, information about cGMP formation in glial cells is scant. In one report fetal cortical astrocytes in culture were shown to accumulate low levels of cGMP after induction of NOS2 (Ding et al., 1997). Our recent results using a similar preparation (T. Sardo´n, M.A. Baltrons and A. Garcı´a, unpublished) show NO donorstimulated cGMP accumulation in human astrocytes in the presence of a phosphodiesterase inhibitor (Fig. 1A and B).

3.3. Regulation of NO-sensitive guanylyl cyclase in astroglial cells Despite the recognition of sGC as a major receptor for NO, mediating numerous of its physiological functions there is little information about the mechanisms that regulate its activity in living cells. Studies in rat cerebellar astrocytes show that sGC located within cells is activated by NO with higher potency and faster activation and deactivation than the purified enzyme. Furthermore, within seconds of adding NO, desensitization rapidly occurs in cells but not with the purified enzyme, suggesting the existence of regulatory factors (Bellamy et al., 2000; Bellamy and Garthwaite, 2001a). These characteristics indicate that in the cellular environment sGC behaves like a neurotransmitter receptor. As occurs after chronic exposure of receptors to their agonists, continuous exposure to NO donors has been shown to down-regulate sGC in different cell types (Ujiie et al., 1994; Papapetropoulos et al., 1996). In smooth muscle cells, this phenomenon that is thought to contribute to nitrovasodilator-induced tolerance, apparently results from a decrease in subunit mRNA stability (Filippov et al., 1997). A similar effect is produced by endogenous NO generated by NOS2 in cells treated with proinflammatory cytokines (Takata et al., 2001). In contrast, in rat astroglial cultures we have shown that chronic treatment with LPS or IL-1b decreases sGC activity and b1 subunit levels in a NO-independent manner

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Fig. 1. NO-stimulated cGMP accumulation in human fetal cortical astrocyte cultures. (A) Cultures stimulated with sodium nitroprusside (SNP; 100 mM, 10 min) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM) were double immunofluorescence labeled with sheep antiformaldehyde fixed cGMP (1:4000) and mouse anti-glial fibrillary acidic protein (GFAP; 1:350), visualized with fluorescein-conjugated anti-sheep and rhodamine-conjugated anti-mouse IgGs, respectively; magnification 630 £ in the negative. (B) Time course of cGMP accumulation in response to diethylamine/NO (2 mM) in the presence or absence of IBMX (1 mM) or the guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin1-one (ODQ; 1 mM) added 10 min before. (C) Cell cultures were treated or not with human recombinant IL-1b (100 U/ml), TNF-a (100 U/ml) or INF-g (200 U/ml) for 72 h, washed and stimulated with SNP in the presence of IBMX.

(Baltrons and Garcı´a, 1999; Pedraza et al., 2002). Down-regulation of the b1 subunit is due to a decrease in the half-life of the protein and requires transcription and protein synthesis, suggesting that the inflammatory agents induce the expression of a protein that is directly or indirectly responsible for sGC degradation. Additionally, LPS and IL1b induce a decrease in sGC a1 and b1 subunit mRNA levels that is NO-dependent. The latter mechanism will contribute to maintain sGC levels low under conditions of prolonged reactive gliosis, associated with high levels of NO production (Fig. 2), and it may explain the long time required for recovery of sGC activity after removal of the inflammatory compound (Baltrons and Garcı´a, 1999). Treatment with b-amyloid peptides that induce astroglial reactivity similarly down-regulates sGC (Baltrons et al., 2002). The decrease in sGC protein and mRNA also occurs in rat brain after intracerebral administration of LPS, IL-1b or b-amyloid peptides (Baltrons et al., 2002; Pedraza et al., 2002). Inflammatory agents may exert a similar effect in human brain since sGC activity was reported to be decreased in brain of Alzheimer’s disease patients (Bonkale et al., 1995). In agreement with this observation, our recent results show that IL-1b and INF-g, but not TNF-a decrease NO-stimulated cGMP formation in human foetal astroglia-enriched cultures (Fig. 1C). The relevance of impaired NOdependent cGMP formation in reactive astroglia during neuroinflammation is difficult to ascertain at present.

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Fig. 2. Schematic representation of LPS and IL-1b effects on NO-dependent cGMP accumulation in astroglial cells. Treatment of rat brain astrocytes with LPS or IL-1b increases NOS2 expression and simultaneously downregulates sGC by two mechanisms: (1) a NO-independent decrease of the half-life of sGC protein that requires transcription and translation; (2) a NO-dependent decrease in sGC mRNA levels that will contribute to maintain sGC levels low during prolonged periods of time. Additionally, IL-1b increases transporter-mediated efflux of cGMP.

4. Cyclic GMP inactivation in astroglial cells 4.1. Cyclic GMP phosphodiesterases Cyclic nucleotide phosphodiesterase hydrolysis of cGMP is the major mechanism underlying the clearance of the nucleotide. Eleven families of PDEs have been identified that comprise more than 50 isoforms encoded by 21 genes (Soderling and Beavo, 2000; Lucas et al., 2000). PDEs differ in their primary structure, affinity for cyclic nucleotides, sensitivity to calcium and other inhibitors and tissue distribution. According to the specificity for the nucleotide determined in cell free systems, PDEs are divided in three groups: PDEs hydrolyzing cAMP and cGMP (PDE1, PDE2, PDE3, PDE10 and PDE11), PDEs hydrolyzing cAMP (PDE4, PDE7 and PDE8) and PDEs highly specific for cGMP. Among the latter PDE6 is unique to photoreceptors and plays a crucial role in the visual transduction cascade, while PDE5 and PDE9 are differentially expressed in rat brain regions. In situ hybridization and immunocytochemical studies indicate that both are

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strongly expressed in the Purkinje cell layer of the rat cerebellum (Andreeva et al., 2001; Kotera et al., 1997), where local administration of the PDE-5/PDE-9 inhibitor zaprinast facilitates cGMP regulated long-term depression (Hartell, 1996). PDE9A, the isoform that shows the highest affinity for cGMP, is also expressed in the forebrain, hippocampus and olfactory bulb, with a distribution similar but not identical to NOS1 and sGC (Andreeva et al., 2001). Additionally, early studies indicated that the predominant cGMP degrading activity in brain is a highly active calcium/calmodulin-dependent PDE1 (Shenolikar et al., 1985; Mayer et al., 1992), and various isoforms of this group have been localized in neuronal structures (Kincaid et al., 1987; Yan et al., 1994; Furuyama et al., 1994). No mention was made in any of these reports about a glial localization of PDE isoforms. However, immunoreactivity for cGMP is potentiated by the non-specific PDE inhibitor IBMX in neurons and astrocytes in different rat brain regions, indicating that both cells types contain cGMP-PDE activities (De Vente and Steinbusch, 2000). Potentiation by IBMX of NO-stimulated cGMP accumulation is also observed in cultured astrocytes from rat (Baltrons et al., 1997) and human brain (Fig. 1A and B). In rat cerebellar granule cells and astrocytes we observed that agents that increase intracellular calcium decrease cGMP accumulation in a calmodulin-dependent manner (Baltrons et al., 1997). Activity measurements in homogenates confirmed that a calcium/ calmodulin-dependent PDE or PDE1 of similar biochemical and pharmacological characteristics was present in both cell types (Agullo´ and Garcı´a, 1997). The calcium concentration required for half-maximal stimulation of PDE1 and NOS1 was similar, suggesting that larger NO-stimulated cGMP accumulations will occur in cell compartments different from those where NO is generated in a calcium-dependent manner. In agreement with this hypothesis, immunocytochemical studies in cerebellar slices show that stimulation of neuronal NO production by NMDA increases cGMP more in astrocytes than in neurons, whereas NO donors produce more generalized increases in cGMP (De Vente and Steinbusch, 1992). In contrast to our results, Bellamy and Garthwaite (2001b) using acutely dissociated astrocytes from 8-day-old rat cerebellum showed by pharmacological means that cGMP was predominantly hydrolyzed by PDE5, with a minor contribution from PDE4 and no detectable calcium-dependent PDE activity. Since PDEs are developmentally regulated (Billingsley et al., 1990; Kotera et al., 1997), a difference in the developmental stage of the two cell preparations may explain the discrepancy. Nevertheless, our recent results also indicate that a PDE5 is present in rat cerebellar astroglial cultures. An increase in this activity appears to be responsible for the decrease in NO-dependent cGMP accumulation that is induced by long-term treatment with the HIV-1 virus coat protein gp120 (Navarra et al., 2002).

4.2. Cyclic GMP efflux Release of cGMP after stimulation of GC by NO or natriuretic peptides has been shown in several tissues, including brain (Kapoor and Krishna, 1977; Tjo¨rnhammar et al., 1986; Schultz et al., 1998). This efflux has been generally considered a mechanism that in conjunction with PDEs would return intracellular cGMP to basal levels. Additionally, extracellular cGMP may play a role in cell – cell communication by regulating the activity

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of membrane proteins through interaction with extracellular sites. For instance, in cerebellar granule neurons extracellular cGMP inhibits kainate receptors (Pouloupoulou and Nowak, 1998) and protects against glutamate-induced toxicity (Montoliu et al., 1999). In rat cortical astrocytes, cGMP released after stimulation with natriuretic peptides was reported to inhibit a Naþ/Hþ exchanger leading to a decrease in intracellular pH, an effect that could modulate important astroglial functions such as Kþ conductance or cell proliferation (Touyz et al., 1997). We have found that treatment of cerebellar astroglial cells with IL-1b induces efflux of cGMP formed after induction of NO release by the cytokine or by LPS (Pedraza et al., 2001). Like the primary active transport system for cGMP described in erythrocyte membranes (Schultz et al., 1998), cGMP extrusion in astrocytes was blocked by inhibitors of organic anion transporters (Touyz et al., 1997; Pedraza et al., 2001). Recently, cGMP was shown to be a high affinity substrate for the multidrug resistance protein isoform, MRP5 (Jedlitschky et al., 2000), and this organic anion transporter is expressed in astrocytes (Hirrlinger et al., 2002). Thus it is tempting to speculate that IL-1b may regulate the activity/expression of MRP5 or a similar transporter in astrocytes, and it will be interesting to investigate possible actions of extracellular cGMP in the context of neuroinflammation.

5. Targets and actions of cGMP in glial cells All three recognized molecular targets for cGMP, i.e., PKGs, cGMP-regulated PDEs and cGMP-regulated ion channels, have been found in CNS structures, but only in the case of PKGs is there any information about its expression and function in glial cells. The two isoforms of PKG, PKGI (splice variants Ia and Ib) and PKGII are expressed in brain cells. PKGI, a cytosolic enzyme, is particularly concentrated in Purkinje cells of the cerebellum and in sensory neurons (El-Husseini et al., 1999), while PKGII, a membrane bound enzyme, shows a widespread distribution in brain, often co-localizing with NO-stimulated cGMP formation, and is associated with neurons, astrocytes and oligodendrocytes (De Vente et al., 2001). Although PKGs are thought to be the major intracellular receptor proteins for cGMP and mediate many of the actions of the nucleotide in brain cells very few specific substrates have been demonstrated (Wang and Robinson, 1997; Lucas et al., 2000). In astrocytes the NO-cGMP-PKG pathway has been implicated in the generation of calcium waves (see chapter by Cornell-Bell et al.). Addition of NO to forebrain astrocytes was shown to increase intracellular calcium by mobilization from ryanodine-sensitive stores (see chapter by Scapignini et al.) and calcium influx and to produce intercellular calcium waves via cGMP and PKG. Mechanical stress of individual astrocytes also resulted in intercellular calcium waves which were NO, sGC and PKG dependent (Willmott et al., 2000). Amplification of the NO effect by activation of astroglial calcium-dependent NOS could contribute to the propagation of calcium waves. This mechanism may be relevant for the bi-directional communication between neurons and astroglia and the active participation of astroglial cells in synaptic integration and processing of information. It is now evident that neuronal activity can trigger calcium increases in astrocytes that are followed by exocytotic release of glutamate which feeds back to neuronal targets

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(reviewed in Vesce et al., 2001; Araque et al., 2001). This is thought to be due to synaptically released neurotransmitters, such as glutamate or noradrenaline, and these transmitters can both stimulate calcium-dependent NO and cGMP formation in astrocytes (Agullo´ et al., 1995; Baltrons and Garcı´a, 1997). Interestingly, in astrocytes of the visual cortex it was shown that the frequency of calcium oscillations was altered by glutamate in a NO-dependent manner, but the production of NO by neurons was not necessary, suggesting that astroglial NO was involved (Pasti et al., 1995). On the other hand, a receptorindependent, but NO-dependent, calcium influx has recently been demonstrated in Bergmann glia after stimulation of parallel fibers, an effect that could have implications for the induction of long-term depression in the cerebellum (Matyash et al., 2001). Synergistic effects of calcium and NO on gene transcription may be involved in the regulation of neuronal differentiation and survival and synaptic plasticity (Peunova and Enikolopov, 1995; Gudi et al., 1999). Recently it was reported that the NO and cGMP induced activation of the fos promoter mediated by membrane-bound, extranuclear PKGII, requires cell-specific factors that are found in cells of neuronal and astroglial origin (Gudi et al., 1999). The NO/cGMP pathway in astrocytes may also affect neuronal development by regulating NGF release (Xiong et al., 1999). Compared to the large amount of studies on the mechanisms of NO-induced cell death, much less is known about the implication of cGMP in neurotoxicity/neuroprotection mechanisms. Recent evidence suggests a protective role of cGMP in neural cells (Kim et al., 1999). In cortical astrocytes cGMP via PKG has been shown to prevent apoptosis by inhibiting the mitochondrial transition pore (Takuma et al., 2001). In an immortalized oligodendrocyte cell line derived from O-2A progenitors, cGMP via PKG also protected from kainate-induced cell death by decreasing calcium influx (Yoshioka et al., 2000).

6. Concluding remarks Existing evidence indicates that besides neurons astroglial cells are the only CNS parenchymal cell type where all the molecular components required for NO-cGMP signaling are expressed. NO can be formed in astrocytes by the three known NOS isoforms and NO can stimulate sCG, resulting in increased intracellular cGMP and PKG-dependent phosphorylation of cell proteins. In contrast, only NOS2 expression has been documented in microglial cells and, as in their peripheral counterparts the macrophages, NO hence produced seems to be implicated in defense mechanisms and neuroinflammation associated cell death. On the other hand, information about the NO/cGMP system in oligodendrocytes is scarce and controversial in some respects. Although no direct evidence has so far been reported, regulation of astroglial function by NO and cGMP is likely to contribute to some of the important neuromodulatory actions in which NO has been implicated. Three important aspects of astroglial physiology—calcium homeostasis, gene expression and survival—that are relevant for neuronal function, are regulated by the NO-cGMP-PKG pathway. Additionally, cGMP extruded from astroglial cells may affect the activity of neuronal membrane proteins. In the next few years we will probably witness significant advances in the understanding of how the NO-cGMP pathway participates in

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astroglial regulation of neuronal development and in synaptic integration and processing of information. Tools like the transgenic mice expressing GFAP promoter-controlled green fluorescent protein (Nolte et al., 2001) will probably be very valuable to investigate this fundamental aspect of glial cell function under normal and pathological conditions.

Acknowledgements We want to thank Carlos E. Pedraza, Teresa Sardo´n and Michele Navarra who generated part of the results of our laboratory reviewed here and Francisca Garcı´a and Annabel Segura for technical assistance. We also thank Dr Nuria Toran for providing human brain tissue from 20 to 22-week-old therapeutically aborted fetuses, obtained by a protocol approved by the Ethical Committee of the Hospital de la Vall d’Hebron (Barcelona, Spain). The sheep anti-formaldehyde fixed cGMP antibody was kindly provided by Dr J. de Vente (European Graduate School of Neuroscience, Maastricht University, The Netherlands). This work was supported by SAF2001-2540, Fundacio´ La Marato´ TV3 1008/97 and SGR2001-212 grants.

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Potassium homeostasis in the brain at the organ and cell level Wolfgang Walz Department of Physiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Sask., Canada S7N 5E5 Correspondence address: Tel.: þ 1-306-966-6535; fax: þ 1-306-966-6532. E-mail: [email protected](W. W.)

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction Blood – brain barrier Choroid plexus Potassium levels in the extracellular space Effect of excess extracellular potassium on neuronal processing Astrocytes as the site of potassium regulatory mechanisms Astrocytes as spatial buffers Astrocytes as transient storage sites for potassium Reactive astrocytes Concluding remarks

Active neurons release potassium ions into the extracellular space. The resulting increase in extracellular potassium concentration is high enough to interfere with ion channel gating and therefore information transfer in neurons. The excess potassium is not released across the blood –brain barrier into the blood. Astrocytes are now acknowledged to be the major site of the clearance of excess extracellular potassium. The major astrocytic mechanism is uptake of KCl via Na/K ATPase and electroneutral Na,K,2Cl carrier. Spatial buffering by potassium currents across astrocytic membranes works only over short distances due to the small length constant and does therefore not contribute significantly. The only exception might be the periphery of the retina. After injury, reactive astrocytes express chloride conductance(s), which allow KCl accumulation via Donnan forces in addition to the carrier-mediated accumulation. Thus, KCl accumulation into astrocytes by active and passive (after injury) means is now a well established cellular mechanism in the nervous system. Advances in Molecular and Cell Biology, Vol. 31, pages 595–609 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction The potassium ion has a crucial role in neuronal excitability. Its distribution across neuronal membranes determines the resting membrane potential features, transmitter release and the kinetics of voltage-gated ion channels. Thus, small absolute changes of the normally low extracellular but not necessarily of the higher intracellular concentration of potassium can play havoc with excitability and neuronal information transfer. Therefore, mechanisms exist which control potassium concentrations in the brain extracellular space (ECS) and cerebrospinal fluid (CSF). There are two sources of potassium instability: (i) potassium concentration in the plasma varies due to dietary intake or other factors, and the brain as an organ must be sheltered from these changes; and (ii) neurones release potassium to the ECS during action and synaptic potentials and due to the low extracellular Kþ concentration and small dimension of the ECS accumulation of released Kþ can lead to large relative changes. The homeostatic mechanisms of the neurones alone are not sufficient to prevent activity-induced increases in extracellular Kþ concentration, and therefore non-neuronal elements bear most of the responsibility for potassium homeostasis. 2. Blood – brain barrier Increases or decreases of the potassium concentration in the blood plasma have no effect on the corresponding concentration in the ECS or CSF (Jones and Keep, 1987). This applies to both, acute and chronic changes (Stummer et al., 1995). Since the total content of brain potassium is not changed during plasma concentration changes, it is unlikely that astrocytes play a role in regulation of whole brain Kþ, when the systemic potassium concentration is changed, because the potassium content of the astrocytic compartment is not altered. Rather it must be the blood – brain barrier itself, which is responsible for this homeostatic mechanism (Keep, 2002). The regulation is so effective, that even an experimental breach of the blood –brain barrier did not raise extracellular Kþ in brain parenchyma, despite the fact that this breach led to dye penetration (Somjen et al., 1991). Raising blood plasma potassium concentration from its normal level of 5 up to 17 mM with a breached blood – brain barrier caused waves of spreading depression with associated changes of the potassium concentration, but no long term increase in extracellular potassium. The Na/K ATPase activity of the endothelial cell is polarized: there is a much higher activity on the interstitial than on the luminal surface (Somjen, 2002). There are also indications of different isoforms of Na/K ATPase existing on luminal versus abluminal surfaces (Keep, 2002). The turnover rate of brain potassium across the blood – brain barrier is approx. 2%/h. This compares with 35%/h for sodium and 20%/h for chloride (Keep, 2002). These 10 –20 times lower permeability rates mean that the barrier for potassium is very effective in shutting plasma potassium out of the brain. Whatever transport exists is out of the brain via the Na/K ATPase, using the ATP created by the high density of mitochondria in the endothelial cells (Oldendorf et al., 1977). Another question is if the brain releases excess local extracellular potassium into the plasma either during normal operation or during pathological events. There is no evidence for transport of potassium out into the blood during normal brain function. The situation is,

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however, different in brain injury. During focal ischemia, when extracellular Kþ may reach very high levels (see below), the Na/K ATPase seems to lead to a translocation of potassium from brain parenchyma to blood, coupled with an import of sodium into the parenchyma (Betz et al., 1989, 1994). In this context it is of interest that the Na/K ATPase of the endothelial cells has sufficiently high affinity for Kþ to be stimulated by increased potassium concentrations (Schielke et al., 1990). 3. Choroid plexus The choroid plexus is responsible for production of CSF and its secretion into the ventricles (see chapter by Weaver et al.). Unlike the situation in most other epithelia, the Na/K ATPase is located on the apical side, the side that faces the CSF, as is a Na,K,2Cl cotransporter. The most comprehensive study to date about the potassium homeostatic role of this epithelium was carried out by Husted and Reed (1976). They found only small increases of potassium transport from blood to CSF when blood serum potassium was raised. However, increases in CSF Kþ were effectively regulated by (i) a reduction of potassium content of the CSF fluid secreted without any effect on secretion rate and (ii) an increased Kþ re-absorption rate out of the CSF. Wu et al. (1998) confirmed that Na/K ATPase and a Na,K,2Cl cotransporter on the CSF side are responsible for removing potassium from the CSF. It is unclear which transport mechanism is responsible for potassium transport across the basolateral membrane back into blood. Wu et al. found evidence for a barium-sensitive potassium conductance without locating it. Hung et al. (1993) located potassium channels in the apical membrane, but did not investigate the basolateral side. A KCC3 (KCl) cotransporter was however localized by Pearson et al. (2001) in the basolateral membrane. Its role was not investigated. Whatever the mechanism, although the choroid plexus is responsible for secretion of CSF into the ventricles, it seems to have the ability to extract excess potassium from the CSF without reducing its capacity for CSF production. In contrast elevated Kþ in blood only increases potassium transport slightly. 4. Potassium levels in the extracellular space The resting level of Kþ in the ECS of the central nervous system is lower than that in blood or other organs. It varies between 2.7 and 3.5 mM (Somjen, 2002). The most frequently used method of estimating increases in the external potassium concentration is the use of potassium-sensitive double-barreled microelectrodes. This method is accurate enough for the estimation of widespread or massive potassium release. However, release by point sources will be underestimated. This is due to the creation of a dead space around the tip of the electrode, whose diameter is several times the width of the extracellular space. Therefore any limited amount of potassium released (as the one during the passage of a single action potential) will be severely underestimated. In these cases one will get more reliable estimates using indirect methods like the amplitude of the afterhyperpolarization of the action potential, which in many axons is a phase of exclusive potassium permeability of the membrane (Frankenhaeuser and Hodgkin, 1956). Using this

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kind of estimate, most authors agree that there is a transient increase in the extracellular potassium concentration after the passage of a single action potential that amounts to nearly 1 mM above the resting level of 3 mM (Adelman and Fitzhugh, 1975). The magnitude of the increase in extracellular potassium depends upon stimulation frequency and number of neuronal elements that contribute to the release of Kþ (Sykova, 1991). Both action potentials and synaptic potentials are the source of this activity-dependent increase in extracellular potassium. Another factor contributing to the increase of extracellular Kþ is a decrease in the volume of the extracellular space under most conditions of increased activity (Ransom et al., 1986). Accordingly, during artificial, intensive stimulation of neuronal pathways extracellular Kþ may increase by as much as nearly 5 mM above the resting level, but it does not exceed this level. During seizures, the accumulation of extracellular potassium is further enhanced, but again there seems to be a ceiling level of 12 mM (Heinemann and Lux, 1977). Only during injury (hypoxia/ischemia, trauma, hypoglycaemia) is this ceiling level disrupted and concentrations of up to 25 mM are reached (Hansen, 1985). An extreme case is waves of spreading depression, which result in transient elevations of the extracellular potassium concentration of 30 –80 mM in the intact nervous tissue (Irwin and Walz, 1999). 5. Effect of excess extracellular potassium on neuronal processing There is only a need for potassium homeostasis if values of excess potassium in the range encountered above are able to significantly change the excitability of neurons. That this is the case has repeatedly been shown. These concentrations affect transmitter release (Gage and Quastel, 1965; Erulkar and Weight, 1977) and electrical properties of axons (Malenka et al., 1981). More specifically, it was shown that increases of the extracellular potassium concentration to 5 mM change the action potential threshold of hippocampal neurons, leading to hyperexcitability (Voskuyl and Ter Keurs, 1981; Balestrino et al., 1986; Kreisman and Smith, 1993). Increases in the extracellular potassium concentration, that exceed 5 mM, increase, in addition, the efficacy of synaptic transmission in hippocampal neurons (Balestrino et al., 1986; Rausche et al., 1990). The underlying mechanisms seem to be a combination of potassium evoked changes in the membrane potential and a direct gating effects of the potassium ion on some of the participating ion channels (Leech and Stanfield, 1981). 6. Astrocytes as the site of potassium regulatory mechanisms One would expect that immediate re-uptake of Kþ into neurons and diffusion in the extracellular space would be sufficient to prevent a build-up of excess extracellular potassium released during neuronal activity. There must, however, be potassium removal sites, which do not resident in neurones, because iontophoretically applied extracellular potassium is removed as efficiently as the potassium that is released from neurons. Only in the latter case is there a simultaneous accumulation of sodium ions into neurones that stimulate the neuronal Na/K pump. Kinetic analysis of potassium transients and their sensitivity to temperature changes and strophantidin, led Ransom et al. (2000) to conclude

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that a fast initial removal is based on glial Na/K ATPases, and a slower, sustained process is due to stimulation of the neuronal enzyme. In addition, the existence of a ceiling level of 12 mM under normal conditions as well as the observed undershoot below the normal resting potassium concentration after a transient phase of extracellular potassium accumulation are strong arguments in favor of active removal processes situated in non-neuronal cells (Heinemann and Lux, 1977; Heinemann et al., 1983). If Kþ is iontophoretically applied into the extracellular space, the diffusion curves appeared normal, but the volume fraction routinely exceeds 100% of the ECS (Rice and Nicholson, 1991). This is physically impossible and the only explanation is that the exit of potassium from the extracellular space through adjacent cell membranes is facilitated by auxiliary mechanisms. The existence of two different potassium clearance mechanisms residing in astrocytes was suggested a long time ago. The first mechanism is removal of excess potassium by uptake and accumulation into adjacent astrocytes, including its transient storage and subsequent release back into the extracellular space (Hertz, 1965). The second mechanism is re-distribution of excess potassium by current loops through the astrocytic syncytium (‘spatial buffering’), which would not lead to potassium accumulation in astrocytes, since for each potassium ion entering another one would leave the syncytium at a site distant from the active neurons (Orkand et al., 1966). For some time these two mechanisms were seen as exclusive alternatives. However, this does not need to be so, since they could co-exist. Accordingly the question arises whether they might be differently distributed and which one is likely to play the dominant role in gray matter. Recently, a study in the dentate gyrus employing 8 mM of potassium did not find much evidence for spatial buffering, but suggested that Na/K ATPase-mediated removal plays a major role (Xiong and Stringer, 2000). Along similar lines D’Ambrosio et al. (2002) used selective blockers and controlled neuronal stimulation in slices of CA3 stratum pyramidale. They concluded that spatial buffering did not affect potassium clearance rates but that Na/K ATPases are responsible for determining the rate of recovery. Thus, these two recent studies support a potassium accumulation rather than spatial buffering as the major mechanism of potassium clearance. However, a differentiation between neuronal and glial Na/K ATPases was not possible in these studies. In the retina, where spatial buffering has been most convincingly demonstrated (see below) Kþ uptake by spatial buffering in Muller cells seems to be restricted to the plexiform layers. In the central retina active uptake seems to dominate (Skatchkov et al., 1999). The spatial buffer concept will be discussed in considerable detail below, since it played a dominant role during several decades, since spatial buffering does seem to be important for Kþ clearance in the retina and occurs over short distances in brain and spinal cord, and since estimates of its contribution to Kþ homeostasis at the cellular level of the CNS can only be evaluated when the underlying principles are understood.

7. Astrocytes as spatial buffers The spatial buffer concept (Orkand et al., 1966) assumes a glial syncytium, coupled by gap junctions (see chapter by Scemes and Spray), in which extracellular potassium is selectively increased in one region. In a syncytium, the membrane potential of neighboring

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cells has a tendency to attain a similar value. Therefore the region experiencing an increased extracellular potassium concentration will have a potassium equilibrium potential that is less negative than the membrane potential. This will lead to an inward driving force for potassium, and since the membrane is highly permeable to potassium, it will enter the cell via a passive current. The current loop has to be closed and the potassium-mediated current will immediately reach the other parts of the syncytium via the gap junctions. In regions further removed from the area exposed to high extracellular potassium concentrations, the potassium equilibrium potential is more negative than the membrane potential due to the tendency of the syncytium to remain isopotential. Therefore at these areas there is an outwardly directed driving force for potassium. Thus the current into the cells (at regions with high extracellular potassium), inside the syncytium and out of the cells (at regions distant from local elevations of potassium), is almost completely carried by potassium as the dominant intracellular ion and an ion which can pass through gap junctions. The loop is closed by a return current in the extracellular space. This current is carried mainly by sodium and chloride ions (the dominant ions in the ECS). Therefore, despite the closed current loop, potassium does not cycle but is passively transported away from an extracellular location with high potassium concentrations and dispersed to extracellular areas with lower potassium. The original concept allowed for flexibility, since any part of the syncytium could be the source of the potassium current, depending solely on the location of the elevated extracellular potassium concentration. In order for astrocytes to act as spatial buffers, the following criteria must be met: (i) When neuronal elements are active, neighboring astrocytes must always be depolarized. (ii) A large enough population of astrocytes has to undergo that depolarization at the same time. (iii) There has to be electrotonic continuity of spatially extended astrocytes and/or the syncytium from the active to the inactive region. (iv) The length constant (see below) of the glial syncytium has to be compatible with the spread of current. Criterion (i) seems to present no problem. In general, astrocytes have a 20 mV more negative membrane resting potential than neighboring neurons, i.e., at least 2 90 mV. This is mainly due to the relatively lower sodium permeability of astrocytes (Somjen, 1995). Whether or not the astrocytic membrane has a significant chloride permeability in situ has been recently reviewed (Walz, 2002) and will not be repeated here. Normal astrocytes seem to have no chloride permeability, but activation into a reactive sub-type induces such a permeability. An almost selective potassium permeability is the reason why astrocytes are more sensitive to increases in extracellular potassium than neurons (Somjen, 2002). The relationship between extracellular Kþ concentration and membrane potential is under most conditions somewhat sub-Nernstian, i.e., the amplitude is less than predicted from the Nernst equation. This can be explained by the simultaneous potassium uptake leading to an increased intracellular potassium concentration rather than a stationary intracellular Kþ, by the modulatory effects of electrogenic uptake and by ionotropic responses of transmitters (see below). In situ, all transmitter agonists, which cause changes in ionic conductance in astrocytes, lead to a depolarization. Examples are Kainate, GABA and dopamine (Murdick-Donnon et al., 1993; Jabs et al., 1997; Bekar et al., 1999). The depolarization is due to three factors: changes in ion equilibrium potentials and in ion channel conductances (Jabs et al., 1997) as well as electrogenic transmitter uptake (Bergles and Jahr, 1997). Thus,

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substances other than potassium released from active neurons could contribute to astrocytic membrane potential changes, and astrocytes always react with a depolarization to neuronal activity. Criterion (ii) does also not present a large challenge. Orthodromic stimulation in a variety of preparations leads to a depolarization of astrocytes that persists longer than the stimulation or the activity of neurons, including synaptic potentials (Somjen, 1975). Lothman and Somjen (1975) studied the relationship of stimulation-induced extracellular Kþ increases and glial membrane potentials in the spinal cord. They found potassium increases up to 8 mM with corresponding glial depolarizations of 25 mV. An amplitude of 25– 30 mV seems to be the maximal astrocytic depolarization achievable by stimulation of neuronal pathways in vivo and in brain slices (Ballanyi et al., 1987). If more physiological stimuli are used, like light flashes, glial depolarizations are much smaller (8 mV) as are the simultaneous rises in extracellular potassium, but there is never any hyperpolarization of glial cells (Karwoski and Proenza, 1977; Kelly and Van Essen, 1974). During seizure-like events, the glial depolarizations may reach 35 mV, which corresponds to the 10 – 12 mM ceiling level (Heinemann and Lux, 1977). Studies using membrane potential-sensitive dyes in glial cells in vivo are lacking; thus, it is not possible to estimate the extent of the direct depolarizations of glial cells by axon bundles or groups of neurons. Presumably, the extent of the direct depolarization is limited to the region that contains raised potassium levels. Criterion (iii) is easily fulfilled since there undoubtedly is extensive dye- and electrical coupling of astrocytes. A dye can appear in as many as 100 astrocytes, when injected into one (Ransom, 1995). The spread of the dye seems to be spherical outward from the injected cell. There is every reason to assume that astrocytes that are dye-coupled are also coupled electrically (Dermietzel et al., 1991). In contrast, criterion (iv) is the major problem for efficient distribution of spatial buffering to potassium homeostasis at the cellular level. The length constant is the distance along a process to the site, where a voltage amplitude has decayed to 37% of its value due to the leakage of current across the cell membrane. It is obvious that a low specific resistance will decrease the length constant and therefore the length that a significant amount of current can travel in the processes of the glial syncytium. Since astrocytes have a relatively low membrane resistance, the issue of a restricted length constant is the most serious argument against the spatial buffer function (Heinemann and Walz, 1998). There are now several studies that estimated the length constant and found it to be too small in all types of astrocytes or astrocyte-like cells tested with the possible exception of those in the retinal periphery. Barres et al. (1990) measured the length constant of an acutely isolated astrocyte from rat optic nerve as 100 mm, whereas the whole length of the cell was 400 mm. Skatchkov et al. (1999) found 70 mm in Muller cells of the frog retina. This was not long enough for long central cells, but sufficient for short peripheral cells. Using data from rat cerebral cortex, Chen and Nicholson (2000) concluded that spatial buffering, as constrained by the leaky cable properties, does not appear to be able to operate over long distances. Thus a major deficiency of the spatial buffer theory seems to be that spatial buffering can only operate across short distances. Evidence from in situ systems support the concept of spatial buffering. Nicholson and Phillips (1981) studied diffusion in rat cerebellum using ion-selective microelectrodes and

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iontophoretic point sources. Assuming an extracellular space of 20% they found for a variety of anions and cations a close correlation of their movement with the laws of macroscopic diffusion. However, there was one ion that did not fit into the scheme: the movement of potassium in an electrical gradient is more keeping with an extracellular space that occupies more than 100% of the brain volume. The anomalous nature of the potassium migration can be explained by assuming that the ion does not remain in the extracellular space, but is in fact, the major current carrier across cell membranes. Hounsgaard and Nicholson (1983) analyzed the possibility in more detail. They measured changes in the extracellular potassium concentration in the vicinity of Purkinje cells in guinea pig cerebellar slices. No extracellular potassium changes were seen when the Purkinje cells were hyperpolarized with current passage or during sub-threshold depolarizations. Only during spike activity was there a rise in extracellular potassium levels. In the vicinity of glial cells, however, a hyperpolarizing current injected into glia reduced the outside potassium concentration in a symmetrical manner, while depolarizing current injection induced a rise in the outside potassium levels. These results demonstrate that the potassium ions are crossing the glial cell membrane during their movement in an electrical gradient. They also provide additional evidence supporting the view that glial cells function as spatial buffer for potassium (see also chapter by Laming). Thanks to its easy accessibility and layered structure, the retina is the part of the CNS that is best characterized in terms of spatial buffering. Light stimulation results in external potassium increases in the two plexiform layers and decreases in the sub-retinal space (Reichenbach et al., 1998—see also chapter by Bringmann et al.). In order to buffer the extracellular Kþ changes, potassium currents enter the Muller cells in the two plexiform layers, where external potassium is high, whereas potassium efflux occurs into the vitreous humor at the endfoot and into the sub-retinal space. In a vascularized retina further efflux can occur at the blood vessels. These currents, K(IR), use inwardly rectifying potassium channels, which open at 2 70 to 2 90 mV (Chao et al., 1994), mediate both inward and outward currents (Skatchkov et al., 1995) and increase their conductance when external potassium is raised (Newman, 1993). The Muller cells have high densities of K(IR) at regions, which act as sinks for buffering currents on the endfoot (Brew et al., 1986). It has been shown, that blockade of these channels with barium raises external potassium amplitudes and increases clearance time (Oakley et al., 1992). Newman (1986) showed with single Muller cells that these cells are capable of carrying potassium currents and releasing substantial amounts at their endfeet. However, the aforementioned work by Skatchkov et al. (1999) seems to show that the length constant permits efficient Kþ clearance by spatial buffering only in the periphery, where the distances are shorter, and that Na/K pump mediated processes must be involved in long distance clearance of Kþ. Whether a similar mechanism as in Muller cells operates in cerebral astrocytes is still not clear. There are indications from hippocampal astrocytes that only a sub-population of the cells has Kþ inward rectifier channels (D’Ambrosio et al., 1998; Zhou and Kimelberg, 2000). These studies support the view that different astrocytes are responsible for inward and outward currents. Also there seems to exist a dynamic coupling and uncoupling of astrocytes, caused by unknown factors, which would add another complexity to such a buffering system (McKhann et al., 1997). However, one should keep in mind the previous mentioned estimates, that potassium buffering in the cortex is only suited for operation

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over very short distances (Chen and Nicholson, 2000) and that compared with the Na/K ATPase mediated removal of potassium, spatial buffering plays a minor role in Kþ homeostasis (Xiong and Stringer, 2000; D’Ambrosio et al., 2002). Thus, one has to conclude that spatial buffering can take place locally but not in an extended area.

8. Astrocytes as transient storage sites for potassium The spatial buffer current does not mediate cellular storage of potassium ions: for every potassium ion entering the syncytium, one is leaving at the same time, although at a different location. Thus no significant accumulation of potassium inside astrocytes will take place, and any observed accumulation can be seen as an indication of a mechanism other than operation of a spatial buffer. In all preparations tested (cultured astrocytes: Walz et al., 1984; cultured oligodendrocytes: Kettenmann, 1987; brain slices: Ballanyi et al., 1987; drone retina: Coles and Orkand, 1983; leech CNS: Wuttke, 1990; reactive astrocytes in situ: Walz and Wuttke, 1999) glial cells accumulate potassium ions, when the extracellular potassium concentration increases. They release potassium again as soon as the extracellular concentration is lowered. This transient accumulation or storage was observed regardless whether the extracellular potassium concentration was increased artificially in a uniform way (a situation where spatial buffer currents would not arise) or whether the activity of neighboring neurons was stimulated by physiological or nonphysiological means. It was found in the optic nerve (Ransom et al., 2000; MacVicar et al., 2002) and hippocampal slices (Xiong and Stringer, 2000; D’Ambrosio et al., 2002) that active uptake via a transporter is more efficient than spatial buffering. This conclusion is strengthened by the aforementioned observations by various authors that the amount of accumulated potassium is similar when the neurons are stimulated and when the external potassium is experimentally increased to levels observed during the neuronal stimulation. This is important since neuronal stimulation increases not only the internal sodium and external potassium concentrations but also significantly changes calcium and proton concentrations as well as transmitter and metabolite levels (Ballanyi, 1995; Ransom, 1992). The increase in glial intracellular potassium is therefore exclusively a response to the increased extracellular potassium levels; the other changes might modify this response but do not cause it. The estimated maximal increase in the glial intracellular potassium concentration during neuronal activity amounts to approximately 10 mM in the experiments mentioned above. If the extracellular potassium concentration is increased artificially to the levels observed during neuronal stimulation, the increase in the glial Kþ concentration is higher (20 – 25 mM). The difference is probably due to the uniform increase around the glial syncytium in the latter case as compared to a more localized increase during stimulation. The storage of potassium in astrocytes is transient and depends on the extracellular potassium levels: as soon as the extracellular potassium concentration decreases, the glial cells start releasing the accumulated potassium. If the external levels again reach the normal value, no storage of excess potassium occurs. The release mechanism is unknown. In cell culture the increase in terms of concentration can amount to a doubling if all the external sodium ions are replaced by potassium ions (Walz, 1987). Since at this stage

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massive swelling occurs, the absolute amount of potassium taken up is about three times the content at resting levels. Presumably such levels play a role following anoxia/hypoxia and spreading depression in vivo (Erecinska and Silver, 1994). Two alternate uptake mechanisms exist, which can complement each other and work in parallel (Walz and Wuttke, 1999). They are discussed below. The intracellular sodium and potassium concentrations are controlled by the activity of the Na/K pump or Na/K ATPase (Walz and Wuttke, 1999; Walz et al., 1993). However, although part of the potassium uptake is sensitive to ouabain, the prototypical Na/K pump blocker, at high uptake rates for potassium, there would simply not be enough sodium in the cells to account for a 1:1, let alone a 3:2 exchange of sodium with potassium. The internal sodium concentration of astrocytes is around 10– 15 mM (Rose and Ransom, 1996) and does not change during potassium uptake (Walz and Hertz, 1983). This can be explained by operation of a transmembrane sodium cycle suggested by Walz and Hinks (1986): potassium is moved inward by the Na/K pump in exchange with the sodium ion, and a decline in intracellular sodium concentration is prevented by a simultaneous stimulation of the electroneutral bumetanide-sensitive Na –K –Cl cotransporter, which passively follows the combined driving forces of all three ions, leading to an inward flow of these ions. Sodium is extruded by the Na/K pump and replenished by the Na –K – Cl carrier. The net effect is KCl accumulation and swelling due to the osmolyte increase (Walz, 1987). The evidence for such a mechanism is based on radiotracer analysis in cultured astrocytes and the fact that inhibition of either pathway was not additive, but showed considerable overlap between both. In addition, there is no evidence for different kinetic properties of the glial and neuronal Na/K pump (Sweadner, 1995—see, however, also chapter by Peng et al.). This is a puzzling finding, which could mean that the glial Na/ K pump is actually stimulated by intracellular sodium, which enters through the Na –K – Cl cotransporter. This would explain the close involvement of an ouabain-sensitive component despite the fact that the main properties of the pump seem to show no kinetic differences between astrocytes and neurons. This view was supported by findings by Rose and Ransom (1996) that the bumetanide-sensitive cotransporter was an efficient mechanism to replenish sodium ions that were pumped out during potassium uptake into astrocytes. A kinetic investigation in the colon (Payne et al., 1995) showed that the Na –K – Cl cotransporter is well suited for potassium uptake due to the combined strong inward driving force of all participating ions and a high affinity for extracellular potassium. That this kinetic arrangement is indeed responsible for astrocytic potassium uptake also in situ was confirmed by MacVicar et al. (2002). Another alternative for Kþ uptake in astrocytes is passive uptake of KCl through ion channels. The driving force for such an uptake is Donnan forces and it will continue until the equilibrium potentials for both chloride and potassium are identical and have the same value as the membrane potential. This accumulation, however, is crucially dependent upon a significant chloride permeability of the cell membrane. It therefore contradicts the paradigms of the spatial buffer mechanisms, which assumes selective potassium permeability. The issue was recently reviewed (Walz, 2002) and it was concluded that there is little evidence for such a Donnan-mediated KCl mechanism in astrocytes in normal tissue. There is a porin in the cell membrane of cultured astrocytes, similar to those found in mitochondrial membranes (Dermietzel et al., 1994). At positive membrane potentials

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these porins would activate their large unitary conductance. However, their exact activation mechanism and the occurrence of this porin in situ still has to be confirmed. It is of interest, however, that during spreading depression a large anion conductance is transiently mediating chloride uptake into cells (Phillips and Nicholson, 1979). 9. Reactive astrocytes High chloride conductances exist in the cell membrane of astrocytes in pathologically altered tissue. These could assist in Donnan-mediated KCl uptake. There is evidence from cell culture studies that transformation of the astrocytes into a more reactive shape and state is activating various chloride conductances (Lascola et al., 1998; Fava et al., 2001). In reactive gliosis in situ such chloride conductances mediate KCl uptake in parallel with the carrier-assisted mechanism (Walz and Wuttke, 1999). In case of a blockade of the carriers the Donnan-mediated KCl uptake can substitute fully. Thus, it is suggested that this channel-mediated KCl uptake comes into play in reactive astrocytes but not in normal cells. Moreover, voltage-gated potassium channels are altered in reactive astrocytes in situ (Jabs et al., 1997; Francke et al., 1997; D’Ambrosio et al., 1999; Bordey et al., 2001). All these studies seem to confirm a down-regulation of glial inward (and some outward potassium) currents. This seems to interfere with spatial buffering, but apparently not with Donnan-mediated KCl uptake (Walz and Wuttke, 1999). 10. Concluding remarks The notion that astrocytes are the major site of potassium homeostasis in the brain seems to have matured to the status of a scientific fact more than 35 years after it was first suggested by Hertz (1965). Moreover, all in situ studies designed to test the importance of buffering versus active uptake for Kþ homeostasis at the cellular level of the CNS constantly reach the conclusion that the carrier-mediated uptake is by far the most important component at all locations with the possible exception of the peripheral retina. After injury astrocytes undergo changes in their mechanism(s) for ion homeostasis, but they are still fully capable of potassium clearance, which maybe now becomes even more important than in healthy tissue. Acknowledgement Supported by operating funds from the Heart and Stroke Foundation of Saskatchewan.

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Potassium and glia-derived slow potential shifts in relation to behaviour Peter R. Laming Medical Biology Centre, School of Biology and Biochemistry, Queen’s University of Belfast, 97, Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK Correspondence address: Tel.: þ 44-1232-272269; fax: þ44-1232-236505 E-mail: [email protected](P.R.L.)

Contents 1. 2. 3. 4. 5. 6. 7. 8.

9. 10.

Introduction Spatial buffering of potassium by glial cells Slow potential shift origins in intact animals Slow potential shifts and behaviour SPS responses during arousal and attention SPS responses associated with changes in motivation Effects of potassium dynamics on motivation The effect of imposed DC shifts on neuronal responsivity and behaviour 8.1. Neuronal responsivity 8.2. Behaviour Changes in the SPS during habituation Concluding remarks

This chapter examines evidence that in fish and amphibians glial cells respond to changes in extracellular potassium ([Kþ]e) in ways that contribute to modulation of neuronal activity and thereby behaviour. Glial cells spatially (and probably directionally) redistribute potassium from regions of increasing concentration to those with a lesser concentration. This redistribution is largely responsible for slow potential shifts associated with behavioural responses of animals. These slow shifts are related in amplitude to the level of ‘arousal’ of an animal, and its motivational state. In addition, glia, especially astrocytes, respond to changes in [Kþ]e. Simulating these effects by imposing potassium loads and by DC stimulation interacts with previous motivational state to alter neuronal responses and behaviour. The responses of glia to changes in extracellular potassium after neuronal activity have been associated with at least some forms of learning, including habituation. The implication of these effects of potassium signalling in the brain is that there is considerable involvement of glia in a number of processes crucial to neuronal activity. Advances in Molecular and Cell Biology, Vol. 31, pages 611–633 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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Glia may also form another route for information distribution in the brain that is at least bidirectional, though less specific than its neuronal counterparts. It is evident that the Neuroscience of the future will have to incorporate much more study of neuron –glial interactions than hitherto. 1. Introduction It was in the 1960s that the first definitive evidence was produced to reveal that glial cells in vivo were depolarised by activity of associated neurones; firstly in the leech (Nicholls and Kuffler, 1964, 1965) and subsequently in the optic nerve of the amphibian, Necturus (Orkand et al., 1966). At about the same time, there were indications that glial cells were uniquely sensitive to extracellular potassium ([Kþ]e). These findings led Galambos (1961) to speculate that glia may modulate neuronal activity by controlling the extracellular milieu. However, it was Hertz (1965) that suggested that glia respond to elevated [Kþ]e by acting as a sensing and conduction medium for information derived from neurones and by depolarising contribute to slow potential shifts (SPSs), associated with behaviour. Since that time, evidence for these suggestions has emerged from a variety of species. Here we will concentrate on that derived from fish and amphibians with especial emphasis on the contributions of glia to SPSs that are associated with behaviour and its modulation. 2. Spatial buffering of potassium by glial cells Activation of neurones releases potassium (Kþ), causing astrocytic or ependymal cell depolarisation (Orkand et al., 1966; Kuffler et al., 1966). In contrast, glial hyperpolarisation results from activation of the electrogenic Naþ/Kþ pump (Walz and Kimelberg, 1985). There is now considerable evidence that extracellular potassium concentration in mammals in vivo may fluctuate from its ‘normal’ level of ca. 3.5 mM to up to 9þ mM in conditions of high neuronal activity (Sykova´, 1983, 1992). In the amphibian tectum [Kþ]e rises from a baseline of 3 mM to 4 mM after a brief light flash has been reported, corresponding to an SPS of 1 mV recorded extracellularly (Roitbak et al., 1992). Elevated [Kþ]e is not restricted to the immediate region of neuronal activity, as glial cells are connected by gap junctions to form a functional syncytium that allows spatial buffering of ions. Thus, a negative SPS induced by electrical stimulation of the tectum or a photic stimulus to a frog Rana esculenta (Roitbak et al., 1992, Fig. 4) or by electrical stimulation of the tectum of a fish, Carassius auratus (Nicol et al., 1993), induces a surface negative SPS that declines with depth and inverts at ca. 400 mm. Since astrocytes are relatively sparse in the tectum of amphibians and teleost fish (Roots and Laming, 1998) it is probable that potassium redistribution and the consequent SPS occurs through radial ependymoglia (Fig. 2, Lazar, 1989). The concept of the spatial buffer function for potassium redistribution assumes a glial syncytium with [Kþ]e increased at one region. The region experiencing increased [Kþ]e will have a more positive Kþ equilibrium potential than the membrane potential, providing an inward driving force for Kþ. Since the membrane is highly permeable to Kþ, it will

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enter the cell via a passive current to be distributed to the other parts of the syncytium via gap junctions. In regions distant from the high Kþ region the Kþ equilibrium potential therefore becomes more negative than the membrane potential, and there is a driving force for Kþ out of the cell. Thus the current flows into the cells at high Kþ regions, through the syncytium, and out of the cells at regions distant from high Kþ locations. This intracellular current is almost completely carried by Kþ. The loop is closed mainly by a return current in the extracellular space (ECS) carried by Naþ and Cl2. A problem with the concept that glia generate the SPS by spatial buffering of potassium lies in measures of the length constant of glial cells. This is the distance along a process to the site, where a voltage amplitude has decayed to 37% of its value, due to leakage of current across the cell membrane. Barres et al. (1990) measured the length constant of an isolated astrocyte of the rat optic nerve for a single process as 100 mm. The overall length of the process is 400 mm, meaning that no significant portion of a current could cross into neighbouring glial cells. However, the length of the glial cell would be sufficient to redistribute Kþ from a site of maximal Kþ accumulation to a remote less active area. Indirect measurements based on Kþ transport induced by an electric field estimated a length constant of around 200 mm for the rat cerebral cortex (Gardner-Medwin, 1983). In the tectum of the frog (Fig. 2), Rana esculenta, the SPS and associated [Kþ]e declined to 37% in 300 mm (Roitbak et al., 1992), whilst in the goldfish the SPS decline to this magnitude was about 250 mm (Nicol et al., 1993). In these animals this is well within distances from surface to depth that could influence the activity of intrinsic tectal neurons, involved in the processing of visual information. Nicholson and Phillips (1981) studied ion diffusion in rat cerebellum using ionselective micropipettes and ionophoretic point sources. They found that the movement of potassium in an electrical gradient behaves as if the ECS volume was more than 100%. This anomaly can be solved by assuming that the ion does not remain in the ECS, but is in fact the major current carrier across cell membranes. Hounsgaard and Nicholson (1983) measured changes in extracellular Kþ concentration in the vicinity of Purkinje cells in guinea-pig cerebellar slices. No extracellular Kþ changes were seen when the cells were hyperpolarised with current passage or during sub-threshold depolarisations. Only during spike activity was there a rise in extracellular Kþ. In the vicinity of glial cells, however, a hyperpolarising current injected into glia reduced extracellular Kþ concentration in a symmetrical manner, while depolarising current injection induced a rise in [Kþ]e. These experiments show that the Kþ ion is mainly using the glial cell membrane during its movement in an electrical gradient. Elevations of [Kþ]e are never restricted to just one layer in a cortical-type structure. Thus, the problem of the restricted length constant of a glial cell syncytium is less severe than just discussed. For example, a number of glial cells may be depolarised at the point of maximal potassium accumulation, so that the depolarisation spreads over a short distance. Potassium released from active neurones will thus be redistributed to the less active surround. There, more spatially extended glial or coupled glial cells could redistribute the potassium further into the surround. The different local currents gradients will then add and contribute to generation of slow potentials over the whole structure. To summarise, changes in the [Kþ]e due to release on neuronal activity causes depolarisation of the glial membrane and Kþ uptake. Electrotonic current flow then occurs

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through glia by spatial buffering, and it may be recorded extracellularly as an SPS. Near the source of neuronal excitation the Kþ potential reflects glial depolarisation and the SPS in mammalian cortex (Roitbak et al., 1987) and in the frog brain (Roitbak et al., 1992).

3. Slow potential shift origins in intact animals Electrical stimulation of the mammalian cortex with a short (0.2 – 0.8 ms) square wave pulse induces a brief (20 – 30 ms) negative wave at the cortical surface. This is known as the dendritic response (Chang, 1951), as it represents the excitatory post-synaptic potentials (EPSPs) of apical dendrites. Such responses can be induced regardless of the polarity of stimulation (Roitbak and Fanardjian, 1981). As the strength of the stimulating current is increased, the amplitude of the dendritic response increases proportionally, and a second, longer duration (250 ms) negativity develops (Chang, 1951; Goldring et al., 1959). The intensity required to provoke the secondary negativity is close to that required to induce depolarisation of deeply situated glial cells identified by their high resting potential and their lack of either spike activity or activity within the EEG range (Roitbak and Fanardjian, 1981). Increasing the frequency of the applied pulses to the cortical surface causes prolonged negativity, associated with summation of the secondary negativity (O’Leary and Goldring, 1964) and glial depolarisation (Karahashi and Goldring, 1966; Ransom and Goldring, 1973a,b; Roitbak and Fanardjian, 1981; Roitbak et al., 1987, Fig. 1). The latter paper suggested that the SPS can be almost entirely accounted for by glial depolarisation, except for the first 300 ms, to which inhibitory postsynaptic potentials (IPSPs) make a major contribution. It is probable that events occurring in the first 500– 700 ms after stimulation include elements of both neuronal and glial, event-related depolarisations, whereas later events are mainly or exclusively glial. Generation of SPSs also occurs with stimulation of the optic tectum of conscious but immobilised anuran amphibia (frogs and toads). The optic tectum is the main integrative region of the brains of these animals and its electrical stimulation by single stimuli provokes dendritic responses (Fig. 1A). The initial negativity of these dendritic responses declines on repeated stimulation, and is replaced by an SPS (Fig. 1B, Laming et al., 1992), which, as in mammals, outlasts the stimulus by seconds (Roitbak and Fanardjian, 1981). The amplitude of SPSs so generated reflects the ‘strength’ of the stimulus, whether this be measured by pulse duration, current or frequency.

4. Slow potential shifts and behaviour To record SPSs in relation to behaviour, natural stimuli and unanaesthetised preparations are required. In unrestrained animals, movement can produce artefactual contamination of recordings, or SPSs induced by the central motor command centres themselves may render interpretation of the sensory response component difficult. Averaging techniques may overcome some of these problems if the response is resistant to habituation (Roughan and Laming, 1998). In spite of these difficulties there has developed a quite substantial literature on mammalian ‘reactive’ SPS responses to sensory stimuli

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Fig. 1. (A) Dendritic response to a 600 ms, 100 mA pulse to the surface of the optic tectum of a frog. Note that the artefact changes polarity with polarity of the stimulus (upper compared to lower trace). (B) With repetition of a 500 ms, 100 mA stimulus at 50 Hz for 1 s, the dendritic responses decline, superimposed on a generated SPS (after Laming et al., 1992).

(Rowland et al., 1985). These responses may have a general distribution in the cortex or may be localised. Lickey and Fox (1966), using a muscle relaxant to immobilise the animal, similarly found surface negative SPS responses to visual, auditory and peripheral electroshock stimuli, localised in the primary sensory region for the stimulus modality used. The occurrence of SPS responses in the mammalian cortex has led many to assume that it is a strictly cortical phenomenon, yet SPSs in response to visual stimulation have been recorded from the hyperstriatum of pigeons, a region lacking a laminated cortical structure (Durkovic and Cohen, 1966, 1968). This led to the proposal that a radially organised structure was not necessary for the expression of SPSs. This concept might be supported by recordings of SPSs from apparently non-radially organised or laminated regions of the fish brain, such as the telencephalon and medulla (Nicol and Laming, 1992). Both fish and amphibians are useful ‘model’ vertebrates for the study of relationships between sensory neuronal activity and SPS responses, as most visual sensory experience is integrated in the midbrain optic tectum (Fig. 3) and auditory information in the underlying torus semicircularis. In anuran amphibians (frogs and toads), the retinal ganglion cell output projects via the optic nerve to the surface of the 9-layered contralateral tectum (Fig. 2), where information is processed from surface to depth. Microelectrodes can be used to simultaneously record both neuronal and SPS responses from the tectum (Fig. 3), and these can be monitored separately by selective filtration and amplification of the recorded signal. Similarly, the electrode can be moved to maximise the amplitude of the signal of one neuron (a unit) whilst smaller signals are filtered out. Recordings of this nature have

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Fig. 2. Schematic representation of the layers and types of cell in the optic tectum of an anuran. Together, the depth of all the layers amounts to approximately 0.5 mm. The left illustrates the tectal cytoarchitecture and fibre patterns. Numbers on the extreme left indicate layers; A to G are sub-divisions of layer 9. Unmyelinated fibres in sub-layers A, C, E are not drawn. Arachnoid and pia are meninges of these names. 1,2—large pear-shaped neuron; 3—large pyramidal neuron; 4—large ganglionic neuron; 5—small pear-shaped neuron; 6—bipolar neuron; 7—stellate neuron; 8—ependymo-glial cell (modified from Roitbak et al., 1992).

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Fig. 3. Schematic drawing of an anuran brain to illustrate positions of recording and stimulating electrodes described in Sections 8.1 and 8.2. Tel ¼ telencephalon; electrodes used in immobilised animals; þ ¼ recording microelectrode, a, p: anterior and posterior ball electrodes for both DC stimulation and SPS recording. Stimulating electrode positions for recording behavioural responses; r ¼ right, l ¼ left.

allowed neuronal units to be classified into types based both on the responses they make to a range of visual stimuli presented to the animal and on the size of their receptive field. The main target for retinal afferents is the midbrain optic tectum which has a laminated cortex-like structure (Fig. 2). The innermost layer (layer 1) is composed of cell bodies of ependymoglial cells. Layers 2 –9 are alternately cellular and fibrous layers, the outermost ones of which (layers 8 and 9) receive retinal input. Retinal fibres, exclusively from the contralateral eye, terminate on the tectum with a retinotopic distribution. Four types of retinal cell input to the tectum have been identified on the basis of their response properties to visual stimuli. All are responsive to moving visual stimuli with circular receptive fields (with sizes of 2– 4, 8 and 10†15 degrees of visual angle) and ’on, on– off and off’ responses to illumination change. Although these units respond to moving visual stimuli, their activity does not show the configurational selectivity of prey-catching behaviour, so the basis for this must lie in cellular elements beyond the retina. Although the tectum has a laminated appearance, it is composed of cells, neurons and glia, which have a column-like organisation, with their processes oriented perpendicular to the surface (Fig. 2). These intrinsic tectal units have been identified on the basis of their physiological responses to moving visual stimuli. Seven major classes of tectal (T) neurons have been identified in this manner, though some have been sub-categorised. Of these units, the T5 units and their sub-classes, T51, T52 and T53, have provoked most interest because they show sensitivity and, in the case of T52, selectivity for ‘worm-like’ stimulus properties

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mirroring those which provoke prey-catching behaviour. T5 units are sensitive to the area of a moving stimulus, T51 units to its extension in the direction of movement and T53 units to its extension perpendicular to its direction of movement. Most significantly, T52 units are sensitive to both so that they are selective for a worm-like stimulus. They have a response pattern of worm preference which reflects the probability that the stimulus will be treated as ‘prey’ (Ewert, 1989). Evidence has accumulated to emphasise that the SPS recorded intracranially is a general expression of sensory activity in vertebrates as a whole. In response to direct electrical stimulation, or sensory stimuli, the negative SPS reflects the strength of the stimulus or the activity of local neurons, respectively. Thus, in anurans, the SPS amplitude is correlated with visual unit activity at the tectal surface, where it is presumably the retinal ganglion cell input (neuron #4 in Fig. 2) that releases Kþ and thus generates the SPS (Laming and Ewert, 1984). In response to the onset of illumination or to electrical stimulation of the tectal surface SPS also closely reflects changes in [Kþ]e (Roitbak et al., 1992, Fig. 4). The SPS at the surface is probably a reflection of excitation

Fig. 4. Plot of mean ^ SEM ðn ¼ 10Þ of changes in SPS and Kþ potential (potential recorded with a potassium selective electrode) to electrical stimulation of the frog tectal surface. Note that the SPS is maximal at 50 mm, the Kþ potential at 200 mm (after Roitbak et al., 1992). A=A0 ¼ the proportion of a value ðAÞ compared to the maximum value obtained ðA0 Þ:

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of unmyelinated retinal afferent terminals in layer 9. These and deeper intrinsic tectal neurons contribute to the rise in [Kþ]e which is maximal at a depth of between 100 and 200 mm. In mammals also, the reactive SPS to sensory stimulation is fairly closely correlated with neuronal activity (Rowland, 1968). Reactive SPSs are always negative at the source of their generation, but may be reflected elsewhere in the brain by either positivity or negativity. In the teleost, Carassius auratus (goldfish), a negative SPS is evoked in the midbrain in response to visual or pressure-wave stimuli, whilst in the foreand hindbrain regions, the SPS is initially positive (Quick and Laming, 1990). In the common toad, Bufo bufo, SPS responses were first recorded in conscious but immobilised animals in response to stimuli, which emulated natural prey objects (Laming and Ewert, 1984). In the optic tectum, these stimuli caused activation of visual units, an increase in EEG frequency and amplitude and a monophasic negative SPS, these being recorded with the same electrode. At the tectal surface, the region of retinal fibre input, the unit activity preceded EEG changes and the SPS, but in deeper layers of the sensory processing system, the SPS marginally preceded the EEG change but significantly pre-empted the activity of local units. This led to the suggestion illustrated in Fig. 5, that the radial glial potassium buffering currents were acting to translocate Kþ to deep layers of the sensory processing system prior to the onset of activity in the neurones themselves (Laming, 1989a). A system of this nature is made possible by the lack of synaptic delay in the glial syncytium, and would be adaptive, in that it would ‘prime’ neurones likely to be imminently in receipt of visual input. Spatial buffering would make this sensitisation mechanism passive, but it could be more dramatic as at least mammalian glia have active uptake of Kþ (see chapter by Walz) and participate in glutamate metabolism (see chapter by Schousboe and Waagepetersen). In sensory systems that are organised topographically to represent spatial sensory experience, like the visual system, neurones in areas close to the part of the sensory map being stimulated would also be sensitised, and responses to moving objects would be enhanced. The release of [Kþ]e in response to sensory stimulation, as implied by this hypothesis, is evidenced by SPSs and changes in [Kþ]e recorded in response to a visual stimulus in frogs. These have a similar depth profile as that induced by tectal electrical stimulation (Roitbak et al., 1992, Fig. 4). Thus, in anurans at least, the column of neurones responding to a particular retinotopic projection would seem to be associated also with a parallel oriented radial glial cell or cells, participating in the responses of the neural information processing system, much as envisioned by Hertz (1965). The toad tectum derives much of its ability to discriminate prey (e.g., an elongate piece of black card moving along its long axis against a contrasting background, mimicking a worm) from non-prey (other configurations of the black card) due to inhibitory projections arising from the pretectal thalamus that are activated by non-prey stimuli. If appropriately positioned small lesions are made in the pretectal thalamus, the toad will treat almost any moving object as prey (Ewert, 1989). In an immobilised animal, a similar lesion causes enhanced neuronal responses to visual stimuli, and the selectivity of responding units for the configuration of the visual stimulus is lost. In spite of the greatly enhanced neuronal activity in the tectum, the SPS is considerably attenuated (Laming and Ewert, 1983). In contrast, telencephalic ligature causes declines in both unit and SPS responses

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Fig. 5. A mechanism by which glia may sensitise neurones by relocation of potassium in a radially organised sensory system, like the toad tectum. On arrival of the sensory input, active neurones, e.g., A1, release Kþ, which is translocated by glia to the regions of A2 and A3, which are therefore partially depolarised prior to being reached by the specific neuronal activity through the A1/A2 and A2/A3 synapses. Spatial buffering would make this sensitisation mechanism passive, but it could be more dramatic as glia have active uptake of Kþ and participate in glutamate metabolism. In sensory systems that are organised topographically to represent spatial sensory experience, like the visual system, neurones in areas close to the part of the sensory map being stimulated would also be sensitised (B1, B2, B3) and responses to moving objects would be enhanced (after Laming, 1989a).

(Laming et al., 1984b). It would thus seem that there are experimentally contrived conditions when the SPS is not a simple mirror of local neuronal activity in toads. In behaving toads, both the frequency of tectal visual unit responses and the amplitude of the SPS response to a visual stimulus is greater than in immobilised animals, and it is associated with prey-catching behaviour. However, if simultaneous defensive behaviour is elicited by a tactile stimulus, then visual unit activity is minimal, whilst the SPS recorded from the same electrode is larger than from visual stimulation alone (Laming et al., 1984a). In this case the SPS may derive from deeper midbrain regions responding to the tactile stimulus, emphasising that an important aspect of ionic currents through glia is that they enable conduction in any direction in which they are connected by gap junctions.

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Thus, deep activation could potentially act to sensitise more superficial regions, a role previously ascribed to reticular formation projections. Presuming the SPS to be due to spatial buffering currents derived from glia, these results suggest that glia may act to integrate signals derived from a number of stimuli or stimulus modalities. When a number of SPS recording electrodes are employed to examine responses of behaving animals to a sensory event, quite complex waveforms may emerge. Thus, toads respond to prey-like objects in their frontal visual field with an initially negative SPS in the corresponding retinal projection (anterio-lateral) region of the tectum. The response in the posterior tectum, which is not directly activated by this visual stimulus, is initially positive. In the behaving animal, observation of the prey leads to orienting (see below) and approach behaviour (Ewert, 1989), during which time (ca. 4 s) the polarity of the regional waves becomes reversed. If the same animals are immobilised, they still exhibit anterior negativity and posterior positivity in tectal SPS responses to the prey stimulus. However, under such conditions these responses are monophasic, suggesting that some aspect of the behaviour itself might explain ‘rebound’ in SPS polarity. One explanation is that the movement of the toad causes visual input to the whole tectum to be activated and thus causes many different sources and sinks to be generated (Laming et al., 1995). Of perhaps greater interest was the finding that in immobilised animals with no visual stimulation of the posterior tectal projection region, but visual stimulation of the anterior tectum, a positive SPS was recorded in the posterior tectal projection region, which mirrored the negative SPS in the stimulated region of the anterior tectum. This implies that sinks and sources for current may be present across the surface of the tectum as well as through its depth. If the conduction pathway through the depth of the optic tectum is via radially oriented glia, then that across the surface may be via tangentially oriented glia. Glial processes are often tangentially oriented at the brain surface, which itself is comprised of a sheet of ependymal glial cells or radial glial end feet as part of the glia limitans, which separates cerebrospinal fluid (CSF) of the sub-arachnoid space from the fluid of the ECS of the brain. The apparent separations between sources and sinks (a distance of 1 mm) found in these immobilised animals, would suggest a low resistance pathway for current spread, potentially available through the CSF, along the glia limitans, which express high density of gap junctions, or possibly even via meningeal (pial) gap junction-coupled cells covering the surface of the tectum as shown in Fig. 2 (see also chapter by Mercier and Hatton). It would be interesting to determine whether the movement of ions, especially Kþ, between ECS and CSF has the buffering properties ascribed to similar Kþ movements in the retina, between vitreous humour and Muller cells (Newman, 1986—see also chapter by Bringmann et al. and by Scemes and Spray) and whether such properties might form part of a mechanism for controlling ECS constituents.

5. SPS responses during arousal and attention The reactive responses of animals to sensory input, as described above, vary according to their behavioural state. This may provide clues to the origin of that state.

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Animals are not in a constant state of alertness, and even when they are awake, there are periods when neural activity is increased and the behaviour of the animal is activated. The animal may be described as being ‘aroused’. External factors, which induce arousal, are stimuli, which have biological relevance, because they are novel, or stimuli which are relevant, either due to an endogenous trigger (food, mate), or because they have acquired relevance through experience. The response to such relevant stimuli is the ‘orientation reaction’ (Sokolov, 1963), initially comprising a non-directed alerting response (arousal), associated with generalised increases in neuronal activity, changes in EEG frequencies and amplitudes, a general reduction in sensory thresholds, development of SPSs and changes in measures of peripheral physiology, such as heart and ventilatory rates. This initial generalised response is brief, especially in mammals, and it is followed by a more directed behaviour towards the source of the stimulus and a reduction in the expression of the responses in those regions of the brain, which are not associated with further assessment of the stimulus. This secondary phase may be described as ‘attention’ and includes behavioural orienting towards the stimulus source (Laming, 1989a,b). The magnitude of the SPS (in terms of amplitude and/or duration) appears to reflect the level of arousal or activation of the brain in mammals (Rowland, 1968). In fish, a transient bradycardia (reduction in heart rate) provides a quantifiable measure of the arousal response to a novel stimulus and of the decline of that response with repetition of the stimulus (Laming and Savage, 1980). Situations that evoke a bradycardia, like the onset of increased illumination, also induce SPSs, recorded with implanted electrodes in the midbrain, hindbrain and forebrain (Quick and Laming, 1990). In response to a moving visual stimulus, the onset of illumination, or a tap to the side of the aquarium, the resulting 4 – 8 s SPSs were predominantly negative in the midbrain, positive in the forebrain and mildly positive in the hindbrain. During early presentations of the tap stimulus, the cardiac arousal responses were large and strongly related, as evaluated by regression analysis, to the amplitude of the midbrain SPS response to the tap stimulus. Interest in this relationship provoked further studies of cardiac arousal and SPS responses, this time with Ag/AgCl electrodes on the telencephalic, anterior tectal, cerebellar and medullary surfaces (Nicol and Laming, 1992). Initial presentation of the onset of illumination to fish in a darkened enclosure evoked a bradycardiac arousal response, accompanied by a predominantly positive SPS on the medullary surface, a predominantly negative SPS on the cerebellum, and an SPS that was initially negative (1 –2 s), then positive and eventually returning to negativity, on the anterior tectal and the telencephalic surfaces (Nicol and Laming, 1992). An SPS response to an arousing stimulus can thus be recorded in many regions of the fish brain, not only those most directly involved in processing the primary stimulus input, though such regions are associated with SPS responses of greatest amplitude. During arousal, all brain responses are considered to be amplified, whereas during attention there is selective response inhibition (Lynn, 1966). In rabbits, visual evoked potentials are enhanced in amplitude if preceded by cortical negativity, leading to the conclusion that the amplitude of the evoked potentials could be changed by reinforcement (Richter et al., 1992). We have performed studies on fish to determine if evoked potentials in response to a relatively neutral stimulus (sound) were affected by the prior presentation

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of an arousing, SPS-evoking stimulus. Initial presentation of a non-acoustic stimulus to fish may cause an increase or a decrease in the acoustic evoked potential (AEP) response in the brainstem to subsequently presented ‘click’ stimuli (Laming and Brooks, 1985). When more localised recordings were made, it was found that it is the peak to peak amplitude that is most affected by the preceding priming stimulus (Laming and Bullock, 1991). Although tenuous relationships have been found between the broad-band increase in EEG power in response to the non-acoustic stimulus and the change in the AEP, these were stimulus-specific and could not alone account for the change in sensory evoked activity (Laming et al., 1991a). Sustained potential shift responses and changes in AEP amplitude were thus simultaneously recorded in the midbrain tectum and torus semicircularis in response to water or saline applied to the flank of a carp. A 4 s negative SPS wave was followed by a positive wave in response to either type of priming stimulus alone (Laming et al., 1991b). Trains of six clicks delivered in the absence of a priming stimulus showed that the highest amplitude AEP was that evoked by the first click in the train. With a priming stimulus, this AEP was the most attenuated, indicating an attentional rather than an arousal response type (since arousal as mentioned above is associated with increased evoked potential amplitudes). These changes in AEP amplitude did not relate to the SPS in the acoustic-response region in which these were recorded. Rather, they showed relationships with the SPS in the other region from which recordings were made (i.e., torus or tectum). Although these studies suggested links between mechanisms generating the SPS and mechanisms causing changes in sensory evoked responses, they were not shown to be causally related and were coincidentally timed so that the SPS was going from negative to positive at a time when the AEP was attenuated. Further experiments were therefore performed, in which the fish was subjected to continuous background auditory stimulation (clicks delivered at 1 click/s), with averaging of AEP response changes, subsequent to the induction of SPSs on the telencephalic, posterior tectal and medullary surfaces in response to the onset of illumination. The posterior midbrain response was similar to that of the cerebellum reported previously, i.e., a largely monophasic, 10 s long negative wave; the medulla showed a small positive wave and the wave on the telencephalic surface was smaller still, and positive (Nicol and Laming, 1993). Attenuation of AEPs was recorded in response to illumination onset as the priming stimulus, again suggesting an attentional response. However, least attenuation was associated with large cardiac responses, indicating that both facilitatory and inhibitory (arousal and attention) mechanisms might be simultaneously active. Again, the amplitudes of these SPSs were closely related across the different regions. Correlations were also found between stimulus-evoked changes in AEP amplitude and simultaneously recorded SPSs during initial presentations of the stimulus. In the telencephalon SPS amplitude and changes in AEP amplitude were significantly correlated, and changes in midbrain AEP amplitude were significantly correlated with SPS amplitudes in the telencephalon and the medulla. These studies have revealed that many modalities of naturalistic stimuli induce SPS responses in most regions of the brains of fish. These responses are often related to cardiac (arousal) responses but they also share features of attention in that they accompany regional attenuation of sensory evoked neuronal activity.

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6. SPS responses associated with changes in motivation In toads, the feeding motivational state of the animal can be tested and quantified by the number of times the animal will perform the behavioural components of the prey catching sequence, when presented with a simulated prey object. The prey is an elongate piece of black card moving along its long axis against a contrasting background; i.e., ‘a worm’. The evoked behaviours are categorised as ‘orient’, ‘approach’, ‘fixate’ and ‘snap’ (Ewert, 1989). After testing for the motivational state, animals were prepared for recording SPSs during subsequent stimulus presentation. Ag/AgCl 0.5 mm ball electrodes were placed bilaterally on the anterior, mid and posterior tectal surfaces through small apertures in the cranium and fixed in place before leaving the animal 24 h to recover. Testing with the simulated prey showed that orientation to prey was associated with a negative shift on the anterior tectal surface, the retinal projection region of the frontal visual field. This negative shift was followed by a positive wave after ca. 4 s. On the surface of the posterior tectum, the reverse occurred, i.e., a positive shift was followed by negativity (Laming et al., 1995). The amplitude of these shifts was related to the prey catching motivation prior to the operation. Animals were then re-anaesthetised and immobilised by a muscle relaxant and retested to the ‘worm’ stimulus. The SPS response now was a monophasic negative wave in the anterior tectum and a monophasic positive wave in the posterior tectum. Again their amplitudes were related to the previously recorded motivational state. In order to determine if the motivational state had been affected by the operation and subsequent testing, animals were again anaesthetised to allow them to recover from the muscle relaxant and they were then tested again. They demonstrated no change in prey catching activity from that exhibited prior to the experiment (Laming et al., 1995). Thus, it can be concluded that the motivational state of the animal measured by prey-catching behaviour, influences the amplitude of SPSs.

7. Effects of potassium dynamics on motivation Section 6 showed that changes in neuronal responsiveness and SPSs are associated with changes in motivation as measured by behaviour in anurans. To test the hypothesis that these changes in motivation were related to changes in potassium dynamics, toads tested for motivation to feed were prepared for tectal recordings of visual units, overall neuronal activity and SPSs in response to application of isotonic artificial CSF to the tectum and subsequent administration of visual stimuli. Though only a few animals were motivated to feed, all were sexually active, which might account for the fact that most of the units found post-operatively responded best to moving square stimuli (T51 unit), which are more likely to represent potential mates than prey. This might account for the high level of overall neuronal responses to this type of stimulus before any solution addition, especially in non-hungry toads. It might also account for the significantly larger overall neuronal and SPS responses made by these animals compared to those of hungry animals. However, all animals showed significant unit, overall neuronal activity and SPS responses to presentation of the moving square stimulus alone at the start of experiments (Laming and Laming, 2003).

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With the exception of 41 mM Kþ solution, which was applied last, all lower concentrations were applied in random order and a summary of the effects of solution addition alone follows. Addition of 0 and 4 mM Kþ isotonic solution to the pia-covered brain surface produced no significant changes in unit or massed unit activity or any SPS. This may be because this potassium concentration is similar or less than that of ‘normal’ extracellular space. Although 7 mM Kþ solution addition showed no overall effect, there was an earlier increase in neuronal activity and an earlier SPS in hungry toads than in nonhungry animals. This is the first indication that the different motivational characteristics of the two groups may be reflected in the manner that the brain (or meninges) deal with a potassium load, marginally reduced sodium or physical trauma. With the addition of 11 and 17 mM Kþ there were significant rises in unit activity and an SPS. With 17 mM Kþ, there was also an increase in massed unit activity and the SPS was more evident in hungry toads. Addition of 27 mM Kþ solution only produced significant unit activity, and the final (non-randomised) addition of 41 mM Kþ only elicited a significant SPS, though at this point in the experiment, all responses appeared erratic, with a high level of massed unit activity. Overall, the concentration range from 7 to 17 mM Kþ produced the most significant responses to solution addition and revealed differences in those responses between the hungry and non-hungry groups. The differential responsiveness of the two groups and the fact that responses were only obtained at these concentrations suggests fairly free permeability of the pia to the ionic constituents. The fact that only these concentrations induced responses might also suggest that the physical trauma of solution addition may not be responsible for the responses themselves, but the lack of relationship between concentration and response would suggest that responses were not entirely due to diffusion characteristics of raised Kþ but may also involve the slightly lowered Naþ concentration. In response to the visual stimulus, there was an increase in overall neuronal activity at all except 7 mM Kþ concentrations. In all cases it was the non-hungry toads that demonstrated the largest responses. This effect was reflected also in the single unit responses at 7 mM Kþ, although a significant unit response was obtained at all concentrations. Orienting behaviour prior to the experiment was positively regressed on overall neuronal activity in response to solution addition and negatively on overall neuronal activity in response to the visual stimulus at both 4 and 7 mM Kþ concentrations. Thus, hungry animals showed more neuronal activity on solution addition and less than nonhungry animals to the visual stimulus. This followed a general trend in that non-hungry animals responded best to the moving square stimulus, but were less obviously sensitive to the addition of solutions. These differences in responsivity were most evident in the concentration range of 4 –17 mM Kþ. What emerges is the probability that the hungry and non-hungry animals all respond to both solution applications to the brain and to a visual stimulus, but in different ways. Non-hungry animals were more sensitive to solution addition, but slower, and more responsive towards the (non-prey) visual stimulus. The latter response suggests that their motivation might be sexual rather than food related. Their later, but more sensitive response to solution addition might reflect a different pial or cellular permeability to potassium. The converse explanation might obtain for the hungry toads that show a reduced responsivity to the non-prey stimulus, but respond faster to

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solution addition, perhaps because of relatively enhanced pial or cellular permeability to potassium and/or sodium. The possibility that permeability changes could contribute to changes in responsiveness and motivation is worthy of greater examination. 8. The effect of imposed DC shifts on neuronal responsivity and behaviour 8.1. Neuronal responsivity One way to emulate the effects of the SPS recorded in the tectum is to generate an SPS by DC stimulation and record it together with its associated neural responses both with and without additional visual stimuli. Although there is considerable evidence that a prolonged (days, weeks) DC stimulation damages neural tissue (Hurlbert et al., 1993), there is no evidence that DC stimulation at levels that mimic those reflected normally as sustained or SPSs has any adverse effect. Nor did the behavioural studies (see below) suggest that any damage had occurred. Toads (Bufo bufo) were tested for their prey catching motivation prior to experiments as described above. All animals showed prey-catching responses of varying degrees and motivation was measured by the number of orienting responses. Experiments involved recording SPS responses from platinum/iridium ball electrodes on the medial anterior and posterior left tectum and neuronal unit responses and SPS from a microelectrode in the anteriolateral right tectum, corresponding to the frontal visual field (Fig. 3, Sterritt et al., 2003). Prior to any DC stimulation, regression analysis showed that the neuronal responses of toads to a prey-like visual stimulus reflected their motivational tendency prior to operations, with a maximum response (, 10 spikes/s) after 4 s (Fig. 6a). Positive DC stimulation in the proximity of an electrode recording from the toad tectum enhanced and accelerated neuronal responses recorded by that electrode to a visual stimulus (Fig. 6b). Negative DC stimulation inhibited those neuronal responses. It would appear from regression analysis that the positive stimulation reinforces the motivational tendency of an individual animal. Although the data showed links between positivity and the neuronal response, this may not be the only interpretation as the visual stimulus was actually presented after the electrical stimulus, during the period when the polarity rebound was occurring, i.e., when the polarity was negative. Thus it can be concluded that DC stimulation interacted with the behavioural measures of motivational tendency to produce enhanced neuronal responses, whilst the potential was going from positive to negative. Recently, experiments have been performed using transcranial DC electrical stimulation in humans, that have revealed that a polarity dependent activation or suppression of cortical activity can be achieved with anodal or cathodal stimulation, respectively (Nitsche et al., 2002). Although those authors recognise the potential therapeutic use of such shifts they do not seem to consider the likelihood of a glial contribution to naturally occurring DC shifts, even though a glial origin of similar shifts has been demonstrated in cats (Roitbak et al., 1987) and amphibians (Roitbak et al., 1992), where the shifts mirror movements of extracellular potassium. It has been suggested that these potassium concentration changes may be a mechanism for modulation of neural excitability (Laming, 2000; Laming et al., 2000). The experiments described in Section 7 are consistent with this concept.

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Fig. 6. Mean ^ SEM of unit responses to the presentation of a moving worm-like stimulus, either (a) alone or (b) after stimulating the anterior tectal ball electrode (see Fig. 3) at þ 1.5 mV for 1 s against the negative posterior electrode, compared to DC stimulation alone (after Sterritt et al., 2003).

8.2. Behaviour The observation that DC currents and a thus imposed DC shift affect neuronal and SPS responses led to the question whether DC shifts also might have a modulatory effect on motivation. Thus, experiments were carried out to examine how DC or equivalent current AC stimulation of the toad tectum might affect the prey catching or avoidance responses

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exhibited by toads exposed to a simulated prey object. As before, toads were tested for their prey catching motivation. Under anaesthesia an operation was then performed to place a stimulating electrode on the anterior surface of each tectum (Fig. 3), and the animal was left for 24 h to recover before stimulating at various, randomly ordered, current strengths of both polarities (Sterritt and Laming, 2003). Prey-like visual stimuli, remotely operated, were presented to the toad in both clockwise and anti-clockwise directions, and the behavioural responses were monitored. The imposition of a DC current to the tectal surface (Fig. 3) caused a current strengthrelated increase in behavioural responses to a visual prey-like stimulus (Fig. 7). In all the experiments there was a large increase in prey-catching behaviour at currents above 50 mA, compared to the control without DC stimulation or the initial DC stimulation of 0.1 mA, and prey-catching behaviour increased as a function of increasing current, regardless of whether a toad started the experiment hungry or not. For orient and crouch

Fig. 7. Mean ^ SEM of orient, approach, (representing prey catching) retreat and crouch (representing avoidance) behaviours in response to 10 s transtectal DC stimulation followed by presentation of a remotely operated worm-like stimulus (3 £ 0.75 cm2 black card moving along its longitudinal axis at 2 cm/s) (after Sterritt and Laming, 2003).

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there was an interaction between right tectal negativity and anti-clockwise response preference, suggesting that there might be an additive effect of right tectal negativity with the negativity biologically generated as an SPS as the worm-like stimulus passes the left eye. In general, best responses for orient, approach and crouch behaviours were when the right tectum was negative, the left tectum positive. Analogously, Bauer et al. (1989) found that their human subjects exposed to epicranial DC responded faster to positive currents than negative. Based on these results they proposed that neurons depolarized to near the firing threshold would be activated or firing delayed, depending on the polarity of the current and spatial arrangement of the cells. At the higher currents visual stimuli in addition elicited defensive behaviours, like crouch and retreat, as well as inappropriate responses like delayed orient or orient in an inappropriate direction. It would seem that at these higher currents the tectal stimulation of the visual system was not always simply additive to the visual stimulus, but it was causing the toad problems in its interpretation of the visual stimulus. In some cases these problems were transient, as shown by the observation that during DC stimulation at higher currents some of the toads crouched and retreated for the first 2– 3 s of the 10 s exposure to the DC stimulation, but then they recovered and orientated and approached before the worm-like visual stimulus had passed the window. Ablation of the optic tectum abolishes all visually guided prey-catching and avoidance movements (Comer and Grobstein, 1981), while AC electrical stimulation of the same structures elicits normal orienting responses (Ewert, 1970). It was therefore interesting to compare the effectiveness of AC versus DC stimulation. It was found that AC stimulation at the current strengths used was particularly ineffective. There are of course no direct comparisons as the energy transfer of DC stimulation is much higher than at an equivalent AC current.

9. Changes in the SPS during habituation During habituation to a variety of arousing stimuli, the decline in the cardiac arousal response of fish to all types of stimuli used has been found to be related to a decline in the positivity of SPS responses in the telencephalon (Quick and Laming, 1990). This is of interest because of the considerable body of evidence linking telencephalic regions (posterior dorso-central) with habituation of arousal responses in fish (see Rooney and Laming, 1986, 1988). Nicol and Laming (1992) found in goldfish that during the period of habituation of the cardiac arousal response to the onset of illumination, SPSs recorded from the telencephalic, tectal and medullary surfaces in response to the same acoustic stimulus also declined in amplitude. In a further study (Nicol and Laming, 1993), it was found that such changes in the amplitudes of SPS responses to an unaltered stimulus are related to concurrent changes in the modulatory effects of the presentation of the arousing stimuli on the amplitudes of AEPs evoked by continuous background auditory stimulation. This was particularly apparent in the telencephalon. In this region, over successive presentations of the visual stimulus, the visually induced attenuation of AEPs declined, and this decline

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was related to the change, over the same series of presentations, in the amplitudes of SPSs recorded from the same site. In toads (Bufo bufo), intrinsic tectal visual unit activity frequency and burst duration habituate on repeated presentation of a prey-like object to immobilised but conscious animals. This habituation may reflect the unobtainability of the simulated prey. SPS amplitude and duration as well as the duration of characteristics associated with arousal in the EEG recorded with the same electrode, similarly decline (Laming and Ewert, 1984). However, the relationship is not a simple one. Over 20 presentations of the stimulus, unit frequency and burst duration declined to 50% of the original values, a similar decline as that of the duration of SPS and EEG responses. The decline in SPS amplitude was much larger, in the order of 70%, suggesting its habituation may be independent of the decline in unit responses. During habituation, the SPS duration was reduced to closely match that of the bursts of the local intrinsic tectal unit, suggesting that initially it was deriving some of its source [Kþ]e from more superficial (retinal input) regions, perhaps by spatial buffering. It would seem that the spatial buffering currents decline in magnitude to a greater degree than the decline in neuronal activity. This may be due to passive build up of [Kþ]e over the repeated stimuli, or to an exponential decline in potentially sensitising, spatial buffering currents. If the former, then it would appear that 1 min is insufficient time for potassium to equilibrate (the time between successive stimulus presentations), if the latter, then it would appear that glia may be actively involved in the process of habituation. 10. Concluding remarks It is apparent that glial cells, especially astrocytes and ependymoglia are highly responsive to elevated extracellular potassium, which triggers its own redistribution by these cells, influencing all cells within range of spatial buffering currents. The extracellular currents, largely caused by spatial buffering through glia, can be recorded as SPSs, associated with behavioural arousal and simple forms of learning in fish and amphibians. The motivational state of an animal is reflected in SPS amplitudes. Experimental changes in extracellular potassium or imposed DC shifts in the brains of amphibia interact with the animal’s motivational state in a way that suggests that ionic dynamics between neurons and glia may contribute to motivation. DC stimulation of the tectum of the common toad, Bufo bufo, enhances prey catching behaviour, though at higher currents it also generates avoidance. The finding that the polarity of the stimulus is relevant both to these behavioural responses and to neuronal activation, suggests that directional ionic movements such as those involved in spatial buffering and active uptake by glia may contribute to neuromodulation and behaviour.

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Regulation of Ca21 stores in glial cells Giovanni Scapagnini,a,b,* Thomas J. Nelsona and Daniel L. Alkona a

Blanchette Rockefeller Neurosciences Institute, West Virginia University, Rockville, MD 20850, USA p Correspondence address: Blanchette Rockefeller Neurosciences Institute, JHU, Academic and Research Building, 9601 Medical Center Drive, Rm. 351, Rockville, MD 20850-3332, Tel.: (301) 294-7191; fax: (301) 294-7007 E-mail: [email protected] b Institute of Neurological Sciences, CNR, 95123 Catania, Italy

Contents 1. 2.

3. 4.

5.

Calcium homeostasis in glial cells Ca2þ storage organelles and intracellular Ca2þ release 2.1. Sarco(endo)plasmic reticulum Ca2þ-ATPases 2.2. Ca2þ-binding proteins 2.3. Calcium release 2.4. Structural complexity and compartmentalization of ER in glial cells 2.5. Mitochondria Capacitative calcium entry Intracellular calcium sensors and effectors 4.1. S-100 4.2. Calcium-binding proteins in Alzheimer’s disease and apoptosis Concluding remarks

Virtually all cell functions are regulated by changes in the cytosolic free Ca2þ concentration. Ca2þ signals within cells can be local or global, can involve waves, oscillations, or even more-complex patterns, and can be modulated in terms of both amplitude and frequency. In astrocytes, calcium signaling is not restricted to single cells, but can cross cell borders via gap junctions, resulting both in intracellular Ca2þ waves, traveling from one glial cell to the next, and in induction of Ca2þ responses in neurons. In mammals, many glial cells contain an elaborate endoplasmic reticulum. Associated with this membrane network are pumps for Ca2þ uptake (the so-called SERCAs), that, as in other cells, are inhibited by thapsigargin and cyclopiazonic acid. The membranous network also contains Ca2þ-binding proteins for Ca2þ storage and channels for Ca2þinduced calcium release. These include the ubiquitous IP3 receptors and, in many cells, the ryanodine receptors. The ryanodine receptors are regulated by a variety of accessory proteins and are sensitive to the classical modulators ryanodine and caffeine. In glia, Advances in Molecular and Cell Biology, Vol. 31, pages 635–660 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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however, the major mechanism for Ca2þ release from internal stores involves activation of inositol 1,4,5-trisphosphate (IP3)-gated Ca2þ release channels (IP3 receptors). Regulation of IP3 receptors is complex with alternative splicing of at least three different isoforms, posttranslational modification of IP3R by phosphorylation, and interaction with adenine nucleotides, calcium, and immunophilins or FK506 binding proteins. The mitochondria and calcium storage proteins also function as high-capacity storage sites for calcium. Other calcium binding proteins play important roles in cell signaling, maintenance of the cytoskeleton, and apoptosis.

1. Calcium homeostasis in glial cells Virtually all cell functions, from cell birth to cell death, are directly or indirectly regulated by changes in the intracellular free Ca2þ concentration [Ca2þ]i, that act as an eclectic second messenger system. Cells have developed specialized machinery to control the spatial and temporal characteristics of these Ca2þ signals. These include transmembrane Ca2þ transporters and Ca2þ-permeable channels, cytoplasmic buffers, and intracellular organelles that are able to accumulate, store, and release Ca2þ. The fundamental importance of Ca2þ for signaling within cells has been established for a multitude of cell types and intracellular compartments. Glial cells express a complex set of molecules controlling Ca2þ signaling, including voltage-gated Ca2þ channels and Ca2þrelease channels from intracellular pools. Moreover, it has become evident that the different types of glial cells, such as cortical astrocytes, oligodendrocytes or microglia, are quite distinct with respect to their repertoire of Ca2þ-signalling mechanisms. This machinery allows these cells to sense, integrate and respond to external environment. Some aspects of Ca2þ-signaling depend directly upon Ca2þ entry from the extracellular fluid. Within the outer cell membrane, a broad range of channels allows the influx of Ca2þ in response to various stimuli; indeed glial cells express a large diversity of membrane receptors coupled to the phosphoinositide breakdown pathway as well as ionotropic receptors and voltage-gated calcium channels (MacVicar, 1984; Berger et al., 1992). Within the membranes of intracellular organelles, such as the endoplasmic reticulum (ER), Golgi apparatus and mitochondria, are additional response elements that control Ca2þ uptake from or release into cytosolic domains (or both). In glia, similar to most cells, ER Ca2þ storage and release contribute crucially to the Ca2þ signals. Net entry of Ca2þ through plasma membrane channels is usually much more limited in magnitude and duration in comparison to the larger, more sustained elevations caused by release from the ER. The ER, in fact, is important not only for buffering Ca2þ, but also for local Ca2þ signaling and for rapidly transmitting Ca2þ signals to the cell interior. Because multiple processes in cells can be influenced simultaneously by changes in [Ca2þ]i, the spatial and temporal patterns of Ca2þ signals is a critical point (Alkon et al., 1998). Indeed, the Ca2þ signals within cells can be local or global, can involve waves, oscillations, or even more-complex patterns, and can be modulated in terms of both amplitude and frequency (see chapter by Shuai et al.). These transient rises of [Ca2þ]i in turn trigger or regulate various intracellular events, including metabolic processes (see chapter by Hertz, Peng et al.), gene expression, and ion transport systems (Bootman et al.,

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1997; Dolmetsch et al., 1998). Moreover, in astrocytes, calcium signaling is not restricted to single cells, but can cross cell borders via gap junctions, resulting in intracellular Ca2þ waves traveling from one glial cell to the next, or the induction of Ca2þ responses in neurons. Calcium signaling might thus be a form of glial excitability, enabling these cells to integrate extracellular signals, communicate with each other and exchange information with neurons. Moreover, since it is apparent that glial Ca2þ signaling is an important pathway of glial –glial and neuronal – glial cross-talk, it is important to understand its role during pathological conditions. 2. Ca21 storage organelles and intracellular Ca21 release In glial cells (Simpson and Russel, 1997; Deitmer et al., 1998), as in all other types of animal cells, stimulation causes an elevation of the [Ca2þ]i, which triggers a large spectrum of physiological responses. Although some of this ‘signal calcium’ may come directly from the extracellular fluid through different types of channels (Verkhratsky et al., 1998), much of the signal Ca2þ comes from the intracellular Ca2þ stores, primarily the ER (Deitmer et al., 1998; Verkhratsky et al., 1998). The role of ER in Ca2þ sequestration was first demonstrated in neurons by Ca2þ flux and electron microprobe studies. Shortly thereafter, the study of Ca2þ signaling was revolutionized by the development of Ca2þsensitive fluorescent probes for use in intact, living cells. Application of these dyes revealed a near-universal distribution of ER Ca2þ-concentrating and Ca2þ-release mechanisms in cells, including glial cells. Nevertheless, to date, in spite of major advances during the last years, little is known about the precise structural and functional organization of the ER Ca2þ store. Direct visualization of ER using electron microscopy and fluorescent staining with lipophilic carbocyanine dyes suggests that the ER is a continuous, interconnecting network of tubules and cisterns (Pozzan et al., 1994; Terasaki et al., 1994). Aplysia glial cells were found to have an unusual analog of ER Ca2þ stores, which may retain an enormously high (up to 50 –100 mM) Ca2þ concentration. These glial cells surrounding the identified giant nerve cell bodies R2 or LP1 of Aplysia punctata were studied by quantitative electron microscopy and found to contain specific, electron-dense but non-osmiophilic membrane-bound granules, approximately 0.3 mm in diameter, called ‘gliagrana’ (Keicher et al., 1991). The density of these gliagrana varies with fluctuations in extracellular Ca2þ ([Ca2þ]o). Increases or decreases in glial calcium depending on [Ca2þ]o suggest the possible involvement of glia in the regulation of [Ca2þ]o. Similar glial granules are more often found in marine than in freshwater molluscs, possibly because they represent a calcium store used to compensate excess Naþ in the extracellular milieu of marine species and to regulate perineuronal calcium concentration (Keicher et al., 1992). In agreement with this hypothesis, the abundance of gliagrana ( ¼ number of glial granules per square micron) is found to be higher in animals adapted to low Ca2þ artificial sea water than in animals kept in high Ca2þ (or low Naþ) conditions. This finding is not observed after 1 week but after 2 weeks of adaptation. Similar stores have been described in frog ependymal glia (Gambetti et al., 1975). In mammals, many glial cells contain an elaborate ER (Privat et al., 1995). Associated with this membrane network are pumps for Ca2þ uptake (the so-called SERCAs), that, as

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in other cells, are inhibited by thapsigargin and cyclopiazonic acid (CPA). The membranous network also contains Ca2þ-binding proteins for Ca2þ storage and channels for Ca2þ release, the ubiquitous inositol triphosphate receptors (IP3R) and, in many cells, also the ryanodine receptors (RyR).

2.1. Sarco(endo)plasmic reticulum Ca2þ-ATPases In order for the cell to utilize Ca2þ as a signaling molecule, Ca2þ gradients across the plasma membrane and Ca2þ stores within intracellular organelles must be maintained. This is accomplished primarily by the activity of several dozen Ca2þ-transporting ATPases encoded by alternatively spliced transcripts from as many as eight different genes. These include three distinct sarco(endo)plasmic reticulum Ca2þ-ATPases (SERCA1 –3) (MacLennan et al., 1997) (human gene nomenclature, ATP2A1– 3), four distinct plasma membrane Ca2þ-ATPases (PMCA1 – 4) (human gene nomenclature, ATP2B1 – 4), and a putative mammalian secretory pathway Ca2þ-ATPase, (human gene nomenclature, ATP2C1). Ca2þ storage in the ER is mediated by a Ca2þ-dependent ATPase that is blocked by agents such as thapsigargin and CPA (Inesi and Sagara, 1994). The discovery of these potent and relatively selective SERCA inhibitors has enabled the widespread study of the roles of SERCA pumps in Ca2þ signaling (Simpson and Russel, 1997). In many cell types, SERCA inhibition leads to elevation of cytosolic [Ca2þ] secondary to leakage of Ca2þ from stores. Both thapsigargin and CPA have previously been reported to be effective in inhibiting SERCA pumps in cultured glia (Blaustein and Golovina, 2001). Some SERCAs (or perhaps non-SERCA Ca2þ pumps), however, are quite resistant to these agents (Tanaka and Tashjian, 1993; Golovina and Blaustein, 2000). The inhibitor-resistant Ca2þ pumps appear to be associated with Ca2þ stores that respond to caffeine and ryanodine, but not to inositol 1,4,5-trisphosphate (IP3) (Golovina and Blaustein, 2000). Three mammalian genes (SERCA1, -2 and -3) encode at least six SERCA isoforms as a result of alternative splicing (Shull, 2000). These gene products are differently expressed in various tissues and exhibit functional differences. SERCA1 consists of two C-terminal variants, both of which are restricted to fast-twitch skeletal muscle. The SERCA2 gene encodes both SERCA2a, the cardiac SR Ca2þ pump, and SERCA2b, the major intracellular Ca2þ pump of smooth muscle and non-muscle tissues. The two variants arise as a result of alternative splicing of sequences encoding their extreme C-termini. SERCA2b is similar to SERCA2a throughout most of its length, but its C-terminus contains an alternative 49- or 50-amino-acid sequence, depending on the species, in place of the last four amino acids found in SERCA2a (Shull, 2000). The extra sequence in SERCA2b contains an additional transmembrane domain, which places the C-terminus in the lumen of the ER, where it apparently interacts with, and is modulated by, calreticulin. The ubiquitous expression of SERCA2b and the relatively limited celltype distribution of the other isoforms suggests that SERCA2b is the major Ca2þ pump serving ER Ca2þ stores in most tissues, included glial cells. Although some cases of thapsigargin-resistance have been observed, treatment of cultured cells with thapsigargin

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usually leads to cell cycle arrest or apoptosis. Thus, it seems likely that SERCA2b plays a housekeeping function that is critical for the long-term viability of most mammalian cell types. In contrast, SERCA2a exhibits a restricted tissue distribution and is expressed at very high levels in cardiac muscle, where it clearly plays an organ-specific function. Contraction of cardiac muscle is initiated when Ca2þ influx across the sarcolemma (SR) triggers release of much larger quantities of Ca2þ from the SR, and relaxation occurs as Ca2þ is resequestered within the SR by SERCA2a and extruded from the cell by the Naþ/Ca2þ exchanger and the plasma membrane Ca2þ pump. Although its importance in cardiac physiology is clear, the degree to which normal cardiac function and long-term health are dependent on the appropriate levels of SERCA2a in heart is uncertain. The function of SERCA3, which has a lower Ca2þ affinity and a higher pH optimum than the other isoforms, is poorly understood. Three C-terminal variants of the enzyme arise by alternative splicing of the primary transcript. In situ hybridization, Western blot, and Northern blot analyses show that SERCA3 is expressed in many tissues, but that its cell-type distribution is quite limited. In all of the cell types in which it is present, SERCA3 appears to be co-expressed with SERCA2b, suggesting that it might play a specialized role in Ca2þ signaling or provide some redundancy in Ca2þ sequestering activity. Several different SERCA subtypes are known to be expressed together in some cells; for example, SERCA2a and SERCA3 are co-expressed with SERCA2b in cerebellar Purkinje neurons (Baba-Aissa et al., 1998). The functional significance of this coexpression, and the question of whether the different isoforms reside on the same or different ER components, remain to be elucidated. A further complication is that other, less well characterized, non-SERCA Ca2þ-ATPases (i.e., products of non-homologous genes) might also play a role in intracellular Ca2þ sequestration.

2.2. Ca2þ-binding proteins ER Ca2þ-binding proteins provide a high capacity buffering mechanism which results in the lowering of the concentration of free Ca2þ in the ER, and thus a reduction in the gradient against which pumps must transport cytoplasmic Ca2þ into the store. They are also thought to be important in localizing Ca2þ to sites of release, and in modulating release activity via protein– protein interactions with release channels (Mackrill, 1999). The best described of these Ca2þ-binding proteins are calsequestrin and calreticulin (CRT). The first of these proteins to be identified was calsequestrin in the striated muscle sarcoplasmic reticulum 19. The very acidic C-terminal domain of calsequestrin binds Ca2þ with low-affinity (average Kd ¼ 1 mM) and high capacity (2 50 Ca2þ-binding sites per molecule). A similar Ca2þ-binding protein, CRT, predominates in most (if not all) non-muscle cells (Krause and Michalak, 1997). In addition to the numerous low-affinity sites of its C-terminal domain, CRT also includes a single high-affinity site positioned towards the middle of the molecule. Localization of calreticulin to InsP3R-containing membrane vesicles has been reported in some cell types using density gradient techniques. The function of this co-expression, however, has remained controversial. Recent reports have indicated that calreticulin may play a role in regulating Ca2þ signals, including

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perhaps serving as a luminal sensor for Ca2þ store depletion. CRT is now recognized to be a multifunctional protein that is associated with cellular responses in many ways. CRT has been reported to interact with the subunit of the integrin receptors, and has been proven to be critical for regulating integrin-mediated cell adhesion in other systems. A recent study highlights the participation of CRT in integrin –ligand interaction in oligodendrocytes (Gudz et al., 2002). This work clearly showed the presence of CRT on the surface of oligodendrocytes. However, data on CRT association with integrins show that it interacts with the cytoplasmic tail of the integrin, and chelating intracellular Ca2þ removes CRT from the integrin – CRT complex, suggesting an intracellular localization. This dichotomy is unresolved, but it has been proposed that two forms of CRT may exist, an endoCRT molecule localized on the intracellular surface of the plasma membrane and an ectoCRT molecule localized on the extracellular surface of cells. Thus, further characterization of the CRT associated with this complex is needed. However, for CRT, extensive binding of, Ca2þ has been shown to take place both in vitro and within the ER of living cells. Such information is still missing for other ubiquitous lumenal proteins, in particular BiP, p94 (endoplasmin), Erp72, PDI and the membrane protein calnexin, all of which bind Ca2þ with low affinity when tested in vitro. Because these proteins do not possess an acidic C-terminal domain, their Ca2þ binding is believed to occur at doublets and triplets of acidic amino acids scattered throughout the molecule. This type of Ca2þ binding is not a general property of the lumenal Ca2þ-binding proteins of the ER. Other such proteins, e.g., reticulocalbin and p55 (calstorin, which is abundant especially in the brain), were shown to include typical EF-hand domains and may bind Ca2þ with higher affinity. Thus, Ca2þ buffering in the ER lumen depends on a host of Ca binding proteins with variable affinities for calcium.

2.3. Calcium release On the ER membrane, two major receptors trigger the release of Ca2þ into the cytosol: the inositol-1,4,5-trisphosphate receptor (IP3R) (Berridge, 1993) and the RyR (Sutko and Airey, 1996). Receptor-mediated activation of phospholipase C (PLC) at the outer cell membrane cleaves phosphatidylinositol bisphosphate (PIP2) to generate 1,2-diacylglyerol (DAG) and IP3, a second messenger which activates the IP3R-mediated release of Ca2þ (IICR) from the ER. Endogenous signaling pathway(s) that activate the RyR-mediated release of Ca2þ from the ER have, until recently, been largely unknown. For levels of [Ca2þ]i $ 10 mM (Buratti et al., 1995), calmodulin (CaM) has been shown to block RyR function (Tripathy et al., 1995), and cyclic ADP ribose (cADPR) at unknown levels can activate the RyR in vitro (Lee et al., 1995). However, neither the endogenous levels nor initial signaling molecules for cADPR have been determined. Nonetheless, it is known that activation of the RyR depends on the levels of Ca2þ already present in the cytosol (Verkhratsky and Shmigol, 1996). There is evidence, however, that IP3R activation also depends on [Ca2þ]i because [Ca2þ]i increases IP3R sensitivity to IP3 (Ehrlich, 1995). In any case, in muscle cells, the RyR does mediate Ca2þ-induced Ca2þ release (CICR) (Dousa et al., 1996). The role of RyR in CICR in glial cells (see below) has not been elucidated completely.

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2.3.1. IICR In glia the major mechanism for Ca2þ release from internal stores involves activation of IP3-gated Ca2þ release channels. (Berridge, 1993; Verkhratsky et al., 1998). The production of IP3, in turn, is achieved by the activation of PLC coupled via G proteins with numerous ‘metabotropic’ plasmalemmal receptors (see Fig. 1). The direct activation of IP3R by photorelease of IP3 from caged compound was shown in cultured astrocytes (Khodakhah and Ogden, 1993; Shao and McCarthy, 1995). Astrocytic IP3 receptors appear to be substantially more sensitive to IP3 than IP3R in Purkinje neurons; the threshold IP3 concentration for activation of the IP3-gated channel in astrocytes was 0.2– 0.5 mM, whereas in Purkinje neurons, it was 9 mM (Khodakhah and Ogden, 1993). The IP3R has been purified from rat cerebellum and localized primarily to the ER (Ross et al., 1989), though nuclear, plasma membrane and neurotransmitter vesicle localizations have also been described (Petersen, 1996). Molecular cloning reveals three different isoforms, encoded by different genes, IP3R1, 2, and 3 (Ross et al., 1992). Regulation of IP3Rs is complex with alternative splicing of at least IP3R1 and posttranslational modification of IP3R by phosphorylation. In addition, there is regulation by adenine nucleotides, calcium, and the immunophilin FK506 binding protein (Sharp et al., 1999). Different isoforms of the receptor may have different affinities for IP3 and different forms of regulation. Receptors in a number of cell lines appear to be heterotetramers composed of more than one isoform (Nucifora et al., 1996), and there is evidence for homotetramers of the IP3R3 isoform in some cultured cells (Nucifora et al., 1996), with the possibility that

Fig. 1. Components of Ca2þ signaling in glial cells. Many glial cells are endowed with voltage gated Ca2þ channels (VGCG) and Ca2þ-permeable ionotropic receptors (IR), which lead to an increase in the intracellular concentration of Ca2þ. In glia the major mechanism for Ca2þ signaling involves the activation of G protein (G) coupled metabotropic receptors (MR), which, through the synthesis of inositol trisphosphate (IP3) by PLC, activate Ca2þ release from the endoplasmic reticulum (ER), trough inositol trisphosphate receptors (IP3R). In some populations of glial cells a calcium induced Ca2þ release through ryanodine receptors (RYR) has also been demonstrated. Depletion of the ER stores might trigger additional Ca2þ entry via capacitative mechanisms, which involve the opening of the store-operated calcium channels (SOC). Regulation of Ca2þ signaling depends also on the intracellular Ca2þ buffering systems that include plasmalemmal Ca2þ ATP-ases (PMCA), the Naþ/Ca2þ exchanger (Na/Ca) and the storage by intracellular organelles by sarco(endo)plasmatic reticulum ATPases (SERCA) in the ER, or Ca2þ uniporters in mitochondria (M). A differential expression or activation of all the above-mentioned components results in a large heterogeneity of Ca2þ responses.

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homotetramers of other isoforms could also exist. Among tissues, IP3R heterotetramers have been demonstrated so far only in liver. Single-channel studies using isolated channel proteins incorporated in lipid bilayers have shown that the kinetic properties of the individual channel subtypes are different (Hagar et al., 1998; Ramos-Franco et al., 1998). The characteristic biophysical properties of the individual IP3R subtypes (Cardy et al., 1997; Miyakawa et al., 1999) expressed in a cell appears to determine the specific temporal and spatial characteristics of the Ca2þ signals that are elicited. Indeed, in the type 2-expressing cell, agonist-evoked [Ca2þ]i signals were oscillatory, the type 1- and 3expressing cells showed more transient Ca2þ responses. This property is believed to be due to the lack of Ca2þ-dependent inactivation of IP3 receptor subtype R2, which unlike the R1 and R3 subtypes are not inactivated at elevated Ca2þ concentrations. The expression of IP3R subtypes 1, 2, and 3 varies with development, and each subtype is expressed by cells in a tissue-specific manner IP3 is highly expressed in brain, IP3R2 apparently highly expressed in spinal cord, and IP3R3 highly expressed in intestine (Ross et al., 1992; Sharp et al., 1999). Different isoforms also predominate in different cultured cell types. The nature of the IP3Rs subtypes in different glial cells is not known in detail and remained for a while controversial. Astrocytes in culture express all three subtypes of IP3R, but this is not necessarily true for astrocytes in situ. In a couple of studies, only type 3 but not type 1 and 2 IP3Rs have been immunolocalized in rat cortical astrocytes, cerebellar Bergmann glial cells (Yamamoto-Hino et al., 1995) and astrocytes of suprachiasmatic nucleus (Hamada et al., 1999). The latter studies used an antibody raised against a peptide differing significantly from the rat sequence and the possibility exists that the IP3R antibody cross-reacts with the rat IP3R2 protein expressed by astrocytes (Sharp et al., 1999). Recently an extensive study has been conducted using more specific antibodies (Holtzclaw et al., 2002). Using dual indirect immunohistochemistry, it has been shown that astrocytes in several adult rat brain areas express only the type 2 isoform of IP3R. In addition IP3R2 labeling was found in cell bodies of oligodendrocyte lineage and in microglial cells. Moreover, in astrocytes receptor labeling appears in punctate patches and extends into the entire network, including fine hair-like branches of processes. The fact that these fine processes showed IP3R2 labeling suggests that ER elements extend into them and that these structures participate in cellular signaling. The unique expression of the IP3R2 ion channels in astrocytes in situ, because of the intrinsic characteristics of this isoform, may allow astrocytes to sustain stimulation at high frequency for extended periods. The patchy distribution of IP3R2 in astrocyte cell bodies and processes may represent clusters of receptors, probably linked to other proteins and organelles involved in Ca2þ signaling, and constituting specialized Ca2þ release sites. Intercellular signaling between astrocytes and between astrocytes and neurons may be supported by this organization of signaling elements in specialized microdomains (see also chapter by Shuai et al.). 2.3.2. CICR The other type of intracellular channel that mediates release of Ca2þ from intracellular stores, the Ca2þ-gated channel or RyR, is also comprised of a family of proteins that share sequence homology and structural similarities (Meissner et al., 1997). For example,

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channels of both families are assembled into heterotetrameric structures to form the functional ion pore. Each subunit consists of large proteins—300 kDa for IP3Rs and 500 kDa for RyRs. Three genes for IP3Rs and three genes (located on different human chromosomes) encode the three types of RyR isoforms (RyR1, RyR2 and RyR3). RyR1, characterized originally in skeletal muscle, is important for excitationcontraction coupling. RyR1 is located on the SR of skeletal myocytes and triggers Ca2þ release following an action potential by its direct interaction with the dihydropyridinesensitive, voltage-operated Ca2þ channel in the plasma membrane. Single-channel recordings, measurements of [45Ca2þ] efflux from SR vesicles, and measurements of binding of [3H]ryanodine have shown that skeletal muscle RyR activity is affected by binding of cations to Ca2þ regulatory sites, as well as by binding of adenine nucleotides and possibly organic polycations (Meissner, 1994). In contrast to RyR1, RyR2 activates excitation – contraction coupling principally in heart muscle cells in response to voltage-dependent Ca2þ influx [through dihydropyridine receptor (DHPR) channels] that accompanies depolarization of the cardiac SR and T-tubules. Here, the DHPR channels are not in physical contact but are in close proximity to the RyR channels. RyR2 amplifies the DHPR signal by means of calcium-induced calcium release (CICR) (Meissner, 1994). Unlike IP3Rs, RyR2 conductance correlates positively with the level of Ca2þ already present in the cytosol, thus producing a positive feedback amplification for Ca2þ signaling. CICR is made possible in myocardial cells by local junctions between the plasma membrane and closely juxtaposed RyRs on the SR. Apparently, this RyR-membrane complex is important for the generation of local [Ca2þ]i oscillations. RyR2 is the most abundant isoform throughout the brain, with the highest levels being found in hippocampal regions such as CA3 and the dentate gyrus, as well as in the cerebellum, olfactory bulb and in certain cortical areas. Although CICR is well documented in peripheral neurons, its function in central neurons has not been established. While CICR has not been as well characterized in neurons as in muscle cells, RyR2plasma membrane juxtaposition within dendritic structures might contribute to local oscillations of [Ca2þ]i, as in myocardial cells. While a number of observations using caffeine, voltage-clamp control of Ca2þ influx and ER depletion do suggest a role for CICR in neurons (Verkhratsky and Shmigol, 1996), a definitive demonstration awaits further study. The RyR3 isoform was originally described in the brain, where it is localized predominantly in CA1 hippocampal neurons. All three RyR isoforms are co-expressed in some neurons. Distinct isoforms might be localized in distinct subcompartments of the ER and thereby provide different sensitivities to ligand- or voltage-based stimuli, or both, particularly within dendritic branches (Furuichi et al., 1994). All three RyR isoforms consist of at least two functional elements: (i) a relatively small transmembrane domain located near the C-terminus region that appears to form a channel, and (ii) a large N-terminal segment that protrudes into the cytosol and is organized into different domains on which reside binding sites for putative modulatory substances. While the recently discovered second messenger, cADPR, does activate some in vitro RyRmediated Ca2þ release (Lee et al., 1995), effective levels of cADPR and upstream transduction pathways have not been identified. In addition to CaM, which binds to the

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C-terminal region of the RyR, another class of putative RyR-modulating proteins includes immunophilins. These low-molecular-weight proteins were characterized originally as receptors of immunosuppressant drugs, such as cyclosporin A, FK506 and rapamycin, and bind both RyR and IP3R and regulate Ca2þ efflux from the ER (Gold, 1997), possibly by maintaining the configuration of the tetrameric structure. The immunophilins are also expressed at higher levels within the brain than in immunocompetent tissues. The expression of RyR in glia has been poorly investigated and is still debatable. Functional Ca2þ-induced Ca2þ release, sensitive to the classical modulators ryanodine and caffeine and activated under physiological conditions, has been demonstrated initially for periaxonal Schwann cells (Lev Ram and Ellisman, 1995) and for freshly isolated Muller glial cells from salamander retina (Keirstead and Miller, 1995). In Bergmann glial cells, studied in cerebellar slices, caffeine and ryanodine triggered a moderate [Ca2þ]i elevation and attenuated [Ca2þ]i transients evoked by kainate. In astrocytes, data on CICR are controversial. Initial studies conducted in cultured and freshly isolated astrocytes failed to detect an obvious caffeine-triggered [Ca2þ]i effect (Charles et al., 1993). Other studies in cultured embryonic cortical astrocytes clearly showed that caffeine triggered a [Ca2þ]i increase (Golovina and Blaustein, 1997). A recent study demonstrated that cultured mouse astrocytes express only the RyR type 3 mRNA, but not Ryr1 or Ryr2 genes (Matyash et al., 2002). Immunolabeling with specific RYR3-antibodies confirmed the distribution of this channel in astrocyte cytoplasm, and its close relation to ER. Similar data were obtained in brain astrocytes in situ. Furthermore, RyR activation with the specific agonist 4-chloro-mcresol triggers an elevation of intracellular Ca2þ in all astrocytes that have been investigated, indicating that astrocytes in the brain express functional RyRs. In the same study, using either pharmacological blockade of RyR by an antagonizing concentration of ryanodine (200 mM) or RyR3 knockout mice, it has been shown that functional RyR is fundamental for astrocyte motility and migration. RyR3 has not been detected in purified microglial cells. Another study identified RyRs by immunocytochemistry with specific antibodies in cultured oligodendrocytes, in type 2 astrocytes and in the bipotential precursor cells (O-2A progenitors) from which oligodendrocytes and type 2 astrocytes can develop (Simpson et al., 1998). Glia acutely isolated from rat brain or in situ in cortical sections were similarly found to express RyRs. Caffeine elicited Ca2þ responses in most cultured type 2 astrocytes and in approximately 50% of cultured oligodendrocytes. Increases of [Ca2þ]i elicited by caffeine were inhibited by pretreatment with ryanodine or thapsigargin, while ionotropic glutamate receptor activation by kainate increased the magnitude of Ca2þ elevation evoked by caffeine in all the cells evaluated.

2.4. Structural complexity and compartmentalization of ER in glial cells Although the ER has been described as a ‘continuous network’ of tubules (Terasaki et al., 1994), some direct studies of Ca2þ store organization provide evidence for heterogeneity and compartmentalization (Golovina and Blaustein, 1997). Cell fractionation studies and immunocytochemical studies also indicate that neuronal ER is heterogeneous. Resolution of the ambiguity about ER compartmentalization requires detailed, direct measurements of the intra-ER Ca2þ concentration ([Ca2þ]ER) with

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sufficiently high spatial resolution to visualize ER subcompartments. Several methods have been employed. Some involve the use of Ca2þ-sensitive fluorochromes such as Furaptra, which do not saturate at the ambient [Ca2þ]ER. Others involve transfection of cells with Ca2þ reporter molecules (aequorin or CaM-linked green fluorescent protein) targeted to specific organelles, or the application of electron microprobe analysis. Anyway, all these methods have limitations, and a wide range of [Ca2þ]ER values (5 mM – 5 mM) has been reported. Probably, to solve the question about compartmentalization, rather than focusing on absolute [Ca2þ]ER levels, relative changes in [Ca2þ]ER have to be considered. Some indirect observations have led to the conclusion that the two ER membrane receptors (IP3R and RyR) release Ca2þ from the same pool (Khodakhah and Armstrong, 1997). However, many other functional studies suggest that the IP3-sensitive and Ry/ caffeine-sensitive Ca2þ stores are independent (Kostyuk and Kirischuk, 1993). For example, imaging of intact snail neurons has revealed spatially distinct cytosolic elevations of [Ca2þ]i in response to caffeine (higher in the subplasmalemmal region) and to IP3 injection (higher in the cell center). Similarly, in adrenal chromaffin cells, IP3 and caffeine-evoked responses appear to be spatially distinct (Cheek et al., 1991). Moreover, in these cells, the two types of stores can be independently emptied: the response to caffeine is unaffected by prior, selective depletion of the IP3-sensitive store in response to either IP3 or thapsigargin. Similarly, studies conducted on cortical astrocytes revealed that ER Ca2þ stores are organized into functionally distinct subcompartments that can be unloaded and refilled independently (Golovina and Blaustein, 1997). The agonists glutamate and ATP released Ca2þ primarily from CPA-sensitive ER Ca2þ stores. Agonist-evoked release was abolished by prior treatment with CPA, but it was unaffected by prior depletion of caffeine/ ryanodine (CAF/RY)-sensitive ER Ca2þ stores. Conversely, prior depletion of the CPAsensitive stores did not interfere with Ca2þ release or reuptake in the CAF/RY-sensitive stores. These studies also addressed the question of whether the functionally independent CPA-sensitive (IP3-sensitive) and CAF/RY-sensitive ER Ca2þ stores were spatially separate. To this end, the ER of living cells was loaded with Furaptra, and the changes in [Ca2þ]ER were studied with high spatial resolution imaging methods to visualize individual small elements of the ER. Image subtraction was then used to demonstrate that some components of the ER unload Ca2þ in the presence of CPA, while the [Ca2þ]ER in other components rises under these circumstances. The components of the ER that load with Ca2þ when CPA is added, are depleted by CAF. This implies that Ca2þ is sequestered in the CAF-sensitive stores by a CPA-insensitive Ca2þ pump. The effects of both CPA and CAF (on the specific stores) are reversible, and there is only a 10 –18% overlap between these two types of stores. Moreover, when Ry (which has a relatively irreversible effect) was used to deplete the CAF/RY-sensitive stores in the presence of CPA, only the CPAsensitive stores refilled after washout (Golovina and Blaustein, 1997). Thus, considering both the functional and spatial information presented in these studies, it is possible that there are two structurally separate (or independent) components of the ER Ca2þ stores in astrocytes. The existence of two different types of ER Ca2þ release mechanisms has been studied also in oligodendrocytes (Haak et al., 2001). Both oligodendrocyte progenitors and myelinating oligodendrocytes are intimately associated with axons, suggesting the

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existence of neuronal signals affecting oligodendrocyte proliferation, migration, and differentiation (Barres and Raff, 1999). Although oligodendrocytes can differentiate without neurons, axons or axon-derived signals enhance myelin protein expression. Axonal signals may also be required for oligodendrocyte survival, and it has been suggested that neuronal electrical activity is linked to myelinogenesis, perhaps by stimulating the release of growth factors and neurotransmitters from axons or from astrocytes or other glial cells (Barres and Raff, 1999). In addition, neurotransmitter receptors are expressed by oligodendrocytes at several stages of differentiation, which suggests that they might participate in oligodendrocyte differentiation. In oligodendrocyte progenitor cells (OPs) activation of either IP3R or RyRs produced kinetically distinct local Ca2þ release events. Spatial and temporal characteristics of Ca2þ signaling, from within microdomains to intracellular waves, have been studied in these particular glial cells that must migrate and proliferate before differentiating into myelinating cells. OPs express specific Ca2þ release channel subtypes: RyR3 and IP3R2. These receptors are expressed in patches along OP processes. RyRs are co-expressed with IP3Rs in some patches, but IP3Rs are also found alone. This differential distribution pattern may underlie the differences in local and global Ca2þ signals mediated by these two receptors. Intracellular Ca2þ waves initiation seems to be dependent on the activation of IP3Rs, while activation of the RyR3 in OPs appears to evoke highly localized Ca2þ signals. Local Ca2þ release from intracellular stores has been shown to be fundamental for cell guidance and migration during brain development (Simpson and Armstrong, 1999). Furthermore, in addition to their separate roles, IP3R and RyRs appear to modulate each other, to tightly regulate Ca2þ release from the ER.

2.5. Mitochondria Mitochondria are another capacious Ca2þ storage site (Pozzan and Rizzuto, 2000; Ganitkevich, 2003). Generally Ca2þ can enter mitochondria through two mechanisms, which have not yet been identified at the molecular level: a saturable low-affinity (10 –20 mM) uniporter and a saturable rapid uptake mode (Gunter and Gunter, 2001). It is clear that most Ca2þ ions entering the mitochondrion are bound and only a very small portion remains free. The Ca2þ-binding ratio in mitochondria ([Ca]total/[Ca]free) was estimated to be in the range of , 4000 to 6000 in the steady-state (Kaftan et al., 2000). Mitochondria do not express significant amounts of Ca2þ binding proteins (e.g., like calreticulin or calsequestrin in ER), so Ca2þ is most likely to be bound to membrane phospholipids and/or to precipitate with phosphate ions (Pivovarova et al., 1999; Thayer et al., 2002). Large amounts of Ca2þ could also be stored in the mitochondrial matrix in the form of insoluble hydroxyapatite, if phosphate ions are available. Ca2þ leaves the mitochondrion mainly through two distinct saturable mechanisms. These are Naþ dependent and Naþ independent transport systems (Pfeiffer et al., 2001). In addition, Ca2þ could be released from mitochondria through a channel known as the permeability transition pore, which is activated with an excessive increase of free Ca2þ in the mitochondrial matrix and trigger cascades of cellular processes leading to cell death (Rizzuto et al., 2000). However, the role of mitochondria in [Ca2þ]i homeostasis in cells,

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and in particular in glia, is little understood. The dissipation of the mitochondrial electrochemical gradient by protonophores [carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone] triggers Ca2þ release in oligodendrocytes (Kirischuk et al., 1995a). CCCP treatment was not able to influence the kinetic parameters of the depolarization-triggered [Ca2þ]i transients (Kirischuk et al., 1995b). This suggests that mitochondrial Ca2þ accumulation does not play an important role in calcium signaling. However, protonophores are not totally selective for mithochondria, and probably do not represent an accurate method of investigation. Other studies highlighted the importance of mitochondrial location with respect to Ca2þ flux in the cytoplasm, to determinate their importance as Ca2þ sink (Collins et al., 2001). In a variety of cell types, the close apposition of mitochondria with ER has been documented (Hajnoczky et al., 2000). An interaction between ER and mitochondria has been implicated in mitochondrial Ca2þ uptake. During Ca2þ release from ER through the opening of IP3-gated channels (Kaftan et al., 2000) or RyRs (Pacher et al., 2002), a high local Ca2þ concentration at sites of close contact between ER and mitochondria is believed to facilitate mitochondrial Ca2þ uptake (Rizzuto et al., 2000). It has been shown that mitochondria closely associated with ER tend to unload Ca2þ preferentially into microdomains, thus facilitating ER Ca2þ reloading (Arnaudeau et al., 2001). In oligodendrocyte processes, mitochondria were always found in association with sites where SERCA expression was elevated. This suggests that high concentrations of SERCA pumps together with other cellular specializations may be important in supporting elevated local Ca2þ-release kinetics in oligodendrocytes. This presumably allows mitochondria to play a specific role that depends on the spatial and temporal profile of cytosolic Ca2þ signals, that is, the mitochondrial contribution to Ca2þ regulation is location and stimulus specific. Therefore, it is not surprising that in some studies no apparent contribution was found of mitochondrial Ca2þ uptake during physiological stimulation.

3. Capacitative calcium entry After stimulation, the temporal and spatial distribution of the increase in [Ca2þ]i in glial cells, especially astrocytes, is remarkably complex. At a given point, the time course of the increase may be a single spike, biphasic with an initial peak followed by a plateau, or oscillations (Verkhratsky and Kettenmann, 1995—see also chapters by Shuai et al., and by Cornell-Bell et al.). The plateau/oscillation phases requires extracellular Ca2þ. The Ca2þ influx pathway activated with metabotropic receptor stimulation is unclear. By analogy with other tissues, the most likely candidate is the store-operated Ca2þ channel, although the existence of this pathway in glial cells has not been completely understood. Store emptying generates a putative signal (Rzigalinski et al., 1999) that induces the opening of the store-operated calcium channel (SOC) at the level of the cell membrane, also known as the calcium release-activated calcium channel (CRAC) (Parekh and Penner, 1997), which allows calcium into the cells from the extracellular space. This channel has been identified as homologous to the transient receptor potential channels in Drosophila (Petersen et al., 1995). Calcium entry through the SOC/CRAC, activated by the depletion of the intracellular calcium stores, is also

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known as capacitative calcium entry (CCE). CCE has been identified in many cells (Parekh and Penner, 1997; Barritt, 1999; Putney, 1999), but the mechanism linking store-depletion and the increased Ca2þ permeability of the plasma membrane remains elusive. Several mechanisms have been proposed. In mast cells, an inward Ca2þ current (ICRAC, Ca2þ release-activated Ca2þ current) is seen following depletion of intracellular Ca2þ stores (Hoth and Penner, 1992), while, in mouse pancreatic acinar cells, CCE results from a ICRANC, Ca2þ release-activated non-selective cation current (Krause et al., 1996). It has been suggested that a Ca2þ influx factor, generated when stores are depleted, diffuses to the plasma membrane where the SOC is located, and activates CCE (Randriamampita and Tsien, 1993). Alternatively, direct physical contact between the plasma membrane and the ER IP3 receptor, which detects depletion of the intracellular Ca2þ stores, might be responsible for activating CCE (Irvine, 1990). Recently, two studies have provided evidence to support this latter hypothesis of an exocytosis-like mechanism of CCE activation (Yao et al., 1999; Patterson et al., 1999). CCE is a mechanism whereby intracellular calcium stores are refilled (Berridge, 1995; Putney, 1999). The maintenance of the filled state of intracellular stores is involved in cell survival, as the prolonged depletion of calcium reservoirs causes cell death and apoptosis. Two hypotheses have been proposed to explain how calcium flowing through the SOC/ CRAC during CCE can be captured and stored in the ER compartments and thus contribute to the spike or the plateau phase of the single calcium transient. Calcium, according to the preferential pathway hypotheses, could be directly sequestered within intracellular stores because of a physical association between the SOC/CRAC and the SERCA –ER complex (Berridge, 1995; Putney, 1999). Alternatively, calcium ions, once admitted into the cytosol by SOC/CRAC opening, might be quickly removed from the cytosol and accumulated in the ER by the SERCA (diffusion pathway) (Hofer et al., 1998). In addition to refilling the intracellular Ca2þ stores, the increased Ca2þ via SOC may also have other cellular functions. For example, Ca2þ in the microdomain of the channel mouth may activate enzymes or target proteins. In glial cells CCE has not been fully characterized, and even the existence of SOC/CRAC channels has not been clearly shown. In a study performed in our lab we evaluated the relation between cytoskeleton rearrangement and CCE in cortical astrocytes (Grimaldi et al., 1999). Cultured conventional astrocytes (occasionally called type-1 astrocytes) express a wide array of second messenger-coupled receptors (Bhat, 1995; Verkhratsky et al., 1998). Among these receptors, P2Y, a subclass of purinergic receptors, activated by ATP (Shao and McCarthy, 1993) and bradykinin (BK) receptors (Chen et al., 1996) are coupled to PLC. Their activation causes calcium mobilization from intracellular calcium stores via PLC-induced IP3 production (Berridge, 1995). In this study, we provided evidence that active and rapid rearrangement of astrocyte morphology induced by activation of the protein kinase A (PKA), is responsible for enhancement of IP3induced cytosolic calcium concentration ([Ca2þ]i) elevation via an enhanced CCE. Enhanced CCE, in turn, is associated with reorganization of the spatial relationship between the outer cell membrane and the ER. In astrocytes, it is likely that the reorganization of the spatial relationship between ER structures and cell membrane may cause a change in the efficiency of calcium mobilization as a result of the improved

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refilling of the intracellular calcium stores. This would, in turn, result in larger calcium transients. A common means of inducing morphological changes in astrocytes has been prolonged application of long lasting cAMP analogues. Astrocytes, which differentiate after prolonged exposure to cAMP analogues, changed from a flat polygonal form to a stellate process-bearing appearance after application of cAMP. Astrocytes differentiated by a long term exposure to cAMP showed changes in biochemical properties such as an increase in the production of IP3 in response to both a1-adrenergic agonists and BK. In addition, longterm cAMP-induced differentiation modifies membrane ionic conductance (Lascola and Kraig, 1996). We assessed the arrangement of the ER in undifferentiated and differentiated cells. In undifferentiated astrocytes, ER structures were largely evident in the periphery of the cells where a tubular network of membrane-delimited structures was identifiable (see Fig. 2). In addition, ER structures and the Golgi complex could be identified in the perinuclear region of the astrocytes. In differentiated cells, perhaps as a consequence of the marked reorganization of the cell shape, the ER was so condensed that areas of the cytoplasm free of ER structures were virtually absent. Such a marked rearrangement may, in turn, increase the availability of calcium flowing from the extracellular space to provide for refilling of the stores during CCE, through a closer association of the outer cell membrane and ER structures. Cell morphology has been previously shown to play a role in agonist-induced calcium mobilization in fibroblasts, in thapsigargin-induced store depletion, and in the associated

Fig. 2. Analysis of the ER distribution and association with cell membrane in control and differentiated astrocytes. ER was labeled in living cells by means of the ER tracker and analyzed with a confocal microscope, equipped with a 633 lens for ER-associated fluorescence. Images were then software zoomed to resolve single astrocytes. In undifferentiated astrocytes (left panel) the density of ER membranes is highest in the perinuclear area, whereas the density of the tubular network is decreased in the peripheral part of the cells. The arrowheads indicate some connections between the tubular ER structures and the cell membrane. Differentiated astrocytes (right panel) showed a more condensed ER organization without appreciable areas free of fluorescence signal across the cell.

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CCE in endothelial cells (Holda and Blatter, 1997). Because cAMP-induced differentiation causes the rearrangement of the actin-formed stress fiber (Goldman and Chiu, 1984), we induced actin depolymerization with cytochalasin D (CytD) before exposing the cells to dibutyryl cAMP. As expected, in the presence of CytD, the cells did not reshape and maintained their flat polygonal appearance, despite the activation of PKA by cAMP. The prevention of the shape changes in dibutyryl cAMP-treated cells by CytD avoided the potentiation of ATP- and thapsigargin-induced [Ca2þ]i elevation (see Fig. 3). Both PKA activity in the cell cytosol and PKA localization at level of the cell membrane, where

Fig. 3. Effect of stress fiber depolymerization on ATP- and thapsigargin-induced [Ca2þ]i elevation. The effect of stress fiber disassembly by means of 10 mM cytochalasin D (CytD) was studied. [Ca2þ]i values obtained at the peak of the response were averaged and graphed as a bar ^ SEM. In panel a, ATP-induced intracellular calcium mobilization in undifferentiated and dibutyryl cAMP-treated astrocytes. Undifferentiated (open bar) and 10 mM CytD pretreated (hatched bar) astrocytes display a similar response to ATP. However, the potentiation of ATP stimulation in dibutyryl cAMP-differentiated astrocytes (solid bar versus open bar) was reversed by CytD pretreatment (square-filled bar). In panel b, the effect of CytD pretreatment on thapsigargin-induced intracellular calcium mobilization in undifferentiated and differentiated astrocytes is displayed. CytD pretreatment (square-filled bar) completely reversed the potentiation of thapsigargin response in differentiated astrocytes (solid bar). * p , 0:05 versus value in undifferentiated cells; * * p , 0:05 versus value in dibutyryl cAMPdifferentiated cells.

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CRAC channels are localized, have been reported not to be affected by CytD pretreatment. Therefore, it seems likely that morphological changes involving the rearrangement of stress fibers play a critical role in the potentiation of the CCE. Another study demonstrated that in rat cerebellar astrocytes, store depletion induced by thapsigargin activates CCE via SOCs (Lo et al., 2002). Furthermore, it has been shown that these channels are also permeable to Naþ but not to Sr2þ and Ba2þ and that their activity is regulated by serine/threonine phosphorylation. A study from our group has shown that 4-aminopyridine, a specific blocker of voltagesensitive Kþ channels (Aronson, 1992), strongly potentiates CCE in astrocytes, but not in neurons (Grimaldi et al., 2001). Because the effect of 4-AP alone on CCE is not as large as when it is triggered by a large calcium mobilization, we believe that other mechanisms must be activated to uncover the potentiation of CCE that we observed. When ICS are depleted, either using an agonist able to cause a large production of IP3, such as ATP or bradykinin or an agent able to completely discharge ICS, such as thapsigargin, a robust signal is generated that triggers the opening of CRAC. Such a signal has not been definitively identified and characterized. However, in cortical astrocytes a soluble factor, probably belonging to the family of the eicosanoid derivatives (Rzigalinski et al., 1999), may be responsible for the opening of CRAC channel. Alternatively, a physical association between SOC/CRAC and the IP3 receptor may be involved in the opening of the CRAC channel after the emptying of ICS (Boulay et al., 1999). Regardless of the signal used to trigger the opening of the CRAC channels, 4-AP causes a considerably larger influx of calcium from the extracellular space than in control cells. CRAC and voltage-sensitive Kþ channels have some similarity in the amino acid sequence (Harteneck et al., 2000); therefore, it is conceivable that 4-AP interacts with the open CRAC channels in a similar manner as with Kþ channels, and thereby increases CCE. This action on CCE may explain some of the therapeutic effects of 4-AP in disorders in which impairment of neurotransmission is involved (Fujihara and Miyoshi, 1998; Andreani et al., 2000; Smith et al., 2000). Moreover, changes in calcium homeostasis induced by 4-AP in astrocytes might cause the release of trophic factors that are likely to support regrowth of neuronal extensions. In microglial cells, a long-lasting activation of capacitative Ca2þ entry after maximal depletion of intracellular Ca2þ stores by stimulation with ATP in the absence of extracellular Ca2þ has been described recently (Toescu et al., 1998). Once activated, the capacitative Ca2þ entry pathway in microglial cells remained operative for tens of minutes, creating a steady-state [Ca2þ]i elevation that dramatically outlasted the period of agonist action.

4. Intracellular calcium sensors and effectors After entering the cytoplasm, Ca2þ binds to a number of proteins that trigger various intracellular signal transduction pathways. Probably the best-known cytoplasmic Ca2þ sensor is CaM, which regulates the functional activity of at least three broad classes of enzymes, namely, CaM-dependent protein kinases, protein phosphatases, and adenylate cyclases. The latter either interact with cytoplasmic enzymes or transfer the signal further down to the nucleus, initiating other pathways responsible for gene expression. Among the

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many molecular targets of internal Ca2þ signaling, PKC isozymes a, b and g are activated and translocated by combination(s) of Ca2þ, DAG and arachidonic acid (AA) (Nishizuka, 1984; Alkon et al., 1998). Elevated [Ca2þ]i also acts on Ca2þ/calmodulin-dependent (type II) kinase(s) (CaM kinases) that, in turn, can regulate voltage-dependent Kþ channels, cholinergic control of neuronal responsiveness, smooth muscle contraction and synaptic transmission. At higher Ca2þ levels (. 10 mM), levels where CaM is known to block RyR function, CaM also regulates a mode of protein trafficking to the nucleus that is similar to, but independent of active nuclear transport regulated by the low-molecularweight GTP-binding protein, ran (ran-mediated transport). Other evidence has also been found, suggesting an effect by CaM kinase(s) on transcription factors that in turn could influence protein synthesis (Gringhuis et al., 1997). An alternative way by which cytoplasmic Ca2þ signals may influence gene expression is the pathway involving Ras proteins (small guanine nucleotide-binding proteins), which after being activated by Ca2þ trigger a cascade of phosphorylation events that lead to a modulation of gene expression. Finally, cytoplasmic Ca2þ signals may propagate to the nucleus, where they directly stimulate the synthesis of immediate early genes as well as structural genes. Unfortunately, little is known of the expression and role of these systems in glial cells; their characterization in glia is an important problem awaiting an experimental solution.

4.1. S-100 S-100 is a highly conserved, low molecular weight (10 – 12 kDa) multifunctional calcium binding protein synthesized in a variety of cells, including astroglia and Schwann cells. The protein is extremely stable and hydrophilic, and 95% of the cellular S-100 is found in the cytosol. The S-100 protein occurs as a homodimer, and it is one of a large family of low-MW EF-hand proteins that includes calcyclin, p9Ka, and the cystic fibrosis serum antigen MRP-8. A large number of functions have been attributed to S-100, including inhibition of protein phosphorylation, promotion of calcium-dependent microtubule dissociation (Fano et al., 1995), and maintenance of the cytoskeleton. Like CaM, S-100 acts by binding to other proteins in a calcium-dependent fashion. For example, S-100 binds to glial fibrillary acidic protein (GFAP) and inhibits GFAP polymerization in a calcium-dependent manner. It also reportedly undergoes calciumdependent binding to a number of targets, including fructose-1,6-bisphosphate aldolase and the RyR, which is involved in calcium-dependent calcium release from the ER, as described above. Inhibition of microtubule assembly was suggested to occur by binding of S-100 to tau proteins, preventing phosphorylation by protein kinase C (PKC). The finding that the gene for S-100 is located in a gene cluster on chromosome 1q21 has suggested possible involvement with Alzheimer’s disease and Down’s syndrome. Indeed, elevated levels of S-100 immunoreactivity have been found in both Down’s syndrome and Alzheimer’s disease patients. In Alzheimer’s disease and AIDS-related progressive neurodegeneration, elevated S-100 levels in extracellular fluids correlate with the extent of the pathological injury. Elevations of S-100 in hippocampus were also found in patients with Alzheimer’s disease.

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Other researchers have found that at high concentrations (0.5 –2 mM), homologous dimers of S-100 produce pathological structural changes within minutes, leading to apoptosis. The effect is preceded by large increases in intracellular calcium. The protein synthesis inhibitor cycloheximide blocks S-100 induced apoptosis. Treatment of rat astrocytes with S100beta produces activation of inducible nitric oxide synthase, which produces nitric oxide resulting in cell death (Hu et al., 1997). However, mice in which S100 is overexpressed show no behavioral abnormalities or pathological changes. Thus, it is still unclear whether the elevations in S-100 are a cause, an epiphenomenon, or part of a compensatory mechanism. Low concentrations of S-100 that are secreted into the extracellular medium act as growth factors or cytokines, inducing proliferation. These effects are mediated by surface receptor RAGE (receptor for advanced glycation endproducts). S-100 applied extracellularly was found to hyperpolarize the resting potential and inhibit spontaneous discharge activity by interacting with potassium channels.

4.2. Calcium-binding proteins in Alzheimer’s disease and apoptosis Calsenilin is another EF-hand containing protein which, although mostly neuronal, is also found in non-neuronal cells. Calsenilin interacts with the C-terminal region of presenilins 1 and 2 (Buxbaum et al., 1998; Leissring et al., 2000). In human neuroglioma (H4) cells transfected with calsenilin, calsenilin expression correlated with the appearance of a proteolytic fragment of presenilin 2 created by the apoptotic protease caspase (Choi et al., 2001). However, calsenilin is not required for the gamma-secretase activity of presenilin 2 (Esler et al., 2002). Calsenilin may also interact with the RyR, because calsenilin enhances apoptosis in the presence of thapsigargin, an inhibitor of Ca-ATP dependent calcium uptake in the ER, by promoting the release of calcium from intracellular stores (Lilliehook et al., 2002). Calsenilin also interacts with voltage-gated potassium channels (Morohashi et al., 2002) and thus shares functional similarity with calexcitin, a learning-related calciumbinding protein found in invertebrates (Nelson et al., 1996). In addition to producing neuronal excitation by its direct inhibitory effect on voltage-dependent potassium (iA) channels, calexcitin binds to and activates the RyR, producing rapid, transient increases in intracellular calcium levels that resemble oscillations (Nelson et al., 1999). Reduced levels of a calexcitin-like protein were also found in cultured cells from Alzheimer’s disease patients (Kim et al., 1995), illustrating one of many similarities that have been noted between pathways involved in learning and Alzheimer’s disease at the biochemical level (Alkon et al., 1998). Although most research on apoptosis induced by the beta-amyloid peptide, the central figure in the amyloidogenic hypothesis of Alzheimer’s disease, has concentrated on neurons, Alzheimer’s disease is also accompanied by changes in white matter, loss of astrocytes and oligodendrocytes and cerebral vasculopathy. Vascular cell degeneration in patients with Alzheimer’s disease complicated by cerebrovascular disease is associated with reduced levels of nitric oxide synthase 3 and increased levels of the apoptosis promoting protein p53. Vasculopathy is characterized by variable amyloid deposition and

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vascular smooth muscle cell apoptosis. Because apoptosis is accompanied by an increase in intracellular calcium, it is not surprising that calcium-buffering proteins such as calbindin-D28k and parvalbumin exert a significant protective effect (Wernyj et al., 1999; Tombal et al., 2002). Calcineurin, a calcium binding protein found in neurons, interacts with the apoptosis protection protein bcl-2. Similarly, neuronal apoptosis inhibitory protein binds to the calcium-binding protein hippocalcin (Lindholm et al., 2002). However, considering the central role played by calcium and calcium-activated proteases, such as calpains in apoptosis (Lu et al., 2002; Tombal et al., 2002), it is likely that any protein that interacts with calcium will either participate in apoptosis directly or exhibit a change in activity or binding behavior during apoptosis. Calcium and hydrogen peroxide levels are inextricably related in the cell. Apoptosis can also be induced by calcium or by oxidative stress induced by hydrogen peroxide or lipid peroxidation products such as 4-hydroxynonenal (Kalinich et al., 2000). Hypoxia and ischemia can trigger changes in calcium homeostasis, allowing increased calcium influx, in a process that requires hydrogen peroxide production by beta-amyloid (Green et al., 2002). In the presence of copper or zinc, beta-amyloid catalytically generates hydrogen peroxide (Opazo et al., 2002), which may mediate the toxicity of beta-amyloid. Hydrogen peroxide and superoxide can alter intracellular calcium levels by their effects on the RyR (Okabe et al., 2000). Presenilin also activates CICR, activating PKC and causing apoptosis (Chan et al., 2002). Inhibition of PKC activity can also induce apoptosis by upregulating H2O2 (Liou et al., 2000). Pharmacological activation of PKC promotes the release of the soluble fragment of APP by activating alpha-secretase, reducing levels of beta-amyloid by depriving beta-secretase of substrate (McLaughlin and Breen, 1999). Although low concentrations of H2O2 activate PKC and the RyR, higher levels are inhibitory due to oxidation of essential sulfhydryl residues.

5. Concluding remarks For a long time, glial cells were considered merely to provide structural and trophic support for neurons. However, this concept is now changing, since they have been shown to play a major role in the regulation of the extracellular pH (Deitmer, 1992—see also chapter by Bevensee and McAlear), Kþ levels (see chapter by Walz), CO2 metabolism (see chapter by Hertz, Peng et al.), redox equilibrium and neurotransmitter uptake (Landis, 1994—see also chapter by Schousboe and Waagepetersen). Ca2þ signalling seems to play a pivotal role in the control of all this functions, and recently one of the major Ca2þ regulatory mechanisms, the capacitative Ca2þ influx, has been implicated in glial cell function (Grimaldi et al., 1999, 2001). Moreover, glial cells have been shown to exhibit an oscillatory Ca2þ response and a propagated Ca2þ wave, which can be transmitted across the gap junction to nearby neurons (Scemes, 2000). Since it is apparent that glial Ca2þ signalling is an important pathway of glia –glia and neuron– glia cross-talk (see chapters by Shuai et al., and by Cornell-Bell et al.), its role during brain pathological conditions has been highlighted. It is well known that injury or brain pathology leads to a complex reaction from the astrocytes and microglial cells (Landis, 1994). For example, elevation of [Ca2þ]i in astrocytes induced by ischemia has been related to astrocyte activation and

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release of growth factors (Duffy and MacVicar, 1996). In addition, it has been suggested that glial Ca2þ waves may be related to certain complex pathological conditions such as migraine headaches (Martins-Ferreira et al., 2000).

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Decoding calcium wave signaling A. H. Cornell-Bell,* P. Jung and V. Trinkaus-Randall Anscans Ivoryton, CT, Ohio University Athens, O, Boston University School of Medicine Boston, MA p Correspondence address: E-mail: [email protected]

Contents 1. 2. 3. 4.

5. 6.

7.

8. 9.

Introduction Glutamate application induces a receptor-mediated response Mediators of intercellular astrocyte calcium wave propagation 3.1. Gap junction blockade can inhibit calcium wave propagation Extracellular ATP as mediators of calcium wave propagation 4.1. Evidence of ATP-mediated propagation 4.2. Purinergic receptors 4.3. ATP release from astrocytes Nitric oxide and PKG as mediators of calcium wave propagation Role of endoplasmic reticulum and mitochondria in Ca2þ signaling 6.1. Endoplasmic reticulum 6.2. Mitochondria Glutamate is involved in neuron-to-astrocyte signaling, but not in astrocytic propagation of calcium waves 7.1. Effects of transmitter glutamate on astrocytes 7.2. Glutamate release does not mediate interastrocytic calcium waves 7.3. Glutamate as the mediator of astrocyte-to-neuron signaling 7.4. Astrocytes can modulate neuronal responses and synaptic transmission Pathology and Ca2þ waves Concluding remarks

Intercellular calcium waves in astrocytes represent a phenomenon whereby a wave of increases in free cytosolic calcium concentration ([Ca2þ]i) spreads from an initially stimulated cell across an astrocytic syncytium. Originally it was believed that the mechanism of the spread was transport of the second messenger inositol trisphosphate (IP3) from cell to cell through connexin-mediated gap junctions, followed by an IP3mediated release of calcium from intracellular stores. Although such a mechanism might participate in some types of calcium waves, calcium wave propagation is not dependent Advances in Molecular and Cell Biology, Vol. 31, pages 661–687 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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upon this mechanism. Rather it depends in most cases upon an autocatalytic release of Ca2þ driven by release of ATP (which is partly Ca2þ-dependent and partly Ca2þ independent, but may be hemiconnexin-dependent) and subsequent autocrine/paracrine stimulation of purinergic P2 receptors, linked to release of Ca2þ from intracellular stores. In addition, NO-mediated signaling mechanisms are involved, but only in some types of calcium waves, especially those triggered by mechanical stimulation. Neuronal activity can induce astrocytic calcium waves by stimulation of metabotropic glutamate receptors on astrocytes. In turn, glutamate release from astrocytes sustaining a calcium wave is capable of triggering neuronal activity.

1. Introduction Three key factors merged to trigger the discovery of calcium waves in the astrocyte syncytium. The first was finding that astrocytes possessed a full array of neurotransmitter receptors, which operated on a millisecond time frame similar to that expected by neurons (Bowman and Kimelberg, 1984; Sontheimer et al., 1988; Backus et al., 1989; Usowicz et al., 1989; reviewed by Cornell-Bell and Finkbeiner, 1991). The activation of these receptors resulted in a Ca2þ influx into astrocytes or intracellular calcium release with subsequent mobilization (Enkvist et al., 1989; Glaum et al., 1990; Inagaki et al., 1991). The second contribution was the many technological advances made in time-lapse video microscopy (Allen et al., 1981; Inoue, 1981, 1986), particularly involving the newly marketed confocal scanning laser microscope, which allowed longer recording periods free of phototoxicity (Brakenhoff et al., 1985; Amos et al., 1987; Inoue, 1995; Pawley, 1995). Lastly, most critical was the development of Ca2þ sensitive fluorescent probes that were ion-specific, which allowed precise quantification of changes in intracellular Ca2þ (Grynkiewicz et al., 1985; Tsien, 1988; Lipscombe et al., 1988; Kao et al., 1989; Harootunian et al., 1991). Early imaging studies of calcium activity in astrocytes identified intracellular oscillations and intercellular Ca2þ waves as key elements in a complicated nonneuronal signaling system (Berridge and Gallione, 1988; Cornell-Bell et al., 1990; Jensen and Chiu, 1990; Charles et al., 1991; Teichberg, 1991). Compelling time-lapse sequences of astrocyte cultures responding to neurotransmitters stimulated speculation about an astrocytic long-range signaling system, which was postulated to be frequency encoded in the form of oscillations. Intracellular Ca2þ responses were thought to possibly synchronize neighboring astrocytes via a unified, long-lasting elevation of free cytosolic Ca2þ concentration ([Ca2þ]i) that occurred during propagation of the long-distance waves over a region of cells. Oscillating cells were seen to dampen as the wave arrived, but then oscillations re-started following the passage of the wave. Initial experiments that defined the Ca2þ wave system recognized that either neurotransmitter receptor activation or mechanical stimulation of a single cell activated at least two separate Ca2þ elevation mechanisms (Cornell-Bell et al., 1990; Charles et al., 1991; Finkbeiner, 1993; Kim et al., 1994). These two distinct mechanisms were (i) Ca2þ entry from outside the cell, which was characteristic for

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kainate-induced Ca2þ waves (Kim et al., 1994); and (ii) IP3-mediated release of Ca2þ from intracellular stores initiated by a mechanical stimulus or metabotropic glutamate receptor stimulation (Charles et al., 1991; Cornell-Bell and Finkbeiner, 1991; McCarthy and Salm, 1991; Venance et al., 1997). During the past decade, a large body of exciting work has unraveled second messenger systems involved in Ca2þ signaling processes. An understanding is emerging of the mechanism for entry of Ca2þ in response to glutamate and ATP, and aspects of what the signaling process actually may encode have emerged. Several laboratories have also investigated what happens when astrocytes are players in a pathological condition such as epilepsy. Different cell types have different signal initiating molecules, and different tissues have different requirements and mechanisms for eliciting Ca2þ waves. In this review we will touch on aspects of each of these topics during the process of dissecting what is currently known about the manner(s) in which glial cells and the neurons they face encode and respond to Ca2þ signals, which regulate responses in the nervous system.

2. Glutamate application induces a receptor-mediated response Astrocytes expressing specific receptor systems linked to calcium regulation remained stable in vitro for several weeks even in the absence of neurons (McCarthy and Salm, 1991). One such receptor is glutamate. Selective agonists pharmacologically defined the glutamate subtypes on glial cells as kainate and quisqualate-preferring receptors, but in general not NMDA preferring receptors (Sontheimer et al., 1988; Usowicz et al., 1989; Backus et al., 1989; Cornell-Bell et al., 1990; Ahmed et al., 1990; Jensen and Chiu, 1990; Glaum et al., 1990; Cornell-Bell and Finkbeiner, 1991). Astrocytes produced Ca2þ spikes at glutamate concentrations of 100 nM or higher, with 50% of the cells in a culture responding at 300 nM (Cornell-Bell et al., 1990). Glutamate concentrations in the superfusate influenced whether oscillations occurred, the frequency of oscillations and whether both intracellular and intercellular waves were observed (Cornell-Bell et al., 1990). The average oscillation period decreased with increasing agonist concentrations. Coupling between oscillations in neighboring cells was not demonstrated, and oscillations within individual astrocytes showed either constant or monotonically decreasing frequency (Cornell-Bell and Finkbeiner, 1991; Finkbeiner, 1993—see also chapter by Shuai et al.). In addition, the pattern of intracellular Ca2þ signaling was dependent upon the concentration of extracellular glutamate. For example, at concentrations of glutamate less than 1 mM small regions of the astrocyte cytoplasm flickered asynchronously, with propagation of the intracellular Ca2þ wave occurring in contained areas of the cytoplasm. If the extracellular glutamate concentrations were increased to between 1 and 10 mM, propagation of Ca2þ in waves was more common, and these waves traveled throughout the cell, often propagating into neighboring cells (Cornell-Bell et al., 1990). Analysis of single astrocytes showed that in more than 80% of the cells studied a gradient of intracellular calcium existed, and the locus from which the wave originated corresponded to the region of highest cytoplasmic Ca2þ concentration (Yagodin et al., 1994). The locus of origin and the regions of calcium spikes with the highest amplitude remained invariant during the propagation of successive intracellular calcium waves

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(Yagodin et al., 1994). Calcium waves were shown to be initiated at discrete regions of the cells where the calcium concentrations were highest, and they were propagated in a saltatory manner through the cytoplasm to other loci, where the rising calcium from the approaching wave front provoked a large Ca2þ increase. Models of intracellular wave propagation indicated that the propagation was dependent upon a Ca2þ-sensitive autocatalytic step (Berridge, 1993; Meyer and Stryer, 1991). Glutamate was shown to stimulate the release of Ca2þ from intracellular stores in astrocytes (Ahmed et al., 1990). One possible mechanism was that an increase in intracellular Ca2þ causes an increase in phospholipase C with resulting increase in IP3 and Ca2þ (Monaghan et al., 1989; Lechleiter et al., 1991; Pearce et al., 1986). Another idea suggested that the calcium ion itself activates further release of calcium from stores by a calcium-induced calcium release (Backx et al., 1989; Charles et al., 1993—see also chapter by Scapagnini et al.). 3. Mediators of intercellular astrocyte calcium wave propagation 3.1. Gap junction blockade can inhibit calcium wave propagation 3.1.1. Gap junction-mediated propagation Early applications of calcium imaging of cultured astrocytes revealed a striking new phenomenon: a cytosolic Ca2þ signal spreading from cell to cell and called a calcium wave (Cornell-Bell et al., 1990; Charles et al., 1991; Smith, 1992). Gap junction channels were identified as an important mediator of intercellular calcium waves (Bennet et al., 1991), since they provide a direct route between cells. Gap junctions are also permeable to calcium and IP3 (Saez et al., 1983). Uncoupling gap junctions resulted in inhibition of the intercellular calcium wave (Anders, 1988; Dermietzel et al., 1991; Finkbeiner, 1992; Enkvist and McCarthy, 1992; Christ et al., 1992; Venance et al., 1995). When octanol and halothane were used to uncouple gap junctions in hippocampal astrocytes the incidence of Ca2þ waves propagating from cell to cell dropped from 50% occurrence to 0 and 5%, respectively (Finkbeiner, 1992). In contrast, the uncoupling did not dramatically affect the pattern or duration of intracellular calcium oscillations or intracellular waves (Finkbeiner, 1992). Normally, C6 glioma cells show no coupling to their neighbors. When C6 glioma cells were transfected with the gap junction protein, connexin43, calcium waves passed from one cell to the next, suggesting that the gap junction was a crucial functional component of the propagating calcium wave (Charles et al., 1992). Despite this concrete evidence of the involvement of gap junctions in wave regulation there were calcium waves propagating in a normal fashion in connexin43 (Cx43) knock-out (KO) mice (Scemes et al., 1998, 2000). Comparison of the strength of coupling between pairs of wildtype (WT) and Cx43 KO spinal cord astrocytes indicated that two-thirds of total coupling is attributable to channels formed by Cx43, with other connexins contributing the remaining one-third of junctional conductance (Scemes et al., 2000—see also chapter by Scemes and Spray). The brains of connexin43 null [Cx43 (2 /2 )] animals were shown to be macroscopically normal and to display patterns of cortical lamination remarkably similar to WT siblings (Dermietzel et al., 2000). Additionally the presence of Cx40 and Cx45 in brains and astrocytes cultured from both Cx43 (2 /2 ) mice and WT littermates was confirmed. Cx30 mRNA was detected in long term (2 weeks) but not in fresh cultures of

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astrocytes. There is also the possibility that Cx26 may be present. These studies reveal that astrocyte gap junctions may be formed of multiple connexins (Dermietzel et al., 2000) providing the exciting possibility that specific roles for each of these proteins may exist in the regulation of calcium waves. It has recently been demonstrated in C6 glioma cells transfected with Cx32 or Cx43 that AMP and ADP and, especially, ATP pass much better through channels formed by Cx43, whereas the opposite applies to adenosine (Goldberg et al., 2000). 3.1.2. Modulation of gap junction-mediated propagation Functional studies in brain slices and cultures have implicated neurotransmitters, cytokines, growth factors and other bioactive compounds in control of gap junctional coupling (Giaume and McCarthy, 1996; Spray et al., 1999). It has recently been demonstrated in various models of astrocyte and astrocyte/neuron cultures that gap junctional communication and expression of Cx43 in astrocytes can be up-regulated, and that the presence of neurons in the cultures enhances gap junction-mediated communication, Cx43 expression and the extent of calcium waves in astrocytes (Rouach et al., 2000). Interestingly, it was demonstrated in these experiments that plating of microglial cells on the astrocyte layer led to a decrease in gap junctional permeability and connexin43 expression, suggesting that several types of cell –cell interactions need to be considered in local control of gap junctions and their contribution to the propagating Ca2þ wave. Prolonged treatments (24 – 72 h) of striatal co-cultures of neurons and astrocytes with tetrodotoxin, picrotoxin, bicuculline and CNQX, an inhibitor of quisqualatepreferring glutamate receptors (Honore and Drejer, 1988; Yamada and Huzel, 1989), resulted in a significant reduction in gap junctional coupling without affecting the expression of connexins as determined by Western blot analysis (Rouach et al., 2000). It thus appears that neuron-induced up-regulation of astrocyte gap junctional coupling is dependent upon synaptic activity in striatal neurons, and it is also related to density and age of cultures (Rouach et al., 2000). Inflammatory cytokines, including IL-1 b and TNF-a, have been shown to downregulate gap junctional connectivity (John et al., 1999; Brosnan et al., 2001). Gap junctions form a molecular link for coordinated long-distance signaling via calcium waves, but there is also an important ATP-mediated extracellular pathway that can be influenced by cytokines (John et al., 1999), as will be described below. Communication in the astrocyte syncytium is sustained by a finely tuned interaction between gap junction-dependent and gap junction-independent mechanisms. When gap junctional coupling was reduced as in Cx43 knock out mice, an increase in the extracellular paracrine/autocrine communication dominated (John et al., 1999; Scemes et al., 2000; John et al., 2000). This interplay between the gap junctions and paracrine/autocrine signaling provides a high degree of plasticity for intercellular communication between astrocytes (Brosnan et al., 2001). The integral membrane components of gap junctions appear to be linked into a macromolecular complex that has been called the Nexus (Spray et al., 1999). Nexus components may vary such that the binding affinities within a Nexus containing an individual connexin may be altered by micro-environmental factors such as cytosolic pH, phosphorylation and changes in binding with other components. For the Cx43 Nexus, binding sites include src homology

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(SH), PSD and zonula occludens (PDZ) binding domains, and the Cx43 molecule. Interactions of Cx43 with proteins containing PDZ, SH2 and SH3 domains is hypothesized to serve as the scaffold on which to assemble components of the intercellular signaling pathway into the multiprotein Nexus complex, which couples their activity to downstream signaling molecules (Brosnan et al., 2001). In the case of Cx43, a proline-rich region of the carboxyl terminus comprising amino acids 273 –285 may bind to the SH3 domain, and a phosphorylated tyrosine at position 265 has been shown to interact with the SH2/SH3 domain proteins v-src and c-src (Swenson et al., 1990). Phosphorylation of Cx43 by v- and c-src is involved in decreased gap junctional conductance (Filson et al., 1990; Brosnan et al., 2001). Studies, such as these, place the Nexus into the framework of second messenger signaling cascades augmenting the importance of these proteins from a conduit between cells to an active regulatory mechanism. 4. Extracellular ATP as mediators of calcium wave propagation 4.1. Evidence of ATP-mediated propagation Ca2þ waves in astrocytes can proceed by an extracellular pathway, as evidenced by the continued propagation of waves between astrocytes in culture even when cells are not in direct contact (Hassinger et al., 1996; Guthrie et al., 1999). Calcium waves cross cell-free gaps, and they are affected by the direction and strength of a perfusion bath (Hassinger et al., 1996). The most compelling evidence for a released extracellular factor is that subsequent calcium waves are produced if naı¨ve cells are exposed to the medium collected from around cells that previously supported a propagating wave (Guthrie et al., 1999; Klepeis et al., 2001). Also, if gap junctional coupling is inhibited, propagation of Ca2þ waves is in several situations unaffected (Naus et al., 1997; Guan et al., 1997; John et al., 1999), at least partly dependent upon the concentration of the inhibitor (Cotrina et al., 1998a,b). Thus, when astrocytes were uncoupled from Muller cells, using octanol, the propagation of Ca2þ waves were maintained, highlighting the importance of the extracellular messenger in wave propagation between these two cell types (Zahs and Newman, 1997). ATP is emerging as the extracellular signaling element that drives astrocytic Ca2þ waves. This applies not only to conditions when the calcium wave crosses an extracellular gap, but also to wave propagation between adjoining cells. Imaging techniques have detected ATP in the saline incubation medium surrounding astrocytes participating in a calcium wave (Guthrie et al., 1999; Stout et al., 2002; Coco et al., 2003). Using a combination of chemiluminescence and Fluo3AM calcium imaging at millisecond temporal resolution it was possible to show that extracellular ATP mediated intercellular calcium wave propagation (Wang et al., 2000). 4.2. Purinergic receptors ATP acts on P2 receptors, whereas P1 receptors are adenosine receptors (see chapter by Hansson and Ro¨nnba¨ck). Multiple subtypes of P2-purinergic receptors exist, as was first proposed by Burnstock (Burnstock and Kennedy, 1985; reviewed by Burnstock, 1997).

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P2Y receptors function as G-protein coupled Ca2þ-mobilizing ATP receptors, operating via stimulation of phospholipase C and formation of inositol trisphosphate (IP3) and diacylglycerol (DAG), the former of which causes release of Ca2þ from intracellular stores on the endoplasmic reticulum (see chapter by Scapagnini et al.); P2X-type receptors act as ligand-gated ion channels; and P2-Z receptors are associated with ATP-induced pore formation (Dubyak and el-Moatassim, 1993). Astrocytes express two subtypes of P2Y receptors, the P2Y1 and the P2Y2 receptor (Zhou and Kimelberg, 2001). It is in support of the concept that ATP is the extracellular messenger for calcium wave propagation that P2 receptor antagonists inhibit Ca2þ wave propagation (Guan et al., 1997; Cotrina et al., 1998a,b; Guthrie et al., 1999; Fam et al., 2000). Also, suramin and PPADS, P2 receptor antagonists both inhibited Ca2þ wave propagation into Mu¨ller cells and astrocytes (Newman, 2001; Zanotti and Charles, 1997; Guan et al., 1997). In addition, wave propagation is reduced if ATP hydrolysis is enhanced with apyrase in cortical or retinal preparations (Guthrie et al., 1999; Cotrina et al., 1998a,b, 2000; Newman, 2001), but not in striatal preparations (Venance et al., 1997). Application of ATP initiated astrocyte calcium waves in cultures from suprachiasmatic nucleus cultures (van den Pol et al., 1992) and retinal cells (Newman and Zahs, 1997). Stimulation of the P2Y receptors on astrocytes was also shown by Kastritsis et al. (1992) to result in inositol phosphate formation and calcium mobilization. Using 45Ca in the presence or absence of LaCl3, an inhibitor of Ca2þ channels, Neary et al. (1998) showed, however, that ATP also stimulates the uptake of extracellular Ca2þ into cultured astrocytes. ATP-stimulated Ca2þ entry might be secondary to stimulation of a P2X receptor, as is the case in microglia (Verderio and Matteoli, 2001) and subsequent opening of voltage-sensitive Ca2þ channels and/or non-specific cation channels (Sun et al., 1999). It is in agreement with this suggestion that an ATP-induced increase in glial [Ca2þ]i in the acutely isolated rat optic nerve can be evoked not only by a P2Y-selective agonist but also by a P2X-selective agonist (James and Butt, 2001). Calcium waves in other cell types are also propagated using ATP as the extracellular messenger (Osipchuk and Cahalan, 1992; Dubyak and el-Moatassim, 1993; Schlosser et al., 1996; Jorgensen et al., 1997; Frame and deFeijter, 1997; Schneider et al., 1994; Klepeis et al., 2001). On the other hand, activation of P2Y receptors on astrocytes have additional effects besides the ability to elicit calcium waves. Thus, P2Y receptors are coupled to the MAP kinases ERK by pathways which are distinct from that leading to an increase in [Ca2þ]i (Neary et al., 1999), a finding which explains how some astrocytes when exposed to ATP respond with increased intracellular Ca2þ and others respond trophically.

4.3. ATP release from astrocytes As mentioned above, ATP is released from astrocytes during propagation of calcium waves (Guthrie et al., 1999; Wang et al., 2000; Stout et al., 2002; Coco et al., 2003). The ability of astrocytes to both release ATP and respond to ATP suggests that ATP may act as an autocrine or paracrine messenger between these glial cells (Quieroz et al., 1999). Two potential pathways exist for exit of ATP from the cell: (i) passage through anion

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channels formed by connexin hemichannels (Cotrina et al., 1998a,b; Stout et al., 2002); and (ii) Ca2þ-dependent and quantal release of vesicularly stored ATP (Coco et al., 2003). If ATP release from astrocytes was a regenerative process, such that the stimulated cell as well as all subsequent cells rapidly released high concentrations of ATP an infinitely propagating wave would result (Guthrie et al., 1999). This is not the case, since propagating calcium waves only travel through approximately 20 cell diameters. Since not all astrocytes respond to ATP with the same dose dependence (Guthrie et al., 1999) or signaling pathways (Neary et al., 1999), and perhaps not all astrocytes release ATP, calcium waves are likely to be limited by extent (Guthrie et al., 1999). Although the study by Wang et al. (2000) showed that extracellular ATP mediated intercellular calcium wave propagation, the release and propagation of ATP was not calcium dependent. Use of the phospholipase C-inhibitor U-73122 blocked the ATP-stimulated Ca2þ wave, while BAPTA and thapsigargin (inhibitors of intracellular Ca2þ release—see chapter by Scapagnini et al.) did not, which implies that products of phospholipase C activity, IP3 or DAG, are directly involved with ATP wave regulation (Wang et al., 2000). This suggestion is supported by observations that the use of caged-IP3 followed by flash photolysis produces an ATP-mediated Ca2þ wave, whereas flash release of caged Ca2þ did not (Leybaert et al., 1998). 4.3.1. Release of cytosolic ATP Ectopic expression of connexins was shown to cause an increase in ATP release with an increase in the radius of the propagating calcium wave (Cotrina et al., 2000). An intact cytoskeleton was needed for Ca2þ wave generation, since cytochalasin D reduced calcium signaling significantly (Cotrina et al., 1998b, 2000). Transjunctional currents between C6 and Cx43 cells were not altered by the inhibitor, confirming that gap junctions remained open (Cotrina et al., 1998a,b). The reason for the dependence on the cytoskeleton is unknown but an intact actin cytoskeleton is also necessary for calcium-dependent secretion in neurons and secretory cells (for review, see Trifaro and Vitale, 1993) and evidence is found that vesicular secretion of ATP contributes to ATP release (see below). Moreover, disruption of the actin cytoskeleton in cultured rat astrocytes suppresses ATPinduced calcium oscillations by reducing capacitative Ca2þ re-entry and store refilling (Sergeeva et al., 2000; see also chapter by Scapagnini et al.). Inhibition of the myosin light chain kinase also resulted in a suppressed Ca2þ signaling without effecting coupling or calcium mobilization (Cotrina et al., 1998b). It is likely that connexin proteins supported purinergic-mediated Ca2þ signaling, not as a substrate for gap junctions, but rather as a facilitator of ATP release. This release may occur through ATP anion flow through connexin hemichannels, and it is enhanced by the removal of extracellular Ca2þ (Cotrina et al., 1998a). Cx-deficient cells can receive but not propagate Ca2þ signals (Cotrina et al., 2000). Recent experiments using real-time bioluminescence imaging of C6 glioma cells have demonstrated that the release of ATP is not uniform across a field, but is restricted to short bursts (Arcuino et al., 2002). The ATP bursts emanate from single cells with transient openings of non-selective channels resulting in a change in membrane permeability (Arcuino et al., 2002). Earlier studies of cystic-fibrosis airway epithelial cells (CFTR), using an atomic force microscope, showed that each affected cell studied contained distinct point sources that were dispersed across

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the epithelium of the cell surface in a random pattern (Schneider et al., 1994). The ATP that is released by the cell from a point source diffuses into the adjacent extracellular medium, or it is hydrolyzed by ecto-ATPases. More ATP molecules are in close proximity to the point source than at a distant location, with a concentration gradient developing as a function of the distance from the point source (Schneider et al., 1994). Increases in intracellular Ca2þ alone were not sufficient to initiate the changes in ATP membrane permeability (Arcuino et al., 2002). C6 –Cx43 cells displayed repeated bursts of ATP release equivalent to the bursts seen for astrocytes. Mock transfected cells with null Cx showed no ATP bursts, further supporting that Cx expression mediates the release of ATP (Arcuino et al., 2002). It is tempting to conclude that ATP is released through functional hemichannels from the cell interior to its outside, secreting cells do express connexins, and in nearly all secretory epithelia intracellular, Ca2þ-activated ion conductances are stimulated by luminal ATP (and other nucleotides) to induce secretion of Cl – , Kþ or HCO2 3 (reviewed by Leipziger, 2003). ATP is highly concentrated in cytoplasm (approximately 2 mM) and in cultured astrocytes (Matz and Hertz, 1989), and efflux of ATP from a single cell potentially can activate several hundred neighboring cells (Arcuino et al., 2002). However, over-expression of Cx43 in C6 – Cx43 cells is also associated with altered expression of other genes and with changes in volume regulation (Naus et al., 2000; Quist et al., 2000). 4.3.2. Release of vesicular ATP Proteins generally involved in regulated exocytosis are expressed in cultured astrocytes (Madison et al., 1996), and they may provide a link between an extracellular component of calcium wave activity and the cytoskeleton (Cotrina et al., 1998b). It is in support of this concept that a recent study by Coco et al. (2003) has convincingly demonstrated vesicular release of ATP from primary cultures of hippocampal rat astrocytes. They reported that besides the described release of cytosolic calcium, which is enhanced during Ca2þ depletion, there is a gap junction-independent, Ca2þ-dependent release of ATP. This release is inhibited by bafilomycin, an inhibitor of ATP uptake into secretory granules, a process requiring an electrochemical proton gradient, maintained by a v-ATPase (for discussion of the v-ATPase, see chapter by Bevensee and McAlear). The vesicles containing ATP were functionally different from those releasing glutamate, as indicated by different inhibitor sensitivity, and the observation that activation of metabotropic glutamate receptors, which strongly evokes glutamate release, had only little effect on release of ATP. 5. Nitric oxide and PKG as mediators of calcium wave propagation Additional signaling pathways mediate intercellular Ca2þ waves as indicated by the recent discovery that the nitric oxide/protein kinase G (PKG) pathway (see chapter by Garcia and Baltrons) has a fundamental role in regulating the initiation and propagation of Ca2þ waves following mechanical stimulation (Willmott et al., 2000). Molsidomine, a nitric oxide (NO) donor as well as an aqueous puff of NO itself produced an increase of [Ca2þ]i and propagating intercellular Ca2þ waves in primary mixed forebrain cultures.

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Entry of extracellular Ca2þ was indicated by the observation that incubation in Ca2þ-free medium or application of an inhibitor of voltage-dependent Ca2þ channels reduced the Ca2þ response. NO is also an inducer of Ca2þ mobilization in several other cell types (Willmott et al., 2000), where Ca2þ was mobilized either through cGMP-dependent protein kinase-coupled activation of ADP-ribosyl cyclase, which produced ADP-ribose (Wilmott et al., 1996; Clementi et al., 1996), or via a direct nitrosylation of regulatory thiol groups of the ryanodine receptors (Stoyanovsky et al., 1997). Synthesis of NO by constitutive nitric oxide synthase is Ca2þ-dependent. It was hypothesized that a rise in intracellular Ca2þ would serve to amplify NO production, thus enhancing NO diffusion, which could contribute to propagation of intracellular and intercellular Ca2þ waves (Willmott et al., 2000). A small response was seen in Ca2þ free saline and no response was seen when just saline was puffed onto cells. Pretreating cells with an NO scavenger (PTIO) arrested the increase in Ca2þ, showing there was a direct relationship between the increase in Ca2þ and in NO. When NO was puffed onto cells treated with ryanodine no resulting [Ca2þ]i response was seen, indicating that Ca2þ is mobilized from intracellular stores (see chapter by Scapalgini et al.). Pretreating cells with a guanylate cyclase inhibitor abolished the NOinduced Ca2þ rise, whereas the PKG inhibitor Rp-8-pCPT-cGMP reduced the NO effect. Thus NO mobilizes Ca2þ from a ryanodine receptor-linked store through the cGMP-PKG signaling pathway in mechanical waves (Willmott et al., 2000). Studies of mechanically induced calcium waves indicated that they shared most essential features with those induced by NO, and an increase in intracellular NO could, indeed, be demonstrated in the calcium waves elicited by mechanical stimulation (Willmott et al., 2000). In contrast, the increase in [Ca2þ]i induced by a bolus of ATP is not mediated via an increased production of NO in glial cells, and it is not dependent upon ryanodine receptorlinked Ca2þ release (Willmott et al., 2000). This is in direct contrast to mechanical wave studies (Simard et al., 1999; Guthrie et al., 1999). There was a significant inhibition, but no elimination, of the ATP-induced Ca2þ response after treatment with the phospholipase C inhibitor U 73122, suggesting that this response is dependent upon intracellular IP3 generation and is independent of NO or ryanodine receptor-linked Ca2þ release (Centemeri et al., 1997; Willmott et al., 2000). It had previously been shown (Venance et al., 1997; Charles et al., 1993) that U 73122 alone was sufficient to completely eliminate the mechanically induced Ca2þ wave. These studies suggest there may be yet again another level of control of the glial Ca2þ signaling system, since the response to ATP presentation by direct application is driven by an IP3-mediated receptor instead of the ryanodine receptor-linked signaling seen in mechanical waves. Evidence using the NOspecific fluoroprobe DAF-2 further confirmed that an ATP-induced Ca2þ rise was not dependent on an NO increase (Willmott et al., 2000). 6. Role of endoplasmic reticulum and mitochondria in Ca21 signaling 6.1. Endoplasmic reticulum Initial studies showed that norepinephrine-induced Ca2þ waves in cortical astrocyte cultures began at discrete initiation loci and from there propagated throughout the cytoplasm in a regenerative manner involving Ca2þ release sites (Sheppard et al., 1997).

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A specific antibody to the type-2 IP3 receptor subtype and specific staining of the endoplasmic reticulum showed domains of elevated Ca2þ response with kinetics defined as high amplitude and rapid rise rate, significantly correlated with high local intense staining of the IP3 receptors (Holtzclaw et al., 2002). Further characterization of the sarcoplasmic – endoplasmic reticulum Ca2þ ATPase (SERCA) of cortical astrocytes and oligodendrocytes showed a slow onset Ca2þ response when SERCA was inhibited (Simpson and Russell, 1997). IP3 receptors and SERCAs sensitive to thapsigargin and CPA (cyclopiazonic acid) are associated with a single Ca2þ store (see chapter by Scapagnini et al.). Inhibition of SERCA activates both Ca2þ release as a wave front and Ca2þ entry via store-operated channels (Simpson and Russell, 1997).

6.2. Mitochondria Mitochondrial buffering of cytoplasm regulates the spread of astrocytic Ca2þ waves. Mitochondrial control of calcium uptake and release from cytoplasm has direct consequences on neuronal and glial Ca2þ responses (Simpson and Russell, 1996, 1998b). In oligodendrocytes, IP3-dependent release of Ca2þ resulted in an elevated Ca2þ signal in the mitochondria, which modified cytosolic Ca2þ wave propagation (Simpson et al., 1997; Simpson and Russell, 1998a,b). Ca2þ release from stores regularly alternated with sites of removal from the cytosol (Laskey et al., 1998). Multiple mechanisms of Ca2þ removal from the cytosol contribute to the negative flux sites. Type-2 IP3 receptors and calreticulin (a calcium binding protein) are expressed with high intensity at Ca2þ wave amplification sites along oligodendrocyte processes, when compared to other cell regions (Simpson et al., 1997). Stationary mitochondria were found at these specialized release sites in close association with high density ER proteins. These findings imply that the propagating Ca2þ wave can be modulated by special Ca2þ-release domains, involving both ER proteins and mitochondria (Simpson et al., 1997). The permeability transition pore forms the major Ca2þ efflux pathway from the mitochondria (Smaili et al., 2001). Ca2þ efflux from the mitochondrial matrix involves reversal of the uniporter and the inner membrane Naþ/Ca2þ exchanger. When mitochondrial function was blocked, there was a measurable decrease in wave speed and puff probability (Haak et al., 2000). An inhibitor of the electron transport chain (antimycin A) increased cytosolic Ca2þ, reduced agonistevoked IP3 production and enhanced PIP2 binding to the Ca2þ-dependent protein, gelsolin (Haak et al., 2000). ATP application to cortical astrocytes mobilized intracellular Ca2þ stores with an increase in Ca2þ over the nucleus as well as cytosol, followed by a delayed increase in mitochondrial Ca2þ, that remained elevated for long periods after nuclear Ca2þ decrease (Boitier et al., 1999). The rise in the mitochondria appeared first at one end of the cell and then progressed in a wave-like pattern to the other end (Boitier et al., 1999). Using a mitochondrial uncoupling agent, FCCP, in conjunction with oligomycin, it was possible to dissipate the proton gradient across the inner mitochondrial membrane, while inhibiting mitochondrial consumption of ATP (Boitier et al., 1999). Collapse of the gradient by FCCP did not alter the peak amplitude of the ATP-induced Ca2þ transient over the nucleus, or the time required to

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reach half of the peak amplitude. However the decay phase was longer, suggesting that mitochondrial buffering plays a role in restoration of the nuclear and cytoplasmic concentrations to basal levels. The Ca2þ wave spreading across the cytoplasm traveled faster, when the mitochondrial gradient was inhibited, implying that mitochondrial uptake exerts a negative feedback action on the propagation rate of the Ca2þ wave (Boitier et al., 1999). These studies indicate that mitochondria could provide a significant regulatory function, modulating propagating intracellular and intercellular Ca2þ waves within the astrocyte syncytium. 7. Glutamate is involved in neuron-to-astrocyte signaling, but not in astrocytic propagation of calcium waves 7.1. Effects of transmitter glutamate on astrocytes 7.1.1. Ca2þ Synaptically released neurotransmitters regulate astrocytic calcium levels, thus making astrocytes sensitive to neuronal signaling (Smith, 1992; Smith, 1994). In further studies, glial cells along axons in the optic nerve were shown to generate Ca2þ signals in response to applications of ATP or glutamate as well as to electrical stimulation of axons (Kriegler and Chiu, 1993). This study of white matter and an earlier study on gray matter (Dani et al., 1992) suggest that both synaptic and non-synaptic regions of a neuron can trigger dynamic glial calcium signaling. Glial Ca2þ spikes were induced by axonal activity in the absence of extracellular Ca2þ (Kriegler and Chiu, 1993). As a model, the release of glutamate from a synapse is vesicular and Ca2þ dependent, whereas release from axons occurs through reversal of a glutamate transporter, following activity-dependent alterations in Naþ and Kþ gradients across the axon membrane. Released glutamate activates metabotropic receptors on glia leading to Ca2þ waves and spiking (Dani et al., 1992). The glial calcium signal may drive metabolic responses (see chapter by Hertz, Peng et al.), and ultimately glutamate receptor activation in astrocytes can induce release of other neurotransmitters (Gallo et al., 1991). 7.1.2. ATP Activation of glutamate receptors on rat cortical astrocytes has also been shown to induce the release of ATP (Quieroz et al., 1999). The release is brought about by activation of any of the three ionotrophic glutamate receptor types, N-methyl-D -aspartate (NMDA), AMPA and kainate receptors (Queiroz et al., 1997). AMPA receptors seem to mediate at least a part of the effect of glutamate, but the additional involvement of the other subtype glutamate receptors cannot be ruled out. Two different mechanisms seem to be involved. The NMDA- and kainate-induced release of ATP requires an influx of calcium, it is not due to neuron-like exocytosis, and is reduced (by an unknown mechanism), but not abolished, by lithium. The AMPA-induced release does not require extracellular calcium, may be mediated by a transmembrane conductance regulator or a mechanism regulated by a transmembrane conductance regulator, and is abolished (by an unknown mechanism) by acute exposure to 1 mM lithium. Glia cells have also been shown to respond

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electrophysiologically to action potential generation in the giant axon by mechanisms involving amino acid neurotransmitter receptors (Lieberman and Hassan, 1988; Lieberman et al., 1989). 7.2. Glutamate release does not mediate interastrocytic calcium waves Both neurons and glia had been shown to stimulate increased Ca2þ in the opposite cell type by release of glutamate (Hassinger et al., 1995; Nedergaard, 1994; Parpura et al., 1994—see also above). This made glutamate a strong candidate for the component likely to be released from astrocytes during the propagation of calcium waves. Historically, there was supporting evidence that astrocytes released glutamate and other neuroactive substances (Martin, 1992). Astrocytes synthesize, metabolize and release neurotransmitters in response to stimuli (Patel and Hunt, 1989; Nicholls and Atwell, 1990; Shain et al., 1986, 1989; Philbert et al., 1988). Pure astrocyte cultures stimulated with 55 mM KCl exhibit a calcium-independent release of D -aspartate, a non-metabolizable glutamate analog, after loading with labeled D -aspartate (Westergaard et al., 1991). The calcium-independence of this release suggests a non-vesicular release that may be due to the reversal of glutamate uptake systems (Szatkowski et al., 1990; Nicholls et al., 1987; Sanchez-Prieto et al., 1996). However, evidence continued to mount that astrocytes also released glutamate in a Ca2þdependent manner (Parpura et al., 1994; Nicholls, 1998). It was reasonable to assume that this released glutamate would play a role as the diffusible element along the path of the calcium wave, providing the regenerative element needed for propagation. Evidence for glutamate providing the regenerative signal for astrocyte intercellular calcium waves, however, is lacking. As early as 1991, Andrew Charles made the critical observation, that the pattern of a mechanical wave initiated repetitively was not affected by the application of glutamate. The path the wave traveled was similar in the presence or absence of glutamate, and it involved a similar percentage of cells suggesting a minimal role for glutamate in the generation of the astrocyte wave process (Charles et al., 1991). Cortical astrocytes in the presence of glutamate receptor antagonists continued to propagate calcium waves between astrocytes, minimizing the role of glutamate might have as the astrocytic stimulus for wave propagation (Hassinger et al., 1995). Glutamate antagonists did however block the propagation of Ca2þ waves from astrocytes to neurons. Taken together, these results indicate that glutamate release was not necessary for the propagation of intercellular astrocyte waves, but had dramatic regulatory effects on neuronal activity. 7.3. Glutamate as the mediator of astrocyte-to-neuron signaling Glutamate is not only released from neurons and stimulating glutamate receptors on astrocytes, but it is also released from astrocytes activating neuronal receptors, i.e., representing a bi-directional communication system between the two classes of cells (Dani et al., 1992; Porter and McCarthy, 1996; Harris-White et al., 1998; Parpura et al., 1994; Charles, 1994; Nedergaard, 1994; Hassinger et al., 1995; Pasti et al., 1997; Newman and Zahs, 1998). Using a NADH-based fluorescence system it was clearly demonstrated that

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glutamate is released in a regenerative manner from astrocytes upon arrival of the calcium wave, with subsequent cells that are involved in the wave releasing additional glutamate (Innocenti et al., 2000). The wave of glutamate release that underlies the NADH fluorescence propagated at a speed of approximately 26 mm/s, which correlated well with the rate of calcium wave progression (10 –30 mm/s). The stimulus for this glutamate release was the increase in cytoplasmic calcium. Local accumulation of glutamate reached 1– 100 mM (Innocenti et al., 2000). It is likely that this concentration range of extracellular glutamate can profoundly modulate the physiology of neighboring neurons (Innocenti et al., 2000). Extracellular glutamate levels in the order of 100 mM are high enough to be toxic (Finkbeiner and Stevens, 1988; Ward et al., 2000) and to have long-term desensitizing effects on other members of the glutamate receptor family (Zorumski et al., 1996; Trussel and Fishbach, 1989). It is provocative to imagine that stimulus-evoked release of glutamate at the synapse is enough to cause the regenerative release of the glutamate wave from astrocytes described by Innocenti et al. (2000) (see also chapter by Shuai et al.), which in turn can have a regulatory effect on neurons over large distances of cortical brain regions. In particular, the concept of long-distance Ca2þ waves regulating and coordinating large regions of cortex becomes appealing. This concept is supported by the demonstration that activity-dependent potentiation of inhibitory synaptic transmission in the hippocampus between interneurons and pyramidal neurons is critically dependent on glutamate release from astrocytes (Kang et al., 1998). Inhibitory interneurons are of great importance for brain function, being the controlling mechanism for modulation of the excitatory activity of neurons in the region.

7.4. Astrocytes can modulate neuronal responses and synaptic transmission Glial contributions to excitatory neurotransmission were studied in single neuron micro-islands where neuronal autaptic and glial responses were recorded during excitatory synaptic events (Mennerick and Zorumski, 1994). The release of glutamate from astrocytes was recently shown to modulate synaptic transmission in cultured hippocampal neurons (Arague et al., 1998a,b), as well as in intact retina (Newman and Zahs, 1998). Can astrocytes modulate electrical and transmission characteristics of neighboring neurons? To test this possibility, focally applied electrical field or laser stimulation was used to initiate a calcium response in astrocyte/neuronal cultures, which resembled waves evoked from other paradigms, including glutamate exposure (Nedergaard, 1994). Neurons lying on contiguous astrocyte monolayers responded to glial signaling with increased intracellular Ca2þ. In contrast, neurons cultured on fibronectin or on astrocytes not involved in a wave did not show corresponding increases in [Ca2þ]i and did not respond to field potentials (Nedergaard, 1994). Repeated stimulation of the same target neuron consistently provided the same results. To test whether the Ca2þ wave was transmitted by extracellular release and diffusion of an astrocyte-derived molecule the perfusion was changed to superfusion with a linear velocity vector opposite to and 20 times faster than the Ca2þ wave, a procedure which prevented any effects on signaling either between astrocyte or between astrocytes and neurons. Inhibition of synaptic mechanisms with tetrodotoxin had no effect on astrocyte to neuron signaling, and the source for the

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increased Ca2þ release appeared to be intracellular stores. Photobleach recovery experiments suggested that local neurons lack a significant degree of gap junction coupling to one another or to local astrocytes. However, gap junctions with unidirectional diffusion capabilities were suggested between heterologous cell types and the Ca2þ signaling from astrocytes to neurons was unidirectional and inhibited by the gap junction blocker octanol (Nedergaard, 1994). Further studies however (Murphy et al., 1993; Blank et al., 1998) did not corroborate the idea of electrotonic coupling between astrocytes and neurons, and glutamate has since been identified as the diffusible chemical messenger, which specifically stimulates neurons (Hassinger et al., 1996; Pasti et al., 2001). Ca2þ oscillations in astrocytes may be the coordinating behavior, which regulates the release of glial glutamate (Pasti et al., 2001). HEK293 cells were transfected with the NMDA receptor to be used as a glutamate biosensor (Pasti et al., 2001). Oscillations of intracellular Ca2þ in astrocytes were shown to trigger synchronous and repetitive Ca2þ oscillations in the sensor HEK cells. Whole cell patch clamp recordings demonstrated activation of NMDA receptors in HEK cells, resulting in inward currents that have extremely fast kinetics, in the order of the NMDA receptor currents in postsynaptic neurons. Agents known to reduce neuronal exocytosis (i.e., tetanus toxin and bafilomycin A) reduced astrocyte Ca2þ oscillations. Ca2þ oscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes (Pasti et al., 2001—see also chapter by Shuai et al.). This system is bi-directional since an increase in the firing rate of neuronal afferents results in an increased frequency of [Ca2þ]i oscillations in astrocytes (Pasti et al., 1997). The frequency of oscillations is under the dynamic control of neuronal activity raising the possibility that it represents a code for the transfer of information from neurons to astrocytes. It was shown that glial uptake removed synaptically released glutamate, thereby contributing to the termination of excitatory synaptic currents. If autaptically associated glial currents represent activation of electrogenic transporters, then under appropriate conditions reverse uptake should produce glial glutamate efflux. A depolarization of glia to þ 50 mV induced a slowly rising neuronal current and accompanying pharmacology showed that the neuronal response was mediated by NMDA receptors, responding to glutamate released from glia (Pasti et al., 2001). Astrocytes respond to neuronal activity through receptor-mediated events as well as electrical events making them important players in information processing in the brain. Other neurotransmitters including, gamma-aminobutyric acid (GABA) (Liu et al., 2000), growth factors (Beattie et al., 2002), amino acids (Baranano et al., 2001), neuroactive peptides (Blondel et al., 2000), and acetylcholine (at the neuromuscular synapse (Jahromi et al., 1992; Reist and Smith, 1992) may also direct astrocyte – neuronal signaling. However, whereas ATP mediates calcium waves between astrocytes, it is unable to trigger astrocytic-neuronal signaling (unpublished experiments). Communication between neurons and glia can occur without the involvement of a synapse. Phosphorylation of myelin basic protein occurs following high frequency axonal firing and this activity-dependent signaling results in release of NO (Atkins et al., 1999). In the PNS, axonal firing was shown to release ATP from non-synaptic regions resulting in activation of the Schwann cell P2Y receptors and stimulation of a Ca2þ-activated signaling pathway (Stevens and Fields, 2000; Fields and Stevens-Graham, 2002).

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8. Pathology and Ca21 waves Disease states are often characterized by chronic imbalances in signaling molecules, which directly affect activity of ion channels, neurotransmitters and transporters, compounding the progression of the pathology. A common feature to most of these conditions is excitotoxity, which is mediated by excessive activation of amino acid receptors with the resulting toxicity ultimately contributing to the degeneration of brain cells (Meldrum and Garthwaite, 1990; Rothstein et al., 1995—see also chapters by Barger, by Brown and Sassoon, by Ghorpade and Gendelman, and by Werner et al.). When concentrations of extracellular neurotransmitters reach excitotoxic levels there are profound effects on the astrocytes in the region of stress. Astrocytes cultured from resected medial temporal lobe epilepsy patients exhibited hyperexcitable responses to glutamate if they were derived from regions of hyperexcitable EEG activity, identified in the neurosurgery operating room (Cornell-Bell and Williamson, 1993). Changes in receptor expression often accompany disease states, as evidenced by up-regulated expression of group I and II metabotropic glutamate receptors in spinal cord of ALS patients (Aronica et al., 2001) and of mGlu receptors 2/3 and 5 in astrocytes from kainate-induced seizures in amygdala of rats (Ulas et al., 2000). The finding of mGlu receptors 2/3, 4 and 8 immunoreactive astrocytes in hippocampus suggested that these receptors are involved in gliosis at the seizure focus (Tang and Lee, 2001). An increase in astrocytic mGlu receptors may directly lead to CNS hyperexcitability as described in the chapter by Shuai et al. Astrocytes from epileptic focus exhibited increased gap junctional coupling (Lee et al., 1995) and altered expression of ion channels (Bordey and Sontheimer, 1998). The resting membrane potential of seizure focus astrocytes from mesial temporal lobe epilepsy were significantly depolarized (approximately 2 55 mV) compared with cortical astrocytes (2 80 mV) and there was a much higher density of Naþ channels, further leading to the excitability of astrocytes. The astroglial glutamate transporters were also impaired in epilepsy tissues, contributing to the elevation of the levels of extracellular glutamate (Ye et al., 1999; Gorter et al., 2002). Electrophysiological changes in astrocytes were also seen to accompany reactive gliosis following scar formation (MacFarlane and Sontheimer, 1997). Moreover hippocampal gene expression is altered following status epilepticus (Hendriksen et al., 2001). Increased levels of extracellular excitatory neurotransmitters including glutamate were directly measured in extracellular space using microdialysis (During and Spencer, 1993) with a loss of glutamate-stimulated GABA release that was secondary to a reduction in the number of GABA transporters (During et al., 1995). During ischemia, citrulline (by-product of NO synthesis), glutamate, glycine, and GABA concentrations were also shown to increase extracellularly (Tan et al., 1996—see also chapter by Ha˚berg and Sonnewald). All of these conditions paint a picture of compromised balance. Once the extracellular environment is thrown out of equilibrium, glutamate levels can soar due to reversal of glutamate transporters (Atwell et al., 1993) and high levels of glutamate (in the millimolar range) can be released from the astrocytes to further injure neurons and escalate damage to glutamate homeostasis even more (Hertz et al., 1988; Szatkowski et al., 1990). Glioma cells release glutamate by cystine – glutamate exchange, which actively kills neurons in the vicinity of a tumor (Ye and Sontheimer, 1999; Ye et al., 1999). These are only a few representative studies of excitotoxic changes occurring in

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diseased tissues. How does the excitotoxic environment disrupt normal calcium wave communication in astrocytes? What other receptors may play a role in astrocyte signaling in diseased tissues and what effects can toxicants released from damaged cells play? In excitotoxic disease states one striking characteristic of time-lapse movies is the distinct lack of long-lasting Ca2þ waves (Jung et al., 2001). Long-lasting signals are replaced by very short-lived elevations in Ca2þ, which rapidly die out and so travel only short distances. In time-lapse studies of calcium activity in a culture of astrocytes from a patient with Tuberous Sclerosis (TS) very short lived signals were noted in response to glutamate application (Jung et al., 2001). In a sequence of subtracted snapshots of calcium activity only local islands of calcium elevations were seen, lacking the large-scale organization observed in cultures of normal rat astrocytes. In these TS cultures periodically a drastic, large-scale organization of Ca2þ activity would develop in the form of a cylindrical wave, which would rapidly entrain nearly all of the cells in the field, indicating at least temporary strong coupling between the astrocytes, leading to a strong, fast wave. During these episodes the cells in the entire culture appear synchronized in their Ca2þ activity. After these events the synchrony is lost, and Ca2þ activity again becomes a local phenomenon. Using a time and space ‘cluster’ calculation as described in the chapter by Shuai et al., it was shown that the entropy for diseased astrocytes was below the entropy measured in normal rat astrocyte cultures. Entropy vanishes if the spatiotemporal patterns or ‘excited states’ belong to only one pattern. In the example of epileptic Ca2þ images, the spatiotemporal pattern prevails of many short lived waves that travel short distances and die out (Jung et al., 2001). Time lapse-imaging studies of astrocytes from neocortical tumor cases and medial temporal lobe epilepsy cases exhibited profound increases in intracellular Ca2þ transients following exposure to glutamate (Cornell-Bell et al., 1992; Cornell-Bell and Williamson, 1993; Lee et al., 1995). Cultures from hyperexcitable cortex and parahippocampus showed significantly higher numbers of intercellular waves when compared to regions exhibiting normal EEG records. Close to 82% of the excitable cortex propagated intercellular waves compared to 19.0% of the normal cells or 16.4% of the hippocampal cells and 5.4% of the peritumoral astrocytes. There was some indication the velocity of these intercellular waves was higher and the lifetimes were shorter (Lee et al., 1995). Astrocytes from a tumor region rarely propagate intercellular waves, which may be an indication of their extreme excitotoxic environment (Ye and Sontheimer, 1999; Ye et al., 1999). There was significantly more gap junctional coupling between cells cultured from seizure foci as well (Lee et al., 1995). These studies of only the pathology of epilepsy clearly indicate that regulatory mechanisms affecting intracellular and intercellular Ca2þ signaling patterns are modified, sometimes rather drastically, by the pathophysiologic changes in the disease environment. Other pathologies including Leao’s spreading depression and hypoxia are characterized by irregular intracellular Ca2þ homeostasis (Do Carmo and Somjen, 1994; Somjen et al., 1993) suggesting that study of the Ca2þ signaling in different diseases may help elucidate common irregularities that lend themselves to therapeutic intervention. One organelle that is targeted by several disease states is the mitochondria (Boitier et al., 1999). Brain mitochondria have been found to be impaired in pathologies including stroke (Sims, 1995); Alzheimer’s disease (Mattson and Fukuawa, 1996) and Parkinson’s disease (Bowling and Beal, 1995). In such states, altered Ca2þ regulation by mitochondria will

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directly impinge on the local and long distance signaling throughout the astrocyte syncytium. Effects of diseased mitochondria on astrocyte signaling as it affects neuronal signaling are implied, but as of yet are not studied. In addition other Ca2þ regulating organelles such as endoplasmic reticulum and the nucleus may be targets for study in cultures of diseased tissues. Other neurotransmitters besides those hitting amino acid receptors may be involved in disease states. Purinergic receptors especially warrant study, since ATP plays such an important regulatory role in astrocyte signaling, providing the extracellular mediator for Ca2þ waves. Extracellular nucleosides and nucleotides have been shown to mediate both proliferative and cytoskeletal alterations on astrocytes (Neary et al., 1996, 1999). This may be directly related to the response of astrocytes to brain injury, which commonly induce hypertrophic and hyperplastic responses (Eng et al., 1987; Norenberg, 1994—see also chapter by Ka´lma´n). Changes in these astrocytes are characterized by elongation of cellular extensions, enhanced expression of GFAP and response to ATP or ATP analogs (Rathbone et al., 1992; Neary et al., 1994; Abbracchio et al., 1994; Bolego et al., 1997; Neary et al., 1999). Astrogliosis is also enhanced by infusion of adenosine analogs into brain (Hindley et al., 1994). Release of purines following injury such as hypoxia may contribute to the gliosis associated with a number of common neurological disorders such as stroke, trauma, degenerative and demyelinative disorders (Bergfeld and Forrester, 1992; Fredholm et al., 1997). Inflammatory mediators such as IL-1b have been shown to regulate Ca2þ wave propagation via P2 receptors and regulation of gap junction coupling (John et al., 1999). IL-1b induces the expression of proinflammatory genes (John et al., 1999; Brosnan et al., 2001). IL-1b also down regulates gap junction connectivity, which could also contribute to the disease state (Brosnan et al., 2001). Add to this growing list the role of astrocyte Ca2þ wave regulation and this opens up the whole field of pathologic changes to astrocyte –astrocyte and astrocyte– neuronal communication, which we are just beginning to appreciate in its complexity.

9. Concluding remarks Few, if any, observations regarding astrocytes have created as much scientific interest and excitement as the demonstration of astrocytic calcium waves. We have now solid information about several basic characteristics of these waves, which can be used as tools in further investigations of the physiological (and pathological) role(s) of astrocytic calcium waves. The ability of neurons to trigger calcium waves in astrocytes, which in turn may activate not only additional astrocytes, but also other, non-synaptically connected, neurons across considerable distances enable astrocytes to play a role in signaling events in the nervous system. The importance of this signaling system may be crucial for CNS function, perhaps especially in connection with global phenomena like mood and attention. It will be an important task for future research to unravel the secrets of this neuronal – astrocytic-neuronal signaling system.

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Mathematical modeling of intracellular and intercellular calcium signaling Jian-Wei Shuai,a Suhita Nadkarni,a Peter Jung,a,* Ann Cornell-Bellb and Vickery Trinkaus-Randallc a

Department of Physics and Astronomy and Quantitative Biology Institute, Ohio University, Athens, OH 45701, USA p Correspondence address: E-mail: [email protected] b ANSCANS, Ivoryton, CT 06442, USA c School of Medicine, Boston University, Boston, MA 02118, USA

Contents 1. 2.

3. 4. 5.

Introduction Modeling of intracellular Ca2þ signaling (IACW) 2.1. Equations used 2.2. Deterministic, non-phenomenological modeling 2.3. Phenomenological modeling 2.4. Stochastic modeling Modeling of intercellular Ca2þ signaling (IRCW) in astrocytes Bidirectional coupling between neurons and astrocytes Concluding remarks

1. Introduction The most complex system in the universe is probably the brain. Billions of neurons, interconnected to a large network, perform numerous cognitive and regulatory tasks. Most work on the modeling of brain functions is based on modeling of neuronal networks. The vast majority of cells in the brain, however, are nonneuronal cells or glial cells; about 90% of all brain cells are glial cells. Among several types of glial cells, the astrocytes are known to carry out many important functions, several of them in interactions with neurons. Astrocytes, in contrast to most neuronal cells, do not fire action potentials due to insufficient Naþ channel density (Bordeay and Sontheimer, 1998b). Documented exceptions are astrocytomas, where an enhanced expression of Naþ channels allows the generation of action potentials (Bordeay and Sontheimer, 1998a). Astrocytes do not connect to other astrocytes or neurons via long processes. Therefore, for many years it has Advances in Molecular and Cell Biology, Vol. 31, pages 689–706 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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been believed that the role of the glia for brain function is to provide structural and chemical support for the neurons. Such support functions include for example uptake and recycling of neurotransmitters. Although it has been known for a long time that synaptic astrocytes respond with depolarization to neuronal action potentials, this was thought to be a passive response caused by the increased extracellular Kþ concentration. The discovery by Porter and McCarthy (1996), that astrocytes respond to neuronal action potentials by glutamate-mediated activation of metabotropic glutamate receptors, has changed the current thinking about the role of astrocytes dramatically. It is now clear that astrocytes listen to neuronal chatter at the synapses and in turn can modulate neuronal dynamics at the same synapse or over some distance. As neurons fire, glutamate is released into the synaptic cleft, which is partially lined by the metabotropic glutamate receptors of the synaptic astrocytes. Upon binding of glutamate to the astrocyte, inositol 1,4,5-triphosphate (IP3) is released into the intracellular space. IP3 in turn binds to the IP3 receptor of the endoplasmic reticulum (ER), and Ca2þ is released from the ER into the cytosol. As described in more detail below, such Ca2þ release can occur in forms of intracellular Ca2þ waves. The Ca2þ wave can propagate across the cell membrane, through the extracellular space into adjacent astrocytes. As will be discussed below, there are several mechanisms that may be involved in this intercellular signaling. Elevated Ca2þ concentrations in synaptic astrocytes generate extracellular glutamate that can modulate the neuronal synapse by generating additional inward currents. This feedback could of course also be inhibitory, if enhanced Ca2þ concentrations are activating inhibitory interneurons. In the remainder of this section, we will review recent progress in mathematical modeling of intra- and intercellular Ca2þ signaling in general and in the context of astrocytes and their control of synaptic plasticity in particular. We would also like to draw the attention of the reader to another recent review on the same topic by Schuster et al. (2002). 2. Modeling of intracellular Ca21 signaling (IACW) 2.1. Equations used Ca2þ is stored in internal stores, most notably the ER. It can be released into the cytosol through release channels and through passive leakage currents driven by the steep concentration gradient between the ER and the cytosol. The release channels, termed IP3 receptor channels (IP3Rs), have been modeled mathematically for more than ten years, and a number of models have been developed. Although the models differ in detail, most models have certain key-elements. Denoting the concentration of Ca2þ in the cytosol by c; conservation of Ca2þ is expressed by the equation of continuity

›c þ 7jc ¼ rðc; x; tÞ; ›t

ð1Þ

with jc denoting the Ca2þ flux density and rðc; x; tÞ the source density of Ca2þ. For not too large gradients in Ca2þ concentration Fick’s law can be used to relate the flux density to

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the Ca2þ concentration, i.e. jc ¼ 2DCa 7c;

ð2Þ

with DCa denoting the diffusion coefficient for calcium. Inserting Eq. (2) into Eq. (1) yields

›c ¼ DCa 72 c þ rCa ðc; x; tÞ: ›t

ð3Þ

The source density describes the influx of Ca2þ into the cytosol per volume and unit time. In the absence of Ca2þ entry from the extracellular space, the Ca2þ concentration in the cytosol can change due to (i) Ca2þ entering from the ER through IP3Rs, (ii) pumpmediated Ca2þ re-uptake by the ER, (iii) leakage of Ca2þ from the ER into the cytosol, (iv) Ca2þ release from mitochondria, and (v) Ca2þ re-uptake by mitochondria, i.e. out rCa ðc; x; tÞ ¼ 2rpump þ rchannel þ rleak 2 rin mito þ rmito :

ð4Þ

The forms of the source density terms in Eq. (4) differ between the models. The pump term is of Hill-type with a Hill-coefficient of 2 in the two-pool model (Goldbeter et al., 1990) k c2 rpump ¼ 2 1 2 ; ð5Þ k2 þ c the single pool model (Somorgyi and Stucki, 2000; Dupont and Goldbeter, 1993), the De Young – Keizer model (De Young and Keizer, 1992) and the Li – Rinzel model (Li and Rinzel, 1994). The mathematical form of the source density due to Ca2þ release from the ER through IP3Rs, rchannel ; differs between the models used for the IP3 receptor. The single and twopool models use a Hill-form for rchannel : In the De Young– Keizer model it is assumed that the IP3 receptor has three independent subunits with three binding sites each: an activating binding site for Ca2þ, and inhibiting binding site for Ca2þ, and a binding site for IP3. A subunit is activated if IP3 is bound and the activating Ca2þ binding site is bound. The channel is open, if all three subunits are activated. We denote the state of each channel by a string ‘abc’ where a; b; and c can assume the values 0 or 1. The first letter indicates occupancy of the IP3 binding site, the second one the occupancy of the activating Ca2þ binding site and the third letter the occupancy of the inactivating Ca2þ binding site. A value of ‘1’ indicates that the binding site is occupied while a value ‘0’ indicates that the binding site is unoccupied. The source density rchannel is thus proportional to the fraction x3abc ¼ x3110 of channels with all three subunits bound with IP3 and Ca2þ

rchannel ¼ v1 c1 x3110 ðcER 2 cÞ;

ð6Þ

where cER is the Ca2þ concentration in the ER. Thus, in this model, the Ca2þ flux through the IP3Rs is driven by the Ca2þ concentration difference between the ER and the cytosol, and it is determined by the fraction of channels, x3110 ; with all subunits activated. The factor c1 accounts for the ratio between the volume of the ER and the volume of

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the cytosol. The leak current is also driven by the concentration difference between the ER and the cytosol and it is assumed to be of the following form in most models

rleak ¼ n2 c1 ðcER 2 cÞ:

ð7Þ

It is only recently that the effect of Ca2þ sequestering by mitochondria has been taken into account for modeling of intracellular Ca2þ signaling (Magnus and Keizer, 1997, 1998a,b). The uptake of Ca2þ by mitochondria through Ca2þ uniporters sets in only when the Ca2þ concentration exceeds a threshold. This threshold-like uptake is modeled by a Hill-type expression with a large Hill-coefficient, i.e. (Mahrl et al., 2000)

rin mito ¼ kin

c8 ; k28 þ c8

ð8Þ

where kin represents the maximum permeability of the Ca2þ uniporter and k2 the halfsaturation for Ca2þ. In other models, such as the one by Falcke et al. (1999), smaller Hillcoefficients have been used. Ca2þ is released from the mitochondria through Naþ/Caþ exchangers and through mitochondrial permeability transition pores. In Mahrl et al. (2000), these two fluxes have been combined into one term, i.e. ! c2 out cm ; rmito ¼ km þ kout 2 ð9Þ K3 þ c2 where cm is the Ca2þ concentration in the mitochondria. Other modelers (Falcke et al., 1999) use a more explicit model that includes the Naþ concentration in the cytosol and thus links Ca2þ signaling directly to transmembrane potentials (see, e.g., Mahrl et al., 1997). Conservation of total Ca2þ requires two additional equations for the Ca2þ concentration in the ER, cER ; and the Ca2þ concentration in the mitochondria cm ; i.e. dcER ðERÞ 2 ¼ rCa ðc; x; tÞ ¼ rpump 2 rchannel 2 rleak þ DCa 7 cER ; dt

ð10Þ

dcm ðmitoÞ 2 out ¼ rin mito 2 rmito þ DCa 7 cm : dt

ð11Þ

and

The second messenger IP3 is generated by binding of agonist and can diffuse relatively quickly through the cell, since it is less buffered than Ca2þ. The dynamics of IP3 is thus described by a diffusion equation with a linear decay-term modeling the observed degradation of IP3, and a production term af ðx; tÞ i.e.

›cIP 1 p ¼ ðc 2 cIP Þ þ af ðx; tÞ þ DIP 72 cIP ; ›t tIP IP

ð12Þ

where cIP is the concentration of IP3, cpIP the equilibrium concentration, 1=tIP the degradation rate of IP3 (for recent values, see Wang et al., 1995), and DIP the intracellular diffusion coefficient.

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2.2. Deterministic, non-phenomenological modeling Typically, these models predict Ca2þ oscillations or oscillatory spikes, if the concentration of IP3 is within a certain interval. For the Li – Rinzel model (Li and Rinzel, 1994), e.g., where concentration gradients and mitochondria are neglected, the bifurcation diagram plotted in Fig. 1 is obtained. For the original parameters in (Li and Rinzel, 1994) one finds a steady state concentration of Ca2þ at IP3 concentrations below 0.345 mM and above 0.642 mM. In between the Ca2þ concentration oscillates between the minimum amplitude described by the lower branch in Fig. 1 and the maximum amplitude described by the upper branch. If the Ca2þ concentration is spatially not uniform (the spatial derivatives in the equations above are now taken into account), the Ca2þ oscillations are organized in terms of non-linear waves. These waves can have the form of concentric rings (target waves), plane waves and rotating spiral waves. Such waves have been observed to exist in Xenopus oocyte (large cells) by Lechleiter and Clapham (1992) and by Lechleiter et al. (1991). Mathematical analysis of these waves have been performed using a two pool model (Dupont and Goldbeter, 1994) or piecewise linear models (Sneyd et al., 1993; Atri et al., 1993). The effect of Ca2þ buffers is reviewed, e.g., by Keener and Sneyd (1998). It has been shown by Smith et al. (1998) for cardiac myocytes that to obtain quantitative agreement between simulations and experiments the diffusible indicator dye, which also acts as a buffer, has to be taken into account. This gives rise to yet another pair of nonlinear partial differential equations coupled to the equations above. The effect of mitochondria on intracellular Ca2þ waves is discussed by Falcke et al. (1999). 2.3. Phenomenological modeling Phenomenological models that are less demanding in compute time than the stochastic models to be discussed below and also serve well in providing an intuitive picture have

Fig. 1. Bifurcation diagram of the Li –Rinzel model (Li and Rinzel, 1994). The two branches in the interval 0.345 , [IP3] , 0.642 mM indicate the minimum and maximum amplitude of the Ca2þ oscillations. Below 0.345 mM and above 0.642 mM oscillations are absent.

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been used to describe intracellular calcium waves with discrete sources. A simple, but very intuitive model is the fire-and-diffuse model (Keizer et al., 1998a; Dawson et al., 1999). In this one-dimensional model, clustered sources of Ca2þ are placed equidistantly on a line. Each source releases a fixed amount of Ca2þ, when the Ca2þ concentration c exceeds a threshold c0 : When Ca2þ is released from a site, it diffuses along the line, increases the Ca2þ concentrations at neighboring sites and—depending on the distance between the clusters and the amount of Ca2þ being released—can cause Ca2þ release at neighboring sites, that in turn can cause release of Ca2þ at their neighboring sites, and so on. The model yields analytic values for the speed of the calcium wave. Another phenomenological model, similar to the fire-and-diffuse model is an excitable cellular-automaton model with discrete release sites (Jung et al., 1998). This model is a two-dimensional array of discrete release sites. Similar to the fire-and-diffuse model, each release site releases a fixed amount of Ca2þ, when the local Ca2þ concentration exceeds a threshold. According to the model, the released Ca2þ diffuses in the intracellular space and approaches instantaneously a stationary, Gaussian profile. In Falcke et al. (2000) and the fire-and-diffuse model, the profiles (although different) are obtained from the reactiondiffusion equations (1) – (12) (with various simplifications) and the one-dimensional diffusion equation, respectively. Qualitatively, the results are similar. If the coupling between the release clusters is small, the waves are abortive. Yet, fluctuations can generate and maintain local patterns spontaneously (Jung and Mayer-Kress, 1995; Jung et al., 1998; Falcke et al., 2000) and aid weak signals in generating a Ca2þ response. Characteristic for these noise-sustained patterns are power-law distributed lifetime and size distributions (Jung et al., 1998; Falcke et al., 2000).

2.4. Stochastic modeling 2.4.1. Clustering of Ca2þ release channels Recently, high-resolution recordings in different types of cells have shed new light on the elementary intracellular Ca2þ release events. It has been observed that the Ca2þ release channels are spatially organized in clusters with only 20 –50 release channels in each cluster, which has a size of about 100 nm. The calcium release through such small clusters is subject to random fluctuations due to thermal open –closed transitions of individual release channels. After Ca2þ is released, it rapidly diffuses within the cluster (within a few ms) and out into the cytosol. There, Ca2þ is absorbed by buffers and pumped back into the ER and out into the extracellular space (not considered for modeling in this paper) resulting in a spatially and temporally limited event that has been termed calcium puff or spark (Cheng et al., 1999; Callamaras et al., 1998; Melamed-Book et al., 1999; Gonzalez et al., 2000). Ca2þ blips arising from the opening of a single release channel have been observed as well (Bootman et al., 1997; Lipp and Niggli, 1998; Sun et al., 1998). Binding of IP3 activates the calcium release channels and additional binding of Ca2þ opens the channels via Ca2þ induced Ca2þ release (Bezprovanny et al., 1991). Puffs remain spatially restricted at low concentration of IP3 stimulus, whereas at high levels of IP3 neighboring clusters become functionally coupled by Ca2þ diffusion and Ca2þ-induced Ca2þ release, so as to support intracellular

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Ca2þ waves that propagate throughout the cell. Therefore, Ca2þ puffs serve as elementary building blocks of intracellular Ca2þ waves. Moreover, puffs can arise spontaneously before a wave is initiated and they can act as the triggers to initiate waves (Bootman et al., 1997). During the last three years, mathematical modeling of intracellular Ca2þ signaling starting at the elementary Ca2þ release of a single cluster have revealed that clustering of the release channels may have profound consequences for the cellular signaling capability (Shuai and Jung, 2003b). 2.4.2. Stochastic models At the level of a single release cluster, stochastic (i.e., random) effects are dominating. The classification of the Ca2þ dynamics in terms of steady state and oscillatory becomes obsolete (Falcke et al., 2000; Shuai and Jung, 2002a,b). The calcium dynamics consists of a strongly random sequence of elementary calcium release events (see Fig. 2). To model intracellular Ca2þ dynamics according to a stochastic model of intracellular Ca2þ signaling with clustered release channels, the membrane of the ER is modeled as a mosaic of passive and active patches, where the active patches represent the release channel clusters and passive patches contain only pumps and leakage channels. The cell is usually assumed to be flat with a uniform Ca2þ concentration across its width in the ER, and another uniform Ca2þ concentration in the cytosol to allow two-dimensional modeling. The discreteness and small size of the Ca2þ release channels gives rise to novel intracellular Ca2þ patterns such as Ca2þ puffs, abortive waves (waves that propagate only a short distance and then die out) and tide waves (Falcke et al. 2000; Ba¨r et al., 2001). Similar modeling studies for muscle cells, where the calcium release channels are Ryanodine receptors have been reported by Keizer and Smith (1998). One key issue in Keizer and Smith (1998) and Falcke et al. (2000) is the transition from local calcium sparks to propagating waves, i.e., the spark-to-wave transition. For IP3 release channels

Fig. 2. Calcium released from a single release cluster with 20 release channels at various IP3 concentrations generated with the stochastic version of the Li –Rinzel Model (Shuai and Jung, 2002a,b). The non-stochastic model predicts Ca2þ oscillations between 0.345 and 0.642 mM IP3 and a steady state Ca2þ concentration anywhere else (see Fig. 1). The unit of the IP3 concentration is mM.

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it has been shown in Falcke et al. (2000) that as the intracellular IP3 concentration is increased, starting from very small sub-threshold values, the calcium patterns change from puffs and abortive waves (sub-threshold) to propagating waves. Figure 3 shows a sequence of space-time plots (Falcke et al., 2000), where the IP3 concentration increases from the left panel to the right panel. In the second panel from the left, one can observe abortive waves that—initiated by spontaneous events—propagate through parts of the system and then abort spontaneously. As IP3 increases, the waves— although still noisy—spread through the entire system and are repetitive, as an oscillatory model (the De Young– Keizer model) has been used.

Fig. 3. Space-time plots obtained from a one-dimensional model for intracellular calcium waves with clustered release channels (Falcke et al., 2000). Lighter gray indicates increasing Ca2þ concentration. The IP3 concentration increases from the left panel to the right panel.

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A systematic classification of the firing patterns with discrete and stochastic Ca2þ release clusters, but neglecting buffers and Ca2þ handling by mitochondria (Shuai and Jung, 2003a) is shown in Fig. 4. The ‘phase-diagram’ of patterns (Ca2þ diffusion coefficient versus IP3 concentration) presented in the figure revealed that essentially all types of observed intracellular Ca2þ release patterns could be reconstructed using Eqs. (1) –(12) supplemented with discrete sources of Ca2þ. In Fig. 5 snapshots can be seen of different Ca2þ transients simulated using this model (Shuai and Jung, 2003a). 2.4.3. Benefit of clustered release channels Recently it has been suggested that the clustering of the IP3Rs may enhance Ca2þ signaling capability (Shuai and Jung, 2003b). To this end, Eqs. (1) – (12), neglecting effects of mitochondria effects and slow buffers, have been supplemented with spatially discrete distributions of a fixed number of Ca2þ release channels with different spatial organization. The stochastic Li –Rinzel model was used to simulate local Ca2þ release through IP3Rs. The concentration of IP3 was kept below the threshold of Ca2þ oscillations observed in Fig. 1. Distributing the release channels homogeneously on the membrane of the ER yielded a cell-averaged low steady-state Ca2þ concentration—as one would expect. Clustering the channels at distances such that the total number of release channels is conserved (i.e., larger cluster-distance goes along with larger clusters), it was found that in a certain range of cluster distances (and corresponding sizes) the cell-averaged Ca2þ concentrations exhibited strong (almost noise-free) oscillations, coding a weak signal (small IP3 concentration), that was not coded, when the clusters where homogeneously

Fig. 4. Phase diagram of intracellular Ca2þ patterns from Shuai and Jung (2003a). The diffusion coefficient of Ca2þ ðDCa Þ is indicated in mm2/s and the concentration of IP3 in mM. The underlying model for the channel fluxes is the spatially extended, stochastic Li –Rinzel model with discrete and clustered Ca2þ release channels. The clusters contain 20 IP3Rs each and are spaced at a distance of 2 mm. The size of the system is 60 mm £ 60 mm. In the notation used, abortive waves are local, non-propagating but almost propagating waves. Snapshots of the different types of Ca2þ transients are shown in Fig. 5.

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Fig. 5. Snapshots of simulated Ca2þ transients as a function of the diffusion coefficient of Ca2þ ðDCa Þ and the IP3 concentration. Lighter gray indicates increasing Ca2þ concentration. The system size is 60 £ 60 mm2. (a) Ca2þ puffs at D ¼ 1 mm2/s and [IP3] ¼ 0.3 mM at a rate of 1 frame/3 s; (b) local Ca2þ waves at D ¼ 2 mm2/s and [IP3] ¼ 0.5 mM at a rate of 1 frame/3 s; (c) abortive Ca2þ wave at D ¼ 10 mm2/s and [IP3] ¼ 0.35 mM at a rate of 1 frame/1.4 s; (d) spreading Ca2þ wave at D ¼ 30 mm2/s and [IP3] ¼ 0.5 mM at a rate of 1 frame/2.4 s; (e) Ca2þ tide wave at D ¼ 10 mm2/s and [IP3] ¼ 0.8 mM at a rate of 1 frame/2 s. (Data taken from Shuai and Jung, 2003a).

distributed (Shuai and Jung, 2003b). This effect is demonstrated in Fig. 6. The left upper panel shows the cell-averaged Ca2þ signal in response to a sub-threshold IP3 concentration, when 14,400 channels were placed homogeneously at a distance of 0.5 mm over an area of 60 mm £ 60 mm. The upper panel of the second column in Fig. 6 shows the cell-averaged Ca2þ response at cluster distance of 3 mm with a corresponding cluster size of 36 IP3Rs. The cell-averaged Ca2þ signal has now a large amplitude and almost perfect phase coherence, although no parameters other than the geometric distribution of the channels have been changed. The small IP3 signal is now well encoded in the Ca2þ signal. Increasing the cluster distance further to 5 mm with a corresponding cluster size of 100 channels, the cell-averaged Ca2þ signal is almost constant with some small fluctuations. Thus the IP3 signal is not encoded.

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Fig. 6. Ca2þ concentrations at two neighboring active sites 0.5 mm apart (A2, A3), 3 mm apart (B2, B3) and 5 mm apart (C2, C3) and the corresponding cell-averaged Ca2þ concentrations (A1, B1, C1). The Ca2þ diffusion coefficient in the cytosol, D ¼ 20 mm2/s and [IP3] ¼ 0.21 mM (Shuai and Jung, 2003b).

Although in most computational studies the clusters of the IP3Rs are assumed to be organized on a regular grid this is not so in cells. Exceptions are the two recent publications by Falcke (2003a,b) on nucleation of calcium waves and the effects of slow buffers. The clusters are typically close to the cell membrane and are clustered themselves forming ‘hot spots’. It remains to be shown whether optimal clustering is robust for these more realistic scenarios.

3. Modeling of intercellular Ca21 signaling (IRCW) in astrocytes Calcium signals can travel through the cell membrane and propagate through many cells. Intercellular Ca2þ waves have been observed in astrocyte cultures (Cornell-Bell et al., 1990; Giaume and Venance, 1998; Charles, 1998), hippocampal slice cultures of mice (Harris-White et al., 1998), cultured glioma cells (Charles et al., 1992), neurons (Charles et al., 1996), and hepatocytes (Combettes et al., 1994; Nathanson and Burgstahler, 1995; Robb-Gaspers and Thomas, 1995; Patel et al., 1999). Although the mechanism for intercellular Ca2þ waves (IRCW) is controversial, it is clear that it is different from the mechanism for intracellular Ca2þ waves (IACW). There exists a substantial amount of indirect evidence that gap junctions connecting neighboring astrocytes are important for the propagation of IRCW, in that they provide permeability for the intracellular messenger IP3. If an increased concentration of IP3 is generated in an astrocyte, it may not only aid in providing intracellular Ca2þ via calcium-induced calcium release in the cell in question, but it also may diffuse through gap junction to neighboring astrocytes and contribute to the generation of an intracellular Ca2þ response. Such a mechanism has been modeled by Sneyd et al. (1995a,b) and Hofer et al. (2002), with

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numerous simplifications. The source density of IP3 has to be supplemented by the divergence of the flux density through the gap-junctions at the cell-boundaries 7jgap ðxÞlcell-boundary :

ð13Þ

Assuming Fick’s law for the flux through the gap junctions (driven by a gradient in IP3 concentration), i.e. jgap ¼ 2Dgap 7cIP ;

ð14Þ

and discretizing the Laplacian in the equation for IP3 (last term on the right-hand side of Eq. (12)), the additional terms due to the gap junctions are 2

Dgap nþ1 ðc 2 2cnIP þ cn21 IP Þ; Dx2 IP

ð15Þ

Fig. 7. A DC stimulus is applied to the neuron during the time interval [0–40 s] (end of stimulation interval is indicated by an arrow) at an IP3 production rate of a ¼ 0:5 in the astrocyte. The lowest (dense) curve depicts the neuronal oscillations and an expanded section of it is shown in the inset. As the neuron fires, the concentration of IP3 in the astrocyte (upper curve) is increasing. If the IP3 concentration is high enough, intracellular Ca2þ oscillations occur. When the stimulus to the neuron is stopped, IP3 degradation overwhelms the positive feedback into the neuron, and the Ca2þ oscillations and the neuronal oscillations disappear.

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if the cell-boundary is perpendicular to this spatial direction. Assuming the cell membranes, separating the two cells is located between xn and xnþ1 ; and a steep gradient of cIP between the cells in comparison to the one within the cells, i.e. n21 < cnIP ; cIP

ð16Þ

one finds 2

Dgap nþ1 Dgap nþ1 n21 n ðc 2 2cnIP þ cIP Þ< ðc 2 cnIP Þ ; Pgap ðcnþ1 IP 2 cIP Þ Dx2 IP Dx2 IP

with the gap-junction permeability Pgap : Gap junction permeability has not been available in the literature and thus its value needs to be inferred indirectly, e.g., by the propagation distance of a wave. Such a diffusive coupling mechanism leads to a diffusive type of IRCW with a speed that would decrease as it spreads. This prediction is in agreement with

Fig. 8. DC stimulus is applied to the neuron during the time interval [0– 40 s] (end of stimulation interval is indicated by an arrow) at the larger IP3 production rate a ¼ 0:8 in the astrocyte. The lowest (dense) curve depicts the neuronal oscillations and an expanded section of it is shown in the inset. As the neuron fires, the concentration of IP3 in the astrocyte (upper curve) is increasing. If the IP3 concentration is high enough, intracellular Ca2þ oscillations occur. When the stimulus to the neuron is stopped, the production rate of IP3 (larger than in Fig. 7) overwhelms the degradation and the Ca2þ oscillations and neuronal oscillations continue indefinitely.

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observations in astrocytes (Sneyd et al., 1995a,b; Harris-White et al., 1998; Jung et al., 1998). It has, however, also been suggested that the diffusion range of IP3 is not large enough to account for the distance an IRCW is propagating (Giaume and Venance, 1998). While there is general agreement regarding the role of IP3 as a messenger (see, however, also chapter by Scemes and Spray), the role of additional extracellular messengers is controversial, and it is a current topic of research. It has been observed that cultured mouse hippocampal astrocytes that were not coupled to other astrocytes by gap junctions, also participate in the IRCW (Hassinger et al., 1996). Using a novel technology to image extracellular ATP it has been reported (Wang et al., 2000) that Ca2þ waves triggered by mechanical stimulation are synchronized with a spreading extracellular ATP signal. Based on the propagation range of this signal it has been concluded by Wang et al. (2000) that the signal is regenerative, although it spreads only across a finite distance, i.e., that cells receiving the external ATP signal (by binding of ATP to purinergic receptors), also generate extracellular ATP in response. It has also been speculated that IP3 traveling through gap junctions may not be the mediator of intercellular Ca2þ waves (Wang et al., 2000). This hypothesis is supported by the report by Bushong et al. (2002) that the astrocytes in most of the brain form isolated domains (see chapter by Scemes and Spray) so that only a diffusible extracellular messenger could facilitate direct signals between the cells. However, Arcuino et al. (2002) challenge the hypothesis of a regenerative wave-like ATP signal, concluding that ATP released in response to stimulation from a group of astrocytes diffuses passively through the extracellular space, and Giaume and Venance (1998) find no evidence that extracellular ATP should be involved in the intercellular Ca2þ wave. Mathematical modeling that includes extracellular messengers has not been carried through according to our knowledge.

4. Bidirectional coupling between neurons and astrocytes Recently our group (Nadkarni and Jung, in preparation) has proposed a model for neuronal dynamics, that takes into account the coupling between neurons and astrocytes. The key elements of the model are coupling of Ca2þ dynamics in the synaptic astrocytes to neuronal dynamics, described by a conductance-based model. An increased Ca2þ concentration in synaptic astrocytes causes an additional inward current in neurons (Parpura and Haydon, 2000), probably due to generation and release of extracellular glutamate from astrocytes. The quantitative relationship between Ca2þ concentration in the astrocyte and the additional inward current in the neuron has been fitted by a curve and added to the ionic conductance model. When the neuron fires, it releases glutamate that binds to the metabotropic glutamate receptors on the astrocytes and causes the generation in the astrocyte of IP3, that in turn contributes to calcium-induced calcium release and an intracellular Ca2þ signal. An equation has been set up to simulate the IP3 concentration (see Eq. (12)), based upon its rates of generation, a; and degradation, 1=tIP (Wang et al., 1995). The rate of generation of IP3 in the astrocyte in response to neuronal firing of an action potential is a free parameter. While there is no experimental value available for this rate, we know that it has to be proportional to the density of metabotropic glutamate receptors on the synaptic astrocytes. One of the important conclusions of this study is that the critical value of

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the injected current into the neuron (sum of all synaptic inputs) to generate repetitive (periodic) neuronal firing is reduced in the presence of the astrocyte feedback-loop (see Figs. 7 and 8). The IP3 production rate a; proportional to the density of mGluR is 0.5 s21 in Fig. 7 and 0.8 s21 in Fig. 8 where spontaneous oscillations remain even after the neuronal stimulation is terminated. As a matter of fact, if the density of metabotropic glutamate receptors on the synaptic astrocytes is large enough (see Fig. 8), oscillations can set off in the absence of external stimulus. In this context it is interesting to note that astrocytes in epileptic foci are known to over-express metabotropic glutamate receptors (Ulas et al., 2000; Aronica et al., 2000; Tang and Lee, 2001). 5. Concluding remarks We reviewed the recent literature on modeling of intracellular and intercellular calcium waves with focus on brain tissue. We have pointed out the importance of stochastic modeling for intracellular calcium signaling in view of the discreteness of the elementary release events even for cellular signaling capability. Mathematical modeling of intracellular Ca2þ signaling of the Ca2þ release channel results in enhanced cellular signaling capability in response to weak stimuli with optimal clustering. While intracellular modeling is advanced and capable of reproducing experimental results, modeling of intercellular signals is still patchy and incomplete. Progress in this field requires a better and more complete understanding of the underlying mechanisms that are currently only poorly understood. Bidirectional interaction of astrocytes with neurons can alter the behavior of the neurons. We have reported on a recent study where it has been shown that the feedback from synaptic astrocytes into the same synapse can have the effect of generating spontaneous neuronal oscillations. Acknowledgements This material is based upon work supported by the National Science Foundation under Grant No. IBN-0078055. References Arcuino, G., Lin, J.H.C., Takano, T., Liu, C., Jiang, L., Gao, Q., Kang, J., Nedergaard, M., 2002. Intercellular calcium signaling mediated by point-source release of ATP. Proc. Natl Acad. Sci. USA 99, 9840– 9845. Aronica, E., van Vliet, E.A., Mayboroda, O.A., Troost, D., da Silva, F.H.L., Gorter, J.A., 2000. Upregulation of metabotrobic glutamate receptor subtype mGluR3 and mGluR5 in reactive astrocytes in a rat model of mesial temporal lobe epilepsy. Eur. J. Neurosci. 12, 2333– 2344. Atri, A., Amundson, J., Clapham, D., Sneyd, J., 1993. A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte. Biophys. J. 65, 1727–1739. Ba¨r, M., Falcke, M., Levine, H., Tsimring, L.S., 2000. Discrete stochastic modeling of calcium channel dynamics. Phys. Rev. Lett. 84, 5664–5667. Bezprovanny, I., Watras, J., Ehrlich, B., 1991. Bell-shaped calcium response curves of Ins(1,4,5) P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 751–754. Bootman, M., Niggli, E., Berridge, M., Lipp, P., 1997. Imaging the hierarchical Ca2þ signaling system in HeLa cells. J. Physiol. 499, 307–314.

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Bordeay, A., Sontheimer, H., 1998a. Electrophysiological properties of human astrocytic tumor cells in situ: enigma of spriking glial cells. J. Neurophysiol. 79, 2782– 2793. Bordeay, A., Sontheimer, H., 1998b. Properties of human glial cells associated with epileptic seizure foci. Epilepsy Res. 32, 286–303. Bushong, E.A., Martone, M.E., Jones, Y.Z., Ellisman, M.H., 2002. Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J.Neurosci. 22, 183– 192. Callamaras, N.J., Marchant, S., Sun, X., Parker, I., 1998. Activation and co-ordination of InsP3 mediated elementary Ca2þ events during global Ca2þ signals in Xenopus oocytes. J. Physiol. 509, 81–91. Charles, A., 1998. Intercellular calcium waves in glia. Glia 24, 39 –49. Charles, A.C., Kodali, S.K., Tyndale, R.F., 1996. Intercellular calcium waves in neurons. Mol. Cell. Neurosci. 7, 337 –353. Charles, A.C., Naus, C.C.G., Zhu, D., Kidder, G.M., Dirksen, E.R., Sanderson, M.J., 1992. Intercellular calcium signaling via gap junctions in glioma cells. J. Cell Biol. 118, 195–201. Cheng, H., Song, L., Shirokova, N., Gonzalez, A., Lakatta, E.G., Rios, E., Stern, M.D., 1999. Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method. Biophys. J. 76, 606–617. Combettes, L., Trans, D., Tordjmann, T., Laurent, M., Berthon, B., Claret, M., 1994. Ca2þ mobilizing hormones induce sequentially ordered Ca2þ signals in multicellular systems of rat hepathocytes. Biochem. J. 304, 585 –594. Cornell-Bell, A.H., Finkbeiner, S.M., Copper, M.S., Smith, S.J., 1990. Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 247, 470–473. Dawson, S.P., Keizer, J., Pearson, J.E., 1999. Fire-diffuse-fire model of dynamics of intracellular calcium waves. Proc. Natl Acad. Sci. USA 96, 6060–6063. De Young, G.W., Keizer, J., 1992. A single-pool inositol 1,4,5-trisphosphate-receptor-based model for agoniststimulated oscillations in Ca2þ concentration. Proc. Natl Acad. Sci. USA 89, 9895–9899. Dupont, G., Goldbeter, A., 1993. One-pool model for Ca2þ oscillations involving Ca2þ and inositol 1,4,5 triphosphate as co-agonist for Ca2þ release. Cell Calcium 14, 311 –322. Dupont, G., Goldbeter, A., 1994. Properties of intracellular Ca2þ waves generated by a model based on Ca2þ induced Ca2þ release. Biophys. J. 67, 2191– 2204. Falcke, M., 2003. On the role of stochastic channel behavior in intracellular Ca2þ dynamics. Biophys. J. 84, 42– 56. Falcke, M., 2003. Buffers and oscillations in intracellular Ca2þ dynamics. Biophys. J. 84, 28–41. Falcke, M., Hudson, J.L., Camacho, P., Lechleiter, J.D., 1999. Impact of mitochondrial Ca2þ cycling on pattern formation and stability. Biophys. J. 77, 37–44. Falcke, M., Tsimring, L., Levine, H., 2000. Stochastic spreading of intracellular Ca2þ release. Phys. Rev. E62, 2636–2643. Giaume, C., Venance, L., 1998. Intercellular calcium signaling and gap junctionional communication in astrocytes. Glia 24, 50–64. Goldbeter, A., Dupont, G., Berridge, M.J., 1990. Minimal model for signal-induced Ca2þ oscillations and for their frequency encoding through protein phosphorylation. Proc. Natl Acad. Sci. USA 87, 1461–1465. Gonzalez, A., Kirsch, W.G., Shirokova, N., Pizarro, G., Brum, G., Pessah, I.N., Stern, M.D., Cheng, H., Rios, E., 2000. Involvement of multiple intracellular release channels in calcium sparks of skeletal muscle. Proc. Natl Acad. Sci. USA 97, 4380–4385. Harris-White, M.E., Zanotti, S.A., Fruatchy, S.A., Charles, A.C., 1998. Spiral intercellular calcium waves in hippocampal slice cultures. J. Neurophysiol. 79, 1045–1052. Hassinger, T.D., Guthrie, P.B., Atkinson, P.B., Bennet, M.V.L., Kater, S.B., 1996. An external signaling component in propagation of astrocytic calcium waves. Proc. Natl Acad. Sci. USA 93, 13268–13273. Hofer, T., Venance, L., Giaume, C., 2002. Control and plasticity of intercellular calcium waves in astrocytes: A modeling approach. J. Neurosci., 22, 4850–4859. Jung, P., Cornell-Bell, A., Madden, K.S., Moss, F., 1998. Noise-induced spiral waves in astrocyte syncytia show evidence of self-organized criticality. J. Neurophysiol. 79, 1098–1101. Jung, P., Mayer-Kress, G., 1995. Spatiotemporal stochastic resonance in excitable media. Phys. Rev. Lett. 74, 2130–2133. Keener, J., Sneyd, J., 1998. Mathematical Physiology. Springer, Berlin.

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pH regulation in non-neuronal brain cells and interstitial fluid Suzanne D. McAlear and Mark O. Bevenseep Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd, 846 MCLM, Birmingham, AL 35294-0005, USA p Correspondence address: Tel.: þ 1-205-975-9084; fax: þ 1-205-975-7679. E-mail: [email protected]

Contents 1. 2.

3.

4.

5.

6.

Introduction Basic principles of pHi physiology 2.1. Chronic acid loading 2.2. pHi-regulating mechanisms 2.3. Steady-state pHi 2.4. Consequences of pHi regulation in brain Importance of pH regulation in brain 3.1. General cellular activity 3.2. Effects of pH on neuronal activity 3.3. Effects of neuronal activity on pHECF pH regulation in glial cells 4.1. Acid-loading conductances 4.2. Acid-loading transporters: HCO2 3 -independent 4.3. Acid-loading transporters: HCO2 3 -dependent 4.4. Acid-extruding transporters: HCO2 3 -independent 4.5. Acid-extruding transporters: HCO2 3 -dependent pH regulation of the CSF 5.1. Overview 5.2. pH regulation by choroid epithelial cells 5.3. pH regulation by endothelial cells of brain capillaries Concluding remarks

pH regulation in brain is important because changes in brain pH can influence neuronal activity, synaptic transmission, and possibly memory and learning. The pH of the cerebrospinal fluid bathing the brain is determined by mechanisms that regulate intracellular pH (pHi) in the choroid plexus epithelium and capillary endothelium. Advances in Molecular and Cell Biology, Vol. 31, pages 707–745 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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In addition, the pH of the extracellular fluid (pHECF) surrounding individual brain cells is determined by pHi-regulating mechanisms of neurons and glia. Changes in pHECF and pHi are therefore intertwined because Hþ and HCO2 3 shuttle between the extraand intracellular compartments. By modulating both pH i and pH ECF, these mechanisms directly influence pH-sensitive neuronal activity. Glial cells play a fundamental role in controlling both pHECF and pHi with the use of powerful acid –base transporters including the Na – H exchanger, the Na/HCO3 cotransporter, and the Cl – HCO3 exchanger. Such transporters are also found in the choroid plexus epithelium and the capillary endothelium.

1. Introduction The regulation of the ionic and chemical environment of brain cells is necessary for normal neuronal activity and brain function. In addition, the regulation of brain pH is important because changes in pH can influence many cellular processes including the activity of cellular enzymes, ion channels, and transporters. Decreases in brain pH associated with pathological conditions such as ischemia, hypoxia, and epileptic events lead to neuronal necrosis and overall brain damage (Katsura and Siesjo, 1998). One of the many functions of glial cells is to regulate the acid –base status of the environment surrounding brain cells. This environment is comprised of two types of fluid: the cerebrospinal fluid (CSF) and the interstitial or extracellular fluid (ECF). The CSF, which comprises the macroenvironment of brain cells, is produced by the choroid plexus and bathes both the internal and external surfaces of the brain. Endothelial cells of the blood –brain barrier also contribute to the composition of the CSF by filtering ions and molecules in the fluid that passes from brain capillaries into the CSF. The second general fluid is the ECF that occupies the space between cells throughout the brain. ECF is in direct contact with brain cells, and therefore comprises their microenvironment. Although the ECF is , 20% of total brain volume, the volume between adjacent cells is very small because cells are in close apposition to one another (, 20 nm apart) (see Ransom, 1992). Thus, the movement of ions (including acid – base equivalents) and other substances across cell membranes can substantially affect extracellular concentrations. For example, a single action potential can be estimated to elevate Kþ in the periaxonal space by as much as 1 mM (Adelman and Fitzhugh, 1975; also see chapter by Walz). The regulation of pHi by brain cells can also lead to large changes in pHECF. The focus of the present chapter is mechanisms by which non-neuronal cells such as glia regulate both pHi and pHECF. We will begin by examining some of the basic principles of cellular pH regulation. Subsequently, we will evaluate the relationship between pH and neuronal activity, and the involvement of glial cells. We will then describe the cellular and molecular physiology of the specific acid –base transport mechanisms identified in glia. These mechanisms will include three of the most powerful acid – base transporters glia use to regulate pHi and pHECF: the Na-H exchanger, the Na/HCO3 cotransporter, and the Cl – HCO3 exchanger. In Section 5 of this chapter, we will examine how some of

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the acid– base transporters found in the choroid plexus and the capillary endothelium contribute to the pH of the CSF. 2. Basic principles of pHi physiology 2.1. Chronic acid loading For a typical cell with a resting membrane potential of 2 60 mV and bathed in a pH-7.3 solution, an electrochemical gradient favors the influx of Hþ (or protonated weak acids) and the efflux of HCO2 3 (or deprotonated weak bases) (see Bevensee and Boron, 1998b). If the plasma membrane of our typical cell is permeable to such acid – base equivalents, then their passive movement will tend to acidify the cell to a pHi of 6.3. In addition, any acid produced by metabolism will tend to remain in the cell. Both the passive movement of acid –base equivalents and metabolic-acid production are regarded as passive acid-loading mechanisms, which subject most cells to a chronic acid load. Nearly all cells have pHi values well above that predicted for Hþ to be in electrochemical equilibrium. To maintain pHi above equilibrium pHi, cells subjected to passive acid-loading mechanisms must therefore expend energy to extrude intracellular acid. 2.2. pHi-regulating mechanisms Nearly all cells use a system of acid – base transporters in their plasma membranes to regulate pHi. A detailed description of the major types of acid-base transporters can be found in Bevensee et al., 2000a. As organized in this review, these transporters can be categorized as acid loaders or acid extruders. Acid loaders move Hþ into cells or bases þ such as HCO2 3 out of cells, whereas acid extruders move H and bases in the opposite directions. In general, acid loaders contribute to pHi recoveries following acute intracellular alkali loads, whereas acid extruders contribute to pHi recoveries following acute intracellular acid loads. As further organized in this review, acid loaders and acid 2 extruders can also be categorized as HCO2 3 -independent or HCO3 -dependent. Acid – extruding transporters are primary, secondary, or tertiary active transporters. Primary active transporters use free energy released from ATP hydrolysis to transport substrate against an electrochemical gradient. An example is the vacuolar-type Hþ pump. Secondary active transporters use free energy released from one substrate moving down an electrochemical gradient to transport another substrate against an electrochemical gradient. An example is the Na –H exchanger. Tertiary active transporters use free energy released from a secondary active transporter. For instance, the transport of monocarboxylates across the basolateral membrane of the kidney proximal tubule is mediated by a Hmonocarboxylate cotransporter; the monocarboxylate gradient is established by the Namonocarboxylate cotransporter in the apical membrane. 2.3. Steady-state pHi The steady-state pHi of a cell is defined by the balance between acidloading mechanisms (e.g., metabolic-acid production, passive movement of acid –base

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equivalents, and acid-loading transporters such as the Cl – HCO3 exchanger) and acidextruding mechanisms (e.g., acid-extruding transporters such as the Na –H exchanger). If there is an imbalance between the two, then pHi will change. As reviewed in detail by Bevensee et al. (2000a), the rate of such pHi change is described by the equation dpHi rðJE 2 JL Þ ¼ dt bT where JE is total acid efflux from all acid-extruding mechanisms, JL is total acid influx from all acid-loading mechanisms, bT is the total proton buffering power of the cell, and r is the cell’s surface area-to-volume ratio. When JE . JL ; pHi will increase at a rate that is proportional to the magnitude of JE 2 JL : When JE , JL ; pHi will decrease at a rate that is proportional to the magnitude of JE 2 JL : The rate of the pHi change is inversely proportional to the ability of the cell to buffer intracellularly introduced acids or bases ðbT Þ: Also, dpHi =dt is proportional to r. When JE and JL are equal, dpHi =dt is zero and the cell is at a steady-state pHi. A range of steady-state pHi values for glial cells has been reported. Steady-state pHi can depend not only on the cell type, but also on other factors such as external pH (pHo), temperature, and the presence vs. absence of CO2/HCO2 3 . In the leech neuropile glial cell for instance, steady-state pHi is 6.9 – 7.0 in the nominal absence of CO2/HCO2 3 (Deitmer and Schlue, 1987; Deitmer, 1992), and approximately 0.3 units higher in the presence of the physiological buffer (Deitmer, 1998). For vertebrate glial cells, reported steady-state pHi values range from 6.7 to 7.3 in the nominal absence of CO2/HCO2 3 , and 6.9– 7.6 in the presence of CO2/HCO2 3 (pHo 7.3– 7.5) (see Rose and Ransom, 1998). Compared to the steady-state pHi values of cultured astrocytes from mouse and rat, the values are higher for rat C6 glioma cells in both the presence and absence of CO2/HCO2 3. 2.4. Consequences of pHi regulation in brain pHi regulation by cells in close proximity has two important consequences. First, the movement of acid –base equivalents across the plasma membrane of a cell will not only change that cell’s pH in one direction, but will also change pHo in the opposite direction. As an example, acid extrusion from an astrocyte will increase pHi of the astrocyte and simultaneously lower pHo. The second important consequence is that the change in pHo will also influence the activity of acid– base transporters that are pHo-sensitive in adjacent cells. Thus, in our example of acid extrusion from an astrocyte, the lower pHo will likely stimulate acid loaders and inhibit acid extruders in nearby neurons and glia. 3. Importance of pH regulation in brain 3.1. General cellular activity Many cellular processes are sensitive to changes in pH (see Roos and Boron, 1981). For instance, changes in pH can alter enzyme activity. A classic example is the glycolytic enzyme phosphofructokinase, which is inhibited , 90% in vitro by a pH decrease of 0.1

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(Trivedi and Danforth, 1966). A decrease in pHi will also inhibit the phosphorylationinduced conversion of inactive phosphorylase b to active phorphorylase a, which catalyzes the breakdown of glycogen in tissues such as liver and muscle (Danforth, 1965). Other cellular processes that are sensitive to pH changes include growth-factor stimulation (Ganz et al., 1990; Pouysse´gur et al., 1984, 1985), microtubule-dependent changes in cell structure (Parton et al., 1991), and electrical coupling between cells (Spray et al., 1981; O’Beirne et al., 1987). As described in more detail below, changes in pH can alter the activity of ion channels.

3.2. Effects of pH on neuronal activity The relationship between neuronal activity and changes in brain pHECF is complex because they influence one another. As reviewed by Chesler and Kaila (1992) and Ransom (2000), decreases in pHECF generally inhibit neuronal activity, whereas increases generally stimulate activity. In the rat hippocampus, for example, raising pHECF elicits an increase in the amplitude of population spikes evoked by a given stimulus (Balestrino and Somjen, 1988). In addition, as shown in both clinical and animal studies, the generation of epileptiform activity can be stimulated by alkalosis and inhibited by acidosis (Cohen and Kassirer, 1982; Woodbury et al., 1984; Aram and Lodge, 1987; Somjen et al., 1987; Lee et al., 1996). pH-mediated effects on neuronal firing are likely due to their influence on many voltageand ligand-gated channels (Tombaugh and Somjen, 1998; Traynelis, 1998), and neurotransmitter transporters that are sensitive to shifts in pHi and/or pHECF. The NMDA-activated, ionotropic glutamate receptor has a pK in the physiological range and is inhibited by decreases in pHo (Traynelis and Cull-Candy, 1990; Tang et al., 1990). In the hippocampus, an increase in pHECF can augment NMDA-mediated synaptic responses to evoked stimuli (Gottfried and Chesler, 1994; Taira et al., 1993). pH-induced changes in the activity of Hþ-activated channels may also influence synaptic transmission, and possibly plasticity and memory. In trying to elucidate the role of Hþ-activated cation currents in the CNS, Wemmie et al. (2002) (see also Cooke and Lilley, 2002) generated a mouse with a targeted knockout of the gene encoding ASIC1, a Hþ-gated cation channel. ASIC1mediated currents are typically observed at pHo less than 6.9 (see Waldmann et al., 1999). Cultured hippocampal neurons from the ASIC1 knockout mice—in contrast to wild-type neurons—failed to elicit fast depolarizations when stimulated by decreasing pHo (Wemmie et al., 2002). In addition, long-term potentiation was impaired in hippocampal slices from the knockout mice. The authors also found that the knockout mice displayed defects in both hippocampal-dependent memory and hippocampal-independent learning. Therefore, the synaptically expressed channel appears to be involved in both memory and learning. Neuronal activity can be directly influenced by HCO2 3 currents per se, which have the additional effect of changing pHi/pHECF. In the hippocampus, GABA-mediated inhibition of pyramidal-neuron activity is caused by an outwardly directed GABAA-activated Cl2 current. However, GABAA receptors also conduct HCO2 3 , albeit to a lesser extent (Kaila and Voipio, 1987; Kaila et al., 1990). As mentioned above, the electrochemical gradient would favor HCO2 3 efflux (a depolarizing response) in a cell with a typical

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membrane potential. As discussed by Sun and Alkon (2002), significant HCO2 3 efflux in hippocampal neurons can convert an inhibitory GABA response into a stimulatory one. Such HCO2 3 conductances may underlie the observation that the enzyme carbonic anhydrase (CA) can influence synaptic activity and memory. CA catalyzes the general þ 2 reaction CO2 þ H2O $ HCO2 3 þ H and facilitates the formation of intracellular HCO3 . 2 2 2 The specific reaction catalyzed by CA is CO2 þ OH $ HCO3 ; the OH arising from deprotonation of H2O bound to the enzyme (Liljas et al., 1994). Activators of CA (e.g., many amines and amino acids such as phenylalanine) substantially enhance synaptic efficacy, spatial learning, and memory in rat (Sun et al., 2001b). In contrast, inhibitors of CA (e.g., sulfonamides such as acetazolamide) impair spatial learning in rat (Sun et al., 2001a).

3.3. Effects of neuronal activity on pHECF It is well established that neuronal firing elicits changes in pHECF that are often biphasic: an alkaline shift followed by an acid shift (Chesler and Kaila, 1992). The magnitudes of these shifts vary in different brain regions. For example, initial alkaline shifts are prominent in regions of gray matter (e.g., hippocampus and cerebellum), whereas acid shifts are more prevalent in regions of white matter (e.g., optic nerve). With excessive firing, an extended acid shift dominates in all regions, probably due to the metabolic production of acid and/or CO2, and subsequent release into the extracellular space. Alkaline shifts in some cases are caused by activation of a neuronal Ca2þ pump that exchanges intracellular Ca2þ for extracellular Hþ following activity-induced increases in intracellular Ca2þ (Schwiening et al., 1993; Paalasmaa et al., 1994; Smith et al., 1994; Paalasmaa and Kaila, 1996; Trapp et al., 1996). HCO2 3 efflux through GABAA-activated channels will also contribute to increases in pHECF (Chen and Chesler, 1990, 1992a; Kaila et al., 1992). An additional component of the alkaline shift could in principle be mediated by Hþ-coupled uptake of glutamate by the glutamate transporter. The acid shift, which can either attenuate or immediately follow the alkaline shift, is due to activation of acid extruders in neurons and glia, particularly astrocytes (Chesler, 1990). Two prominent transporters in astrocytes that contribute to acid shifts are the electrogenic Na/HCO3 cotransporter and the H-Lactate cotransporter. It should be noted that extracellular acid and alkaline shifts are associated with changes in the interstitial partial pressure of CO2 ðPCO2 Þ: Using CO2/Hþ-sensitive microelectrodes, Voipio and Kaila (1993) demonstrated that substantial PCO2 and pHo gradients exist between the CA1 layer of the rat hippocampal slice and the bath solution. At the cell layer, pHo was reported to be lower (range: 7.24– 7.37) and PCO2 higher (range: 50– 37 mm Hg) compared to values in the perfusion solution (pH ¼ 7.4; PCO2 ¼ 24:3 mm Hg). Such gradients are likely the result of continual metabolic production of intracellular acid that is titrated by HCO2 3 to form CO2, which then diffuses from the cell layer to the bath solution. The authors found that repetitive stimulation in the hippocampal slice elicited an extracellular alkaline shift and decrease in PCO2 ; followed by a pronounced acid shift and increase in PCO2 : Although the alkaline shift was accompanied by an increase in extracellular HCO2 3 , the acid shift occurred at a

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constant HCO2 3 . The acid shift, but not the increase in PCO2 ; was blocked by applying the extracellular CA inhibitor benzolamide. The increase in PCO2 associated with the acid shift is consistent with activity-evoked increases in metabolism that generate intracellular CO2, which then diffuses into the bath. As mentioned above, the activity of acid-extruding mechanisms can account for either an attenuation of the alkaline shift or the generation of the acid shift near the onset of stimulation. Interestingly, HCO2 3 -dependent acid extruders such as the electrogenic Na/HCO3 cotransporter in astrocytes are predicted to cause an increase in extracellular 22 PCO2 during the acid shift if they transport CO22 3 . CO3 transport has been described for the Na/HCO3 cotransporter in gliotic slices (Grichtchenko and Chesler, 1994a), and for the cloned electrogenic Na/HCO3 cotransporter NBCe1, as well as the cloned electroneutral Na-driven Cl-HCO3 exchanger NDCBE expressed in Xenopus oocytes (Grichtchenko and Boron, 2002a,b). The PCO2 increase would arise from two sequential processes. First, 22 CO22 3 transport into cells causes a decrease in extracellular CO3 , which then drives the þ 2 deprotonation of HCO3 and formation of more extracellular CO22 3 and H (pK , 10.3). 22 See Voipio (1998) for a description of pH changes caused by CO3 . Second, because the 2 increase in Hþ is disproportionately larger than the decrease in HCO2 3 , the CO2/HCO3 equilibrium favors the formation of CO2 and H2O. The opposite events would occur inside þ the cell: the transporter-mediated influx of CO22 3 rapidly equilibrates with H to form þ 2 HCO3 . Because the decrease in H is disproportionately greater than the increase 2 in HCO2 3 , the CO2/HCO3 equilibrium favors the hydration of CO2. Thus, intracellular PCO2 decreases. In all likelihood, the non-CO2/HCO2 3 buffering power is lower outside than inside cells, and the increase in extracellular Hþ will be larger than the decrease in intracellular Hþ at similar pH values. Consequently, the increase in extracellular PCO2 will be larger than the decrease in intracellular PCO2 . In principle, the transporter-generated transmembrane PCO2 gradient could lead to transient CO2 diffusion from the extracellular space into the cell, before net CO2 diffusion out of the tissue. 4. pH regulation in glial cells In this section, we will examine the main acid-loading and acid-extruding mechanisms identified in glial cells. These mechanisms contribute to steady-state pHi, as well as pHi recoveries from acid –base perturbations such as acute intracellular acid and alkali loads. The majority of the studies involve invertebrate glia and mammalian astrocytes. Investigators have also examined the pHi physiology of additional vertebrate glia, including oligodendrocytes, microglia, Mu¨ller cells of the retina, and Schwann cells of the peripheral nervous system. Investigators have used several techniques to measure pHi or pHo. For example, the pHi physiology of giant neuropile glial cells of the leech Hirudo medicinalis has been particularly well characterized with the use of double-barreled, pHsensitive microelectrodes. Advantages of this glial preparation are that it is well established (Kuffler and Potter, 1964), and the cells are large enough to tolerate impalement by multiple microelectrodes. Microelectrodes have also been used to examine extracellular pH changes in mammalian brain-slice preparations. In studies on smaller mammalian glia (e.g., astrocytes in culture), most investigators have studied

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pHi-regulating transporters with either radioactive tracers (e.g., 22Naþ) or pH-sensitive dyes (e.g., 20 ,70 -bis(carboxethyl)-5,6-carboxyfluorescein, or BCECF) and ratiometric fluorescence techniques. Below, we will review both functional and molecular studies on acid – base transporters identified in glial cells.

4.1. Acid-loading conductances As described previously, the passive movement of acid – base equivalents can produce a chronic intracellular acid load. However, such movement through channels can also elicit acute decreases in pHi and increases in pHECF. Channels that conduct acid –base equivalents can therefore be described as acid loaders. In the crayfish muscle fiber for 2 channels and causes pHi to instance, HCO2 3 exits through GABAA-stimulated Cl decrease (Kaila and Voipio, 1987; Kaila et al., 1990). Through a similar mechanism, GABA stimulates HCO2 3 efflux from cells in the turtle cerebellum (Chen and Chesler, 1990) and the hippocampal slice (Chen and Chesler, 1992a; Kaila et al., 1992), thereby eliciting a transient extracellular alkaline shift. GABAA receptors have been reported in several types of glial cells, although at lower levels than in neurons (see Riquelme et al., 2002). Functional GABAA receptors have been found in cultured astrocytes (Blankenfeld and Kettenmann, 1992; Fraser et al., 1994; Bovolin et al., 1992; Bormann and Kettenmann, 1988; Blankenfeld et al., 1991), where their activation causes a depolarization, rather than a hyperpolarization, due to the higher intracellular Cl2 concentration than found in neurons (see chapter by Hansson and Ro¨nnba¨ck). These receptors have also been demonstrated in cultured oligodendrocytes (Blankenfeld et al., 1991), and in both astrocytes and Bergmann glial cells in brain slices (Kang et al., 1998; Muller et al., 1994; Riquelme et al., 2002). Furthermore, activation of the GABAA receptor in cultured rat astrocytes does elicit a HCO2 3 -dependent decrease in pHi (Kaila et al., 1991). Another ion conductance that mediates pHi/pHo changes is the voltage-dependent Hþ conductance first described in snail neurons (Thomas and Meech, 1982). In this preparation, the conductance is activated at depolarized membrane potentials close to , 0 mV, thereby favoring Hþ efflux. Voltage-activated Hþ currents with similar properties have been reported in many other cell types including phagocytes, microglia, skeletal muscle, and alveolar epithelia (DeCoursey and Cherny, 1994; Eder and DeCoursey, 2001). In microglia, decreases in pHi and increases in pHo lower the depolarization threshold for channel activation. Hþ movement only occurs in the outward direction when there is a large pHo/pHi gradient. In phagocytes such as neutrophils, these Hþ channels become activated during respiratory bursts when O2 is converted into superoxide anion during phagocytosis (Henderson et al., 1987, 1988a,b). In a similar fashion, these channels might also be activated in phagocytosing microglia, particularly if there are accompanying increases in pHECF. Consequent changes in microglia pH may influence cell migration, ion-channel function, and microglia activation during pathological events (Faff and Nolte, 2000; Eder and DeCoursey, 2001).

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4.2. Acid-loading transporters: HCO2 3 -independent 4.2.1. Plasma membrane Ca2þ pump Functional studies. The plasma membrane Ca2þ-ATPase (PMCA) is an ATP-driven transporter that exchanges intracellular Ca2þ for extracellular Hþ (A, Table 1). A vanadate-sensitive PMCA elicits increases in cell-surface pH of snail neurons following Table 1 Acid-loading and acid-extruding transporters in glia Acid loaders A B

Plasma membrane Ca2þ pump (PMCA) Glutamate transporter (EAAT) Cl-HCO3 exchanger (AE)

Net charge

Inhibitors

–

Vanadate

þ1 in

DL -threo-b-hydroxyasparatate

1:3 Na/HCO3 cotransporter (NBC)

22 out

(putative) Stilbene derivatives, Oxonol dyes Stilbene derivatives

Acid extruders

Active transport

Net charge

F

Na-H exchanger (NHE)

28

–

G

Vacuolar-type Hþ pump

18

þ1 out

H

H-K pump

18

–

I

H/monocarboxylate cotransporter (MCT)

38

–

Amiloride and analogues, benzoylguanidines NEM, bafilomycin A1, dicyclohexylcarbodiimide Omeprazole, some Schering compounds CHC, pCMBS

J

1:2 Na/HCO3 cotransporter (NBC)

28

21 in

Stilbene derivatives

K

Na-driven Cl-HCO3 exchanger (NDCBE)

28

–

Stilbene derivatives

C D

–

Inhibitors

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elevations of intracellular Ca2þ (Schwiening et al., 1993). A similar Ca2þ pump has been described in mammalian hippocampal neurons (Paalasmaa et al., 1994; Smith et al., 1994; Paalasmaa and Kaila, 1996; Trapp et al., 1996), where it is responsible for both an extracellular alkaline shift and an intracellular acid shift following activity-evoked increases of [Ca2þ]i. PMCAs may also couple Ca2þ and pHi changes in glia. Kawai et al. (1989) used an i ultracytochemical technique to identify Ca2þ-pump activity in reactive and non-reactive astrocytes. Applying dry ice to the exposed rat scalp induced astrocyte activation and repair. The reactive astrocytes surrounding the lesion exhibited increased Ca2þ-pump activity compared to basal-level activity of the non-activated astrocytes. Activity of a vanadate-sensitive PMCA has also been described in the leech giant glial cell with the use of the Ca2þ-sensitive dye Fura-2 (Nett and Deitmer, 1998). The authors found that the pump contributes to low basal [Ca2þ]i levels, and is the primary means by which the cells recover from depolarization-induced elevations of intracellular Ca2þ. The functional coupling of changes in [Ca2þ]i and pHi by PMCAs in glia has yet to be explored. Molecular studies. cDNAs encoding four PMCAs (PMCA-1 through PMCA-4) have been identified (see Z˙ylin´ska and Soszyn´ski, 2000). According to both mRNA and protein localization studies, PMCA-1 and PMCA-4 are ubiquitously expressed, whereas PMCA-2 and PMCA-3 are predominantly expressed in brain and heart (Guerini, 1998; Stauffer et al., 1995). Using immunoblotting, immunohistochemical, and RT-PCR approaches, Fresu et al. (1999) identified PMCA-1, PMCA-2, and PMCA-4 in primary cultures of rat cortical astrocytes, and PMCA-1 and PMCA-4 in rat C6 glioma cells. 4.2.2. Glutamate transporter Functional studies. Glutamate transporters are electrogenic, secondary active transporters that exchange extracellular glutamate, 2Naþ, and Hþ for intracellular Kþ (B, Table 1). Glutamate and aspartate are the major excitatory neurotransmitters in the brain, and their extracellular concentrations therefore have to be exquisitely regulated. A prolonged elevation of extracellular glutamate in the brain leads to excitatory neurotoxicity and subsequent cell death due to glutamate-induced cellular entry of Ca2þ (Choi, 1988). As described in detail in the chapter by Schousboe and Waagepetersen, neurons and glia use plasma-membrane glutamate transporters to remove released glutamate from chemical synapses following transmitter release. These transporters in astrocytes are particularly important for neuronal function and survival, because they can help terminate glutamatergic transmission (Danbolt, 2001). From the standpoint of pHi physiology, glutamate transporters are acid loaders because glutamate is cotransported with Hþ usually into the cell. Several studies have documented pHi changes elicited by glutamate transporters in invertebrate glia and mammalian astrocytes. For example, Bouvier et al. (1992) examined glutamate transport in voltage-clamped salamander retinal glia using either pH-sensitive microelectrodes to measure pHo or intracellular BCECF to record pHi. This preparation is ideal for these studies because the glia lack glutamate-gated ion channels that may conduct Hþ. In one series of experiments with the microelectrodes, activating glutamate uptake by

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hyperpolarizing the cells elicited an increase in pHo that was dependent on external glutamate and Naþ. In another series of experiments with BCECF, activating glutamate uptake generated a decrease in pHi that was also Naþ-dependent. In an invertebrate preparation, Deitmer and Schneider (1997) used microelectrodes to record pHi decreases and depolarizations elicited by applying glutamate to leech giant glial cells. These pHi decreases appear to be mediated by the glutamate transporter for the following four reasons. First, similar results were obtained with D -aspartate, which has a low affinity for glutamate receptors but is transported by the glutamate-uptake system. Second, the glutamate/aspartate-induced pHi decreases were unaffected by glutamate-receptor blockers. Third, the responses were Naþ-dependent. Fourth, the responses were partially inhibited by several putative glutamate-uptake inhibitors including DL -threo-b-hydroxyaspartate. Glutamate has been reported to elicit a decrease in the pHi of astrocytes from several brain regions including mouse cerebrum (Brookes and Turner, 1993), rat cerebellum (Brune and Deitmer, 1995), and rat hippocampus (Rose and Ransom, 1996). In cultured rat cerebellar astrocytes for example, glutamate and aspartate caused decreases in pHi that were independent of any increases in intracellular Ca2þ (Brune and Deitmer, 1995). Working on rat hippocampal astrocytes, Rose and Ransom (1996) demonstrated that such glutamate/aspartate-induced decreases in pHi were paralleled by increases in [Naþ]i. These changes were observed even when non-NMDA glutamate receptors, which might conduct Hþ, were inactivated. These data are consistent with glutamate transporters mediating glutamate-induced pHi decreases in astrocytes. Similar results have been obtained from hippocampal cells in the slice preparation (Amato et al., 1994). Molecular studies. There is a considerable body of literature on the identification and localization of the five cloned glutamate transporters (EAAT1-EAAT5). As reviewed by Danbolt (2001), EAAT1 (GLAST) and EAAT2 (GLT) are predominantly found in glia of the normal adult mammalian CNS, whereas EAAT3 (EAAC) is found in several types of neurons. EAAT4 is principally expressed in Purkinje cells of the cerebellum, and EAAT5 appears to be found mainly in retinal cells, including neurons and Mu¨ller cells. 4.3. Acid-loading transporters: HCO2 3 -dependent 4.3.1. Cl – HCO3 exchanger Functional studies. Cl – HCO3 exchangers (also known as anion exchangers, AEs) are acid loaders that normally exchange extracellular Cl2 for intracellular HCO2 3 (C, Table 1). AEs are electroneutral, Cl2-dependent, Naþ-independent, and sensitive to a class of compounds called stilbene derivatives (e.g., 4,40 -diisothiocyanatostilbene-2,20 -disulfonic acid or DIDS), as well as oxonol dyes (Knauf et al., 1995; Alper et al., 1998). Investigators have used several experimental approaches to identify AE activity in glia. Using double-barreled, pH-sensitive microelectrodes, Szatkowski and Schlue (1994) identified a Cl –HCO3 exchange mechanism in connective glial cells of the leech. Recovery from an alkali load induced by removing acetate in the presence of CO2/HCO2 3 was partially dependent on both extracellular Cl2 and HCO2 3 , and was inhibited by

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the stilbene derivative SITS (4-acetamido-40 -isothiocyanostilbene-2,2-disulfonic acid). Furthermore, removing external Cl2 elicited a DIDS-sensitive alkalization that was Naþindependent yet HCO2 3 -dependent. Functional AEs have also been documented in mammalian astrocytes. Based on SITSsensitive 36Cl2 uptake and efflux measurements, Kimelberg et al. (1979) suggested the presence of a Cl – Cl or Cl – HCO3 exchange mechanism in primary astroglial cultures from neonatal rat brain. The following two groups subsequently identified Cl –HCO3 exchange in rat astrocytes using fluorometric techniques with the pH-sensitive dye BCECF. Mellerga˚rd et al. (1993) observed that the pHi of primary cultures of rat cortical astrocytes recovered rapidly from alkali loads elicited by decreasing bath CO2/HCO2 3 at constant pHo. Applying DIDS or removing external Cl2 reduced the rate of recovery. Shrode and Putnam (1994) alkali loaded both C6 cells and primary cultures of rat cortical astrocytes with an acute exposure to the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO). Beforehand, cells were incubated in a Cl2-free, CO2/HCO2 3 solution. Returning the cells to a Cl2-containing solution following the alkali load elicited a DIDS-sensitive pHi decrease that was Naþ-independent. AE activity has been examined in at least two other glial preparations. Primary cultures of Schwann cells from the rat sciatic nerve exhibited a pHi increase in response to removing external Cl2 (Nakhoul et al., 1994). This pHi increase occurred in the absence of external Naþ, and was substantially slower in the nominal absence of CO2/HCO2 3. However, the pHi increase was unaffected by 100 mM DIDS. A similar pHi increase elicited by removing external Cl2 has also been documented in oligodendrocyte progenitors (Boussouf and Gaillard, 2000). As with the Schwann cells, the pHi increase in þ these progenitor cells required HCO2 3 and was unaffected by removing external Na . In contrast to the Schwann cells however, the pHi increase was blocked by DIDS (500 mM). The authors demonstrated the lack of AE activity in the more differentiated prooligodendrocytes, as well as the mature oligodendrocytes. Therefore, Cl –HCO3 exchange activity in oligodendrocytes appears to be developmentally regulated. Molecular studies. cDNAs encoding four Cl – HCO3 exchangers are known: AE1, AE2, AE3, and AE4. The AEs in conjunction with the Na/HCO3 cotransporters (NBCs) and the Na-driven Cl – HCO3 exchangers (NDCBEs) to be described shortly, are members of a superfamily of HCO2 3 transporters. AE4 has been found primarily in kidney collecting duct, gastrointestinal tract, and submandibular gland (Tsuganezawa et al., 2001; Ko et al., 2002) and will therefore not be discussed further. AE1 (or the ‘band-3 protein’) was the first anion-exchanger cloned (Kopito and Lodish, 1985), and is found in vertebrate erythrocytes. AE2 is found on the basolateral membrane of mouse choroid plexus based on in situ hybridization and immunohistochemistry studies (Lindsey et al., 1990). AE3 mRNA is predominantly expressed in brain and heart (Kopito et al., 1989). According to in situ hybridization data, AE3 is present throughout rat brain and is found in nearly all types of neurons. In contrast, AE3 is not detected in many regions with high levels of glial-cell bodies (e.g., corpus callosum and cerebellar peduncles). Both the brain and cardiac isoforms of AE3 are expressed in the rat retina (Kobayashi et al., 1994). Interestingly, the brain isoform is expressed in Mu¨ller cells, whereas the cardiac isoform is found in horizontal cells. The authors found that expression of the brain isoform gradually increases from postnatal day 3 (P3) to P15. In contrast, expression of the cardiac isoform is not

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apparent until P15; after which time its rapid expression coincides with the onset of retinal function. The highest expression of the brain isoform in Mu¨ller cells is seen in basal endfoot processes that contact the vitreous humor and nearby blood vessels. This AE3 isoform may therefore facilitate CO2 removal from the retina to blood vessels near the vitreous humor (Kobayashi et al., 1994). 4.3.2. Na/HCO3 cotransporter Functional studies. Na/bicarbonate cotransporters (NBCs) mediate the cotransport of one Naþ ion and one or more HCO2 3 ions (D and J, Table 1). These transporters have been identified with one of three Naþ:HCO2 3 stoichiometries: 1:1, 1:2, and 1:3. The cloned electrogenic NBCe1 appears to transport CO22 3 based on an elegant series of experiments on the transporter expressed in Xenopus oocytes (Grichtchenko and Boron, 2002a). Similar to the AEs, NBCs are inhibited by stilbene derivatives. The electrogenic NBC in the proximal tubule of salamander kidney was the first NBC to be functionally characterized (Boron and Boulpaep, 1983). In the kidney, this transporter has a 1:3 2 Naþ:HCO2 3 stoichiometry (Soleimani et al., 1987) and is responsible for , 80% of HCO3 þ 2 reabsorption in the proximal tubule. By moving Na and HCO3 across the basolateral membrane from cell to blood, the transporter functions as an acid loader. NBCs with Naþ:HCO2 3 stoichiometries of 1:1 (electroneutral) and 1:2 (electrogenic) found in other cells usually function as acid extruders by transporting Naþ and HCO2 3 into cells (see Boron et al., 1997). As described below under ‘acid-extruding transporters: HCO2 3dependent’, the 1:2 NBC has been identified in many glial cells. In the retinal Mu¨ller cell however, the transporter has a 1:3 Naþ:HCO2 3 stoichiometry. Using the whole-cell, voltage-clamp technique on freshly dissociated Mu¨ller cells, Newman and Astion (Newman and Astion, 1991; Newman, 1991) demonstrated that þ HCO2 3 -induced outward currents required the presence of external Na , but not external 2 Cl . These currents were blocked by either stilbene derivatives (e.g., 4,40 -dinitro stilbene2,20 -disulfonic acid, DNDS) or harmaline. By examining the reversal potentials of the þ DNDS-sensitive HCO2 3 currents at different transmembrane Na gradients, the authors þ 2 computed a Na :HCO3 stoichiometry of 1:3. Interestingly, the NBC-mediated current was an order of magnitude larger at the endfoot near the vitreous humor than at the distal end of the cell. The authors speculated that this polarized distribution of NBC activity may either facilitate CO2 removal from active photoreceptors to the vitreous humor, or contribute to pH-mediated dilation of blood vessels near the endfeet. Molecular studies. The molecular physiology of these transporters will be described below in the NBC section of ‘acid-extruding transporters: HCO2 3 -dependent’ (p. 726). 4.4. Acid-extruding transporters: HCO2 3 -independent 4.4.1. Na – H exchanger Functional studies. Na – H exchangers (NHEs) are ubiquitous acid –base transporters that have been found in nearly all eukaryotic cell types studied to date (see Counillon and Pouysse´gur, 2000). These electroneutral, secondary active transporters exchange

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extracellular Naþ for intracellular Hþ (F, Table 1). Pharmacologically, the isoforms have varied sensitivities to amiloride, amiloride analogs such as ethylisopropyl amiloride (EIPA), and benzoylguanidines including HOE642 (cariporide) and HOE694 (Putney et al., 2002). Cariporide is a particularly potent competitive inhibitor of NHE-1 (Counillon et al., 1993). NHEs have been found in both neuronal and glial cells (Bevensee and Boron, 1998a; Deitmer, 1998; Rose and Ransom, 1998). Na –H exchange activity generally elicits a pHi recovery from an intracellular acid load (usually studied in the nominal absence of þ CO2/HCO2 3 ) that is dependent on external Na and sensitive to amiloride or its analogs. In addition, a functional NHE at steady-state pHi is evident by a decrease in pHi elicited by applying amiloride and unmasking background acid-loading mechanisms (Boyarsky et al., 1990; Sjaastad et al., 1992). In glia, these transporters are potent regulators of pHi during recoveries from acute intracellular acid loads. Using double-barreled, pH-sensitive microelectrodes on leech neuropile glial cells, Deitmer and Schlue (1987) determined that the pHi recovery from an acute acid load in the nominal absence of CO2/HCO2 3 was inhibited , 50% by 2 – 3 mM amiloride and required the presence of external Naþ. These data are consistent with the presence of an amiloride-sensitive NHE. Using the same approach, Szatkowski and Schlue (1992) identified a similar NHE in leech connective glial cells. Na – H exchange activity has been reported in mammalian preparations such as rat C6 glioma cells, as well as mammalian astrocytes. Measuring 22Naþ uptake in C6 glioma cells, Benos and Sapirstein (1983) noted that upon serum starvation for at least 4 h, an amiloride-sensitive Naþ transporter was expressed de novo. Jean et al. (1986) used the 22 Naþ-uptake technique and the pH-sensitive dye BCECF in both the C6 rat glioma cell line and NN hamster astrocytes to identify a similar transporter that was inhibited by EIPA, amiloride, and benzamil. For example, the pHi recovery from an acid load in BCECF-loaded C6 cells was dependent on external Naþ (Fig. 1A) and blocked by 50 mM EIPA (Fig. 1B). NHE activity has been examined further in glioma cell lines including rat C6 (Shrode and Putnam, 1994; McLean et al., 2000), as well as human U-118, U-87, and U-251 cell lines (McLean et al., 2000). As we mentioned previously, gliomas have a higher steady-state pHi than normal astrocytes. McLean et al. (2000) reported that increased NHE-1 activity in malignant rat and human gliomas is responsible for their higher steady-state pHi compared to non-transformed rat astrocytes. NHEs contribute to the pH physiology of rodent astrocytes cultured from multiple brain regions including the hippocampus (Pappas and Ransom, 1993; Pizzonia et al., 1996; Bevensee et al., 1997b), cortex (Chow et al., 1992; Mellerga˚rd et al., 1993; Shrode and Putnam, 1994; McLean et al., 2000), forebrain (Boyarsky et al., 1993), and cerebellum (Brune et al., 1994). It is worth noting that the NHE in cultured astrocytes from rat forebrain (Boyarsky et al., 1993) is insensitive to the amiloride analog EIPA. Thus, NHEs in astrocytes from different brain regions may not be the same, at least pharmacologically. In a similar pattern, some hippocampal neurons have been reported to exhibit no sensitivity to amiloride compounds (see Bevensee and Boron, 1998a). In addition to regulating pHi, NHEs also contribute to cell-volume regulation and are activated by cell shrinkage (see reviews by Hallows and Knauf, 1994; Hoffmann and Simonsen, 1989; Lang et al., 1995). The regulation of astrocyte volume is important

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Fig. 1. EIPA-sensitive NHE in rat C6 glioma cells bathed in the nominal absence of CO2/HCO2 3 . Modified from Jean et al. (1986) with permission from European Journal of Biochemistry. Copyright 1986, Federation of European Biochemical Societies. (A) pHi was measured in a cell population using the pH-sensitive dye BCECF and a commercial spectrometer. Prior to the beginning of the experiment, the cells were incubated in a Naþ-free solution. In the continued absence of external Naþ, the cells had an average steady-state pHi of ,7.05 at the beginning of the experiment. The cells were subsequently acid loaded using the NHþ 4 -prepulse technique (Boron and De Weer, 1976a). Applying 30 mM NH3/NHþ 4 elicited an initial increase in pHi as NH3 entered the cell and þ combined with Hþ to form NHþ 4 . Subsequently, the pHi decreased due to influx of NH4 or activation of acidþ loading processes. Removing external NH3/NH4 elicited a pronounced decrease in pHi because accumulated þ intracellular NHþ 4 dissociated into H (which remained trapped in the cell) and NH3 (which diffused out of the cell). As shown in the figure, the pHi does not recover in the absence of external Naþ. Adding 40 mM Naþ to the bath solution elicited a rapid recovery of pHi to ,7.0. Increasing external Naþ to 80 mM caused a further increase of pHi to the initial steady-state pHi. (B) In a separate experiment in which cells were subjected to the same acidloading protocol, pHi failed to recover in the absence of external Naþ. The pHi scale in part (A) also applies to part (B). Applying 50 mM EIPA had little effect on the pHi recovery in the absence of Naþ. Furthermore, adding 40 mM external Naþ in the continued presence of EIPA produced no appreciable increase in the pHi recovery. Thus, the Naþ-dependent pHi recovery was entirely EIPA sensitive at the lowest pHi after the acid load.

because the brain is confined within a rigid skull, and neurological defects are associated with brain-cell swelling that undoubtedly alters cell – cell contacts and reduces the extracellular volume (Andrew, 1991). Swollen astrocytes can also release excessive amounts of excitatory amino acids that cause neurotoxicity (Kimelberg, 1995). Shrinkageinduced activation of NHE activity has been reported in the rat C6 glioma cell line (Jean et al., 1986; Shrode et al., 1995, 1997), and appears to involve phosphorylation of myosin light chain in confluent cell monolayers (Shrode et al., 1995, 1997). There is evidence for Na – H exchange in mammalian oligodendrocytes, microglia, and Schwann cells. Kettenmann and Schlue (1988) used Hþ-sensitive microelectrodes on cultured oligodendrocytes from mouse spinal cord to identify a HCO2 3 -independent pHi recovery from an acid load that was completely blocked by removing external Naþ or applying 1 mM amiloride. In studies involving the use of BCECF, an amiloride-sensitive

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NHE was found in both immature oligodendrocyte progenitor cells (Boussouf and Gaillard, 2000) and mature oligodendrocytes (Boussouf et al., 1997) from rat. Finally, in both microglia cultured from mouse (Faff et al., 1996) and Schwann cells cultured from the rat sciatic nerve (Nakhoul et al., 1994), pHi recoveries from acid loads in the þ nominal absence of CO2/HCO2 3 required external Na and were inhibited by amiloride compounds. Molecular studies. cDNAs encoding seven Na –H exchangers (NHE-1 through NHE-7) have been identified to date. We will focus our attention on NHE-1 through NHE-5; all of which appear to mediate Na – H exchange at the plasma membrane (Counillon and Pouysse´gur, 2000; Baird et al., 1999; Attaphitaya et al., 1999). NHE-6 is found in recycling endosomes (Brett et al., 2002), whereas NHE-7 is targeted to the trans-Golgi network (Numata and Orlowski, 2001). Ma and Haddad (1997) performed both Northern blot analysis and in situ hybridization to determine mRNA expression of NHE-1 through NHE-4 in rat brain. The authors concluded that NHE expression depends on transporter subtype, brain region, and animal age. NHE-1 is the most abundantly and ubiquitously expressed, with high levels found in the hippocampus, cerebellum, and the second/third layers of the periamygdaloid cortex. NHE-2 and NHE-4 are expressed in low levels mainly in the cerebral cortex and brainstemdiencephalon. Low levels of NHE-4 are seen in glial cells within the cerebellar white matter. Finally, NHE-3 is predominantly found in Purkinje and glial cells of the cerebellum. Regarding developmental profiles, the levels of mRNA for NHE-1, -2 and -4 increase in the cortex, but decrease in the cerebellum as animals age from postnatal day 0 (P0) to P30. In contrast, mRNA encoding NHE-3 increases in the cerebellum from P0 to P30. Based on other mRNA studies, NHE-5 is present in multiple brain regions (Baird et al., 1999), and has been localized to neurons in the dentate gyrus of rat hippocampus (Attaphitaya et al., 1999). Expression of NHE-5 mRNA in glial cells has not been reported. Immunochemical data on NHEs are consistent with the aforementioned mRNA localization. In an animal-development study, Douglas et al. (2001) used immunoblotting with NHE isoform-specific antibodies to examine NHE-1, -2, and -4 expression in cortex, cerebellum, and brainstem-diencephalon. NHE-1 expression levels in all three regions gradually increase during animal development from embryonic day 16 to postnatal day 105. In contrast, NHE-2 and NHE-4 expression levels tend to peak at 3– 4 weeks postnatal and then decline. These data may reflect different functional roles of NHE isoforms during development. Antibodies have been used to examine glial-cell expression of NHEs. Pizzonia et al. (1996) used immunoblotting to identify NHE-1 expression in astrocytes cultured from the rat hippocampus. Different expression profiles of the NHE isoforms may contribute to differences in the pHi physiology of brain cells. 4.4.2. Vacuolar-type Hþ pump Functional studies. Vacuolar-type (or V-type) Hþ-ATPases are present in vesicles and other organellar membranes where they establish the low intraorganellar pH necessary for many organellar functions including enzyme activation, ligand – receptor uncoupling, and

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Hþ-coupled carrier transport. V-type Hþ pumps are also found in the plasma membrane of many cells (e.g., macrophages, neutrophils, and osteoclasts), where they are involved in pHi regulation (see Bevensee et al., 2000a). On the apical membrane of the proximal and distal nephron of the kidney, they contribute to acid secretion into the lumen. These electrogenic pumps are primary active transporters that use the free energy released from ATP hydrolysis to extrude Hþ from the cell (G, Table 1). They are inhibited by N-ethylmaleimide (NEM), dicyclohexylcarbodiimide, and bafilomycin A1. A V-type Hþ pump has been found in both cultured astrocytes and gliomas. Pappas and Ransom (1993) used BCECF in cultured astrocytes from the rat hippocampus to monitor pHi recoveries from acute acid loads in the nominal absence of CO2/HCO2 3 . The authors identified a Naþ-independent component of the pHi recovery that was inhibited by either bafilomycin A1 or NEM. Furthermore, depolarizing the cell by raising external Kþ increased the rate of the pHi recovery—consistent with activation of an electrogenic Hþ pump. There is electrophysiological evidence for a V-type Hþ pump in the C6 glioma cells, as well as in DI TNC1 cells—an immortalized cell line derived from primary cultures of rat diencephalon astrocytes. In voltage-clamp experiments on these cells, bafilomycin A1 inhibited a residual hyperpolarizing current that was unmasked after blocking ion channels (Philippe et al., 2002). Molecular studies. The V-type Hþ pump is composed of a V0 and a V1 domain (see review by Kawasaki-Nishi et al., 2003). The V0 domain, with five different subunits (a,b,c,c0 ,c00 ), is involved in Hþ translocation. The V1 domain, with eight different subunits (A –H), is involved in ATP hydrolysis. Using RT-PCR and immunoblotting techniques with primary astrocyte cultures from rat striata and cortex, as well as cultures of C6 and DI TNC1 cells, Philippe et al. (2002) identified the presence of the a and c subunits (V0 domain) and the A subunit (V1 domain) of the V-type Hþ pump. Mouse genes encoding the G1 and G2 isoforms of the G subunit have recently been identified (Murata et al., 2002). The authors found at the RNA and protein levels that the G1 isoform is ubiquitously expressed in multiple tissues (including brain), and the G2 isoform is predominantly expressed in neurons in the CNS. Based on immunohistochemical studies, the G1 isoform is expressed in both astrocytes and oligodendrocytes cultured from mouse hippocampus. 4.4.3. H – K pump Functional studies. H –K ATPases are primary active transporters that exchange extracellular Kþ and intracellular Hþ (H, Table 1). The gastric H – K pump in parietal cells is responsible for the acidity of gastric secretions (Okamoto and Forte, 2001), and both gastric and non-gastric H – K pumps in the collecting duct of the kidney contribute to Kþ reabsorption (Silver and Soleimani, 1999; Jaisser and Beggah, 1999). These pumps are inhibited by omeprazole and certain Schering compounds. Shirihai et al. (1998) have used an elegant approach to identify an H – K pump in microglia. With ion-selective electrodes in self-referencing mode (generated by rapidly moving between two positions), the authors measured an external Kþ gradient within 10 mm of the cell surface. This gradient appeared to be generated by an H – K pump because it was enhanced by an increase in extracellular Kþ, and was dissipated by

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omeprazole or the Schering compound 28080. Unexpectedly, lowering pHo increased the Kþ gradient, presumably by stimulating the H – K pump. This pump may be particularly active in microglia following brain injury when [Kþ]o rises and pHECF falls.

4.4.4. Monocarboxylate transporter Functional studies. Monocarboxylate transporters (MCTs) are tertiary active transporters that mediate the electroneutral cotransport of Hþ and a monocarboxylate (e.g., lactate or pyruvate) (I, Table 1). Transport inhibitors include a-cyano-4hydroxycinnamate (CHC) and p-chloromercuribenzenesulfonate ( pCMBS). MCTs play an important role in metabolic coupling between glia and neurons because glycolytically produced lactate in astrocytes might be used as an energy source by neurons (however, see chapter by Roberts and Chih). Perhaps more importantly, MCTs are also essential for the transport of pyruvate into mitochondria. We will focus here on the influence of MCTs on the pHi physiology of glial cells. Using an enzyme assay, Walz and Mukerji (1988) measured extracellular lactate efflux from both neurons and astrocytes cultured from rat cortex. Lactate efflux from both cell types was inhibited by pyruvate and the non-transportable analog DL -p-hydrophenyllactate, but not by CHC. Although MCTs normally mediate transport out of glial cells, many investigators have characterized their activity operating in the opposite direction. For example, Nedergaard and Goldman (1993) used BCECF to measure the pHi of astrocytes cultured from the forebrain of embryonic rat. The authors concluded that a lactate transport system is responsible for intracellular acidification rates that are a saturable function of externally applied lactic acid. This transport system has a low KM for lactate (0.4 mM), and is insensitive to CHC and pCMBS. However, in radioactive tracer studies on primary cultures of rat astrocytes, Tildon et al. (1993) reported two lactatetransport systems: both high-affinity (KM ¼ 0:5 mM) and low-affinity (KM ¼ 11 mM) ones. Uptake was stimulated by low external pH, and inhibited by high external pH and CHC. The low-affinity transporter has a similar KM to a CHC-sensitive lactate transporter (KM ¼ 7:7 mM) identified in rat astroglial cells (Bro¨er et al., 1997). According to another report, lactate transport in rat C6 glioma cells and cortical rat astrocytes is insensitive to CHC, but sensitive to quercetin (Volk et al., 1997). Dringen et al. (1995) on the other hand found two lactate-uptake mechanisms in the C6 cells: a CHC-insensitive, non-saturable one, and a CHC-sensitive saturable one. According to the same study, primary cultures of rat astroglial cells only exhibit a CHC-insensitive, non-saturable mechanism. Based on the aforementioned studies, at least two lactate transporters with different lactate affinities have been identified in astrocytes and C6 cells. Molecular studies. cDNA sequences of eight mammalian monocarboxylate transporters (MCT1-MCT8) have been identified. While MCT1 is the predominant isoform found in astrocytes, MCT2 with a higher substrate affinity (at least when expressed in oocytes) is particularly prevalent in the astrocytic foot processes that abut blood vessels (see review by Halestrap and Price, 1999). The MCT2 expression profile may optimize efficient transport of metabolic substrates from the endothelium into astrocytes. MCT1-MCT4 have all been documented in retina.

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4.5. Acid-extruding transporters: HCO2 3 -dependent 4.5.1. Na/HCO3 cotransporter Functional studies. As described previously, the NBC with a 1:3 Naþ:HCO2 3 stoichiometry normally functions as an acid loader (e.g., in the proximal tubule of the kidney), whereas those with 1:1 and 1:2 stoichiometries normally function as secondaryactive acid extruders. Based on many studies, the predominant acid-extruding HCO2 3 transporter in glial cells is an electrogenic NBC with a 1:2 Naþ:HCO2 3 stoichiometry that moves net-negative charge in the direction of ion transport (J, Table 1). Characteristics of an acid-extruding NBC include HCO2 3 -dependent increases in pHi (e.g., following intracellular acid loads) that are Naþ-dependent, Cl2-independent, and usually stilbenesensitive. Electrogenic NBC activity in glia was first reported in the giant neuropile glial cell of the leech. Using pH-sensitive microelectrodes, Deitmer and Schlue (1987) identified a Naþ- and HCO2 3 -dependent pHi recovery from an acid load that was inhibited by the stilbene derivative SITS. In subsequent studies, the authors used ion-sensitive microelectrodes to monitor pHi, intracellular Naþ activity (aNaþ i ), and Vm in the same preparation (Deitmer and Schlue, 1989; Deitmer, 1992). Exposing the cells to a CO2/ HCO2 3 -containing solution elicited an increase in steady-state pHi and a hyperpolarization; both of which were Naþ-dependent and DIDS-sensitive (Deitmer and Schlue, 1989). þ CO2/HCO2 3 also induced a DIDS-sensitive increase in aNai . The authors estimated a 1:2 þ þ 2 2 Na :HCO3 stoichiometry from the Na :HCO3 coupling ratio. Electrogenic NBC activity was particularly evident when Deitmer (1992) exposed the cells to a Naþ-free, þ HCO2 had three HCO2 3 -containing solution. Removing external Na 3 -dependent effects that occurred simultaneously: the cell depolarized, the pHi decreased markedly, 2 and aNaþ i decreased at a faster rate than observed in the absence of HCO3 (Fig. 2). These observations are consistent with NBC operating in the reverse direction þ and mediating the efflux of Naþ, HCO2 3 , and net-negative charge during the Na removal protocol. Electrogenic NBCs were subsequently identified in other glial preparations including connective glial cells from leech (Szatkowski and Schlue, 1992) and astrocytes from the optic nerve of the mudpuppy Necturus (Astion and Orkand, 1988). The transporter was also further characterized in the leech glia. Using the two-electrode, voltage-clamp technique, Munsch and Deitmer (1994) confirmed the stoichiometry of the NBC in the neuropile leech glial cells by evaluating DIDS-sensitive current-voltage relationships. In addition, the authors demonstrated the reversibility of the transporter, which has a reversal potential near the resting Vm of the cell (, 2 75 mV). Normal inward transport can be reversed with only small changes in either Vm ; or the transmembrane Naþ and HCO2 3 gradients. In other words, the NBC can readily function as either an acid extruder or acid loader depending on the prevailing electrical and chemical gradients. NBC activity has been examined in considerable detail in mammalian astrocytes. The transporter has been studied in astrocytes from rat forebrain (Boyarsky et al., 1993), rat hippocampus (Pappas and Ransom, 1994; O’Connor et al., 1994; Bevensee et al., 1997a,b), rat cerebellum (Brune et al., 1994), rat cortex (Shrode and Putnam, 1994), and

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Fig. 2. Electrogenic NBC activity in leech giant neuropile glia. Modified from Deitmer (1992) with permission from Pflu¨gers Archives. Copyright 1992, Springer-Verlag. Ion-sensitive microelectrodes were used to measure membrane potential ðEm Þ; intracellular pH (pHi), and activity of intracellular Naþ (aNai) simultaneously. The experimental protocol involved removing external Naþ from the bath solution either in the nominal absence or 2 presence of CO2/HCO2 3 (constant pHo of 7.4). Compared to results in the absence of HCO3 , the following three observations were made for the cell in the presence of the physiological buffer: (i) The cell depolarized due to netnegative charge leaving the cell; (ii) pHi decreased at a considerably faster rate due to Na-coupled HCO2 3 efflux; þ 2 and (iii) aNaþ i decreased at a faster rate in the absence of external Na (compare dotted lines) due to HCO3 coupled Naþ efflux. These data are consistent with electrogenic NBC transporting Naþ, HCO2 , and net-negative 3 charge out of the cell during the Naþ-removal protocol.

mouse cortex (Brookes and Turner, 1994; Chow et al., 1991). In most of these studies, pHi was measured with BCECF, and transporter activity was evident from one or more of the following observations. The Naþ- and HCO2 3 -dependent transporter contributes to pHi recoveries following intracellular acid loads. The transporter is responsible for increases in steady-state pHi when astrocytes are exposed to CO2/HCO2 3 -containing solutions. Finally, electrogenic NBC activity appears to cause an increase in steady-state pHi when cells þ bathed in CO2/HCO2 3 are depolarized by high [K ]o (termed a depolarization-induced alkalization or DIA). A depolarization increases the electrical driving force for Naþ/ 2HCO2 3 cotransport into cells. DIA was first described by Siebens and Boron (1989a,b) working on the kidney proximal tubule. In this preparation, DIA is mediated by both a SITS-sensitive, Naþ-dependent transporter and a monocarboxylate transport system involving Na-Lactate and H-Lactate cotransporters. Conclusively identifying an electrogenic NBC requires examining two key characteristics of transport: Cl2 independence and electrogenicity. Ruling out the involvement of Cl2 is important to distinguish the transporter from a related one—the Na-driven Cl – HCO3 exchanger. In rat hippocampal astrocytes, transporter-mediated intracellular alkalinizations still occurred after reducing intracellular Cl2 to near zero (Bevensee et al., 1997a). In addition, the transporter could still operate in the reverse direction in the absence of external Cl2. Both observations are consistent with the transporter being an NBC and not a Cl2-dependent process. Examining the electrogenicity is important to distinguish the transporter from electroneutral NBCs. The electrogenicity of the

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transporter in mammalian astrocytes has been examined using patch-clamp techniques (O’Connor et al., 1994; Brune et al., 1994; Bevensee et al., 1997a). In rat hippocampal astrocytes for instance, removing external Naþ elicited a DIDS-sensitive, HCO2 3dependent depolarization (Bevensee et al., 1997a). The magnitude of the mean depolarization compared to the mean HCO2 3 flux in parallel pH experiments is consistent with the transporter having a 1:2 Naþ:HCO2 3 stoichiometry. A functional electrogenic Na/HCO3 cotransporter has also been characterized in the gliotic hippocampal slice. Using pH-sensitive microelectrodes, Grichtchenko and Chesler (1994a,b) found that depolarizing astrocytes by elevating [Kþ]o induced both an extracellular acidification and an astrocyte alkalinization that were both partly due to a Naþ- and HCO2 3 -dependent transporter. These data agree well with the aforementioned NBC studies on cultured astrocytes. There is one discrepancy however: the NBC in gliotic tissue is insensitive to stilbenes. Less is known about NBC activity in oligodendrocytes. Kettenmann and Schlue (1988) identified a Naþ- and HCO2 3 -dependent acid – base transporter in cultured oligodendrocytes from embryonic mouse spinal cord. The transporter does not appear to require Cl2, and therefore may be an NBC. As in the gliotic astrocytes described above, the transporter is not sensitive to stilbene derivatives. It is not clear if the transporter is electrogenic or not. In a more recent study, Boussouf et al. (1997) reported similar DIDSinsensitive transporter activity in cultured mature oligodendrocytes from the rat cerebellum. The transporter appears to be electrogenic because membrane depolarizations (generated by raising [Kþ]o) elicited Naþ- and HCO2 3 -dependent alkalizations. In a later study, the authors obtained similar results on oligodendrocyte progenitors and more differentiated pro-oligodendrocytes (Boussouf and Gaillard, 2000). Interestingly, the authors found that the HCO2 3 transporter in progenitor cells is DIDS-sensitive, whereas the one in mature cells is not. Thus, the DIDS-sensitivity of the HCO2 3 transporter in oligodendrocytes appears to decrease with development. Further identification of an electrogenic NBC in these cells will require a direct measure of transporter-mediated currents or voltage changes. Activity during neuronal firing. Although many acid –base transporters contribute to pHi/pHECF regulation in the brain, the electrogenic NBC occupies an unusual niche. When active, the transporter alters both pHi and pHECF (by moving HCO2 3 ) and Vm (by moving net-negative charge). In addition, the Vm influences the direction and activity of the transporter (Munsch and Deitmer, 1994). Therefore, this transporter may serve as a link between neuronal activity and pH changes in the brain. According to the model shown in Fig. 3, and originally proposed by Chesler (1990) and Ransom (1992), an electrogenic NBC elicits pH changes in response to nerve activity. Neuronal excitability causes an increase in [Kþ]ECF, which depolarizes adjacent astrocytes with a high Kþ conductance (see chapter by Walz). The depolarization stimulates the activity of an electrogenic NBC that transports HCO2 3 from the extracellular space into the astrocyte. The pHi of the þ astrocyte increases due to the CA-catalyzed general reaction: HCO2 3 þ H ! CO2 þ þ H2O, which consumes H . Although somewhat controversial, CA II is found in at least some astrocytes (see review by Ridderstrale and Winstrand, 1998). Simultaneous transport of HCO2 3 out of the extracellular space elicits a decrease in pHECF due to the reaction þ CO2 þ H2O ! Hþ þ HCO2 3 , which generates H . This reaction is catalyzed by

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Fig. 3. A model of changes in pHi and pHECF mediated by an NBC (Na/HCO3 cotransporter) during electrical activity (see model from Ransom, 2000). An action potential in a neuron elicits an increase in extracellular Kþ ([Kþ]ECF) that depolarizes adjacent astrocytes with a high Kþ conductance. Depolarization stimulates an electrogenic NBC to transport Naþ, 2HCO2 3 , and net-negative charge into the cell. The pHi of the astrocyte þ þ increases due to the CA-catalyzed general reaction: HCO2 3 þ H ! CO2 þ H2O, which consumes H . The opposite reaction in the ECF will cause pHECF to decrease. Through several potential mechanisms discussed in the text, a decrease in pHECF will inhibit further neuronal activity. The action potential shown in the model neuron is an actual Vm recording from a spontaneously firing cultured rat hippocampal neuron that was patch clamped under current-clamp conditions (McNicholas-Bevensee C.M. and Bevensee M.O., unpublished).

extracellular CA, which has been found in the brain slice (Chen and Chesler, 1992b; Kaila et al., 1992) and on the surface of both neurons and astrocytes (Svichar and Chesler, 2003). As described previously, the pHECF decrease can inhibit further neuronal excitability by inhibiting many voltage- and ligand-gated channels. This negative-feedback model would be neuroprotective in that excessive neuronal activity (e.g., during epileptic events) would elicit a greater decrease in pHECF, and consequently a greater inhibition of further neuronal activity (Ransom, 2000). There is considerable evidence to support the model presented in Fig. 3. As already mentioned, DIAs have been documented in several glial preparations including the leech neuropile glial cell (see Deitmer, 1998), astrocytes cultured from several brain regions (Boyarsky et al., 1993; Pappas and Ransom, 1994; Shrode and Putnam, 1994; Brookes and Turner, 1994; Chow et al., 1991), and the gliotic hippocampal slice (Grichtchenko and Chesler, 1994b). Depolarizations in the gliotic hippocampal slice also cause decreases in pHECF (Grichtchenko and Chesler, 1994a). Using a very elegant approach, Newman (1996) used BCECF attached to the extracellular surface surrounding retinal Mu¨ller cells to demonstrate that cell depolarizations elicit pHo decreases near the endfeet where NBC is predominantly expressed. Thus, depolarization of isolated glial cells and activation of NBC can elicit decreases in pHo, in addition to increases in pHi. There is developmental evidence highlighting the importance of astrocytes in mediating activity-evoked extracellular acid shifts. In the rat spinal cord, activity-evoked changes in pHo and [Kþ]o are dependent on the age of the animal (Sykova´, 1998).

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Compared to neonatal animals, older animals display smaller [Kþ]o increases and larger acid shifts with spinal-cord stimulation (Jendelova´ and Sykova´, 1991). The larger acid shifts seem to coincide with gliogenesis during postnatal development as the number of mature glial cells increases from 31% at P1 – P3 to 77% at P13 –P15 (Chva´tal et al., 1995). Also, ‘early’ postnatal X-irradiation, which selectively blocks gliogenesis, prevents the development of both larger acid shifts and smaller [Kþ]o increases elicited by stimulation (Sykova´, 1998). Molecular studies. Within the last several years, considerable advances have been made in the molecular identification of cation-coupled HCO2 3 transporters (e.g., NBCs and Na-driven anion exchangers). The first cDNA encoding an NBC was cloned by expression from the kidney of the salamander Ambystoma by Romero et al. (1997). Subsequently, numerous other NBC-related clones were identified from several tissues and species. Electrogenic NBCs have been cloned from human kidney (hkNBC) (Burnham et al., 1997), rat kidney (rkNBC) (Romero et al., 1998), human pancreas/heart (hpNBC, hhNBC) (Abuladze et al., 1998; Choi et al., 1999), rat pancreas (rpNBC) (The´venod et al., 1999), and rat brain (rb1NBC, rb2NBC) (Bevensee et al., 2000b; Giffard et al., 2000). Based on differences at the amino and carboxy termini, these variants can be categorized into one of three groups: NBCe1-A (kidney clones), NBCe1-B (pancreas/heart and rb1NBC clones), and NBCe1-C (rb2NBC clone). Another group of NBCs (NBC4) distinct from the NBCe1s has been identified from human testis and heart (Pushkin et al., 2000a,b; Sassani et al., 2002) and a mixture of human tissue cDNAs (Virkki et al., 2002). At least one of the variants (NBC4c) functions as an electrogenic Na/HCO3 cotransporter (NBCe2) (Sassani et al., 2002; Virkki et al., 2002). Electroneutral NBCs (NBCn1s) have been cloned and characterized from human skeletal muscle (Pushkin et al., 1999) and rat smooth muscle (Choi et al., 2000) after a clone was sequenced from human retina (Ishibashi et al., 1998). The following antibodies were generated to distinguish between the NBCe1-B and NBCe1-C variants that were cloned from rat brain: aNBCe1-A/B and aNBCe1-C (Bevensee et al., 2000b). aNBCe1-A/B is expected to recognize both the A and B variants. Based on immunoblot data, aNBCe1-A/B and aNBCe1-C both recognize protein from rat brain, as well as protein from neurons and astrocytes cultured from rat cortex. Localization studies on brain slices with polynucleotide probes (Giffard et al., 2000; Schmitt et al., 2000) and polyclonal antibodies (Schmitt et al., 2000) have confirmed astrocytic, as well as some neuronal expression of NBCs. NBCe1 is present in both astrocytes and neurons in several regions of the CNS including the cortex and hippocampus. In a preliminary report, aNBCe1-A/B and aNBCe1-C labeled neurons in the hippocampus and cerebellum (Risso Bradley et al., 2001). According to a recent animal-development study using immunoblotting techniques, NBCe1 is expressed more abundantly in the cerebellum and brainstem-diencephalon than in cortex of P33 rats (Douglas et al., 2001). In all three regions, expression increases gradually from embryonic day 16 (when it is 25– 40% of the adult level) to P105. According to Northern blot and RT-PCR analyses, NBCn’s (Pushkin et al., 1999; Choi et al., 2000) and NBCe2 (Pushkin et al., 2000b; Sassani et al., 2002) are also present in brain. NBCn1 protein has been localized to neuronal fibers within the hippocampus (Risso Bradley et al., 2001).

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4.5.2. Na-driven Cl – HCO3 exchanger Functional studies. The Na-driven Cl – bicarbonate exchanger (NDCBE) is an electroneutral secondary active transporter that exchanges extracellular Naþ for intracellular Cl2 (K, Table 1). In the transport process, two acid equivalents are extruded into the cell (Grichtchenko and Boron, from the cell, probably by transport of CO22 3 2002b). A stilbene-sensitive Na-driven Cl – HCO3 exchanger was first identified in two invertebrate neuronal preparations: the squid giant axon (Boron and De Weer, 1976a,b; Russell and Boron, 1976; Boron and Russell, 1983) and the snail neuron (Thomas, 1976a, b, 1977). Subsequently, the transporter has been documented in vertebrate cells including fibroblasts (L’Allemain et al., 1985), kidney mesangial cells (Boyarsky et al., 1988a,b), and Ehrlich mouse ascites tumor cells (Kramhoft et al., 1994). The transporter is the predominant HCO2 3 -dependent acid extruder in rat hippocampal CA1 neurons (Schwiening and Boron, 1994), and likely other mammallian neurons as well (Bevensee and Boron, 1998a). Although an NBC is the primary HCO2 3 -dependent acid extruder in astrocytes, NDCBE activity has also been reported. In rat cortical astrocytes studied with BCECF, the pHi recovery from an acid load was inhibited by applying DIDS, or removing either external Naþ or Cl2 (Mellerga˚rd et al., 1993). Shrode and Putnam (1994) obtained similar results with rat cortical astrocytes: CO2/HCO2 3 -induced alkalinizations that are DIDS-sensitive and HCO2 3 -dependent can be inhibited by pre-incubating the cells in 0 Cl2 for 2 h. Na-driven Cl –HCO3 exchange has also been reported in rat cerebellar astrocytes (Ko et al., 1999). A HCO2 3 -dependent transporter, which might be an NDCBE, also contributes to pHi regulation of mouse cortical microglia (Faff et al., 1996). The authors identified a DIDS2 sensitive, Naþ- and HCO2 3 -dependent pHi recovery from an acid load, which may be Cl dependent. Molecular studies. NDCBEs are members of the HCO2 3 -transporter superfamily that also includes the NBCs and AEs. Both a Na-driven anion exchanger from Drosophila (Romero et al., 2000), and an electroneutral Na-driven Cl – HCO3 exchanger (NDCBE1) from human brain (Grichtchenko et al., 2001) have been cloned and functionally characterized. A partial NDCBE sequence was previously obtained from human NT-2 cells (Amlal et al., 1999), and a full-length one was more recently cloned from mouse kidney cells (Wang et al., 2001). Surprisingly, the corresponding full-length proteins in these two studies are reported to be likely Cl2independent. According to mRNA analyses in the above studies, NDCBE and related transporters are all strongly present in the CNS. In a preliminary immunohistochemical study, NDCBE1 was found in the soma and dendrites of cerebellar Purkinje cells of rat (Risso Bradley et al., 2001). Further localization studies are required to assess NDCBE expression in glia, and to support the aforementioned functional studies. A reported Na-driven Cl-HCO3 exchanger (NCBE) has been cloned from an insulinsecreting cell line, and the mRNA is abundantly expressed in cerebellum and cerebrum of rat (Wang et al., 2000). Further functional studies however are required to confirm the Cl-independence and evaluate the electroneutrality of NCBE.

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5. pH regulation of the CSF 5.1. Overview The choroid plexus is an epithelial structure with a rich network of capillaries that secretes CSF into the brain ventricles (see Johanson, 2003; and chapter by Weaver et al.). The CSF circulates in the ventricles and subarachnoid space before being reabsorbed into the venous sinuses by the arachnoid villi. The CSF has three principle functions: (i) to cushion the brain, (ii) to serve as a fluid reservoir for controlling brain volume, and (iii) to serve as a nutritional source for brain cells. Although nascent CSF is produced by the choroid plexus, approximately one-third of the total CSF in the brain originates from secretion of capillary endothelial cells in brain parenchyma (Pollay and Curl, 1967). Compared to blood plasma, the CSF contains very little protein and significantly less glucose and amino acids. Both epithelial cells in the choroid plexus (the blood –CSF barrier) and endothelial cells in the capillaries in brain parenchyma (the blood – brain barrier) are responsible for the composition of CSF. There are two general reasons why the CSF is not simply an ultrafiltrate of blood plasma. First, the choroid epithelial cells and the capillary endothelial cells possess tight junctions that restrict the movement of ions, small polar molecules, and macromolecules from blood to CSF. Second, these polarized cells have apical and basolateral transporters and channels that regulate the composition of the secreted fluid. 5.2. pH regulation by choroid epithelial cells Mechanisms that regulate the pHi of the choroid epithelial cell will contribute to the pH of the CSF produced by the choroid plexus. According to both in vitro and in vivo studies, the choroid epithelial cell from rat has a pHi of , 7.0 (Johanson, 1978; Johanson et al., 1985). The following two main acid– base transporters are present in the choroid epithelial cell: Na-H exchanger (NHE) and Cl – HCO3 exchanger (AE). Recent molecular evidence is consistent with the presence of a Na/HCO3 cotransporter (NBC) as well. Below, we will review the evidence for each of these transporters in the choroid plexus. NHE contributes to intracellular acid extrusion and Naþ loading on the basolateral side of the epithelium (see Fig. 4). Consequently, NHE facilitates Naþ transport from blood (or interstitial fluid) to the CSF. Using radioactive tracers to measure pHi and [Naþ]i of rat choroid plexus epithelium in vivo, Murphy and Johanson (1990) observed NHE activity that elicited [Naþ] decreases in both the epithelial cells and CSF of acid-loaded rats. In contrast, [Naþ] increases were observed in the cells of alkali-loaded rats. The sidedness of amiloride inhibition is consistent with the transporter being on the basolateral (blood) side of the epithelium. In a subsequent study on rats, the authors found that systemic acetazolamide treatment decreased Naþ i and increased pHi of choroid epithelial cells in vivo, and reduced Naþ movement from plasma to CSF (Johanson and Murphy, 1990). The data are consistent with acetazolamide-induced inhibition of CA in the choroid plexus epithelial cells causing an increase in pHi, which then inhibits basolateral NHE activity. NHE activity has also been reported in vitro. For example, 22Naþ uptake is inhibited by amiloride in rat choroid plexus epithelium in vitro (Murphy and Johanson, 1989b)—a

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Fig. 4. Transporters in the choroid plexus epithelium. (A) The epithelial layer of the choroid plexus surrounding the interstitial fluid (ISF) that bathes a capillary. The tight junctions on the apical membranes define the blood– CSF barrier. The basolateral membranes face the ISF. Modified from Johanson et al. (1985) with permission from American Journal of Physiology. Copyright 1985, American Physiological Society. (B) A single epithelial cell of the choroid plexus. NHE and AE2 reside on the basolateral membrane, whereas a Naþ pump and a Na/K/Cl cotransporter are present on the apical membrane. As mentioned in the text, the Na/K/Cl cotransporter has been reported to transport in either direction. An NBC and/or a HCO2 3 -conducting anion channel on the apical membrane could mediate HCO2 3 movement into the CSF.

result that nicely complements the aforementioned in vivo NHE studies. In cultures of choroid epithelia from rabbit, the pHi recovery from an acid load was Naþ-dependent and inhibited by amiloride compounds at low external Naþ (Mayer and Sanders-Bush, 1993). At higher external Naþ, the transport process was amiloride-insensitive. NHE in the lateral ventricle choroid plexus from several mammals including pig and human has been reported based on radiolabeled amiloride binding (Kalaria et al., 1998). In addition, the authors used RT-PCR techniques to identify the presence of NHE-1 in these preparations. However, Alper et al. (1994) were unsuccessful in identifying NHE-1 expression in the lateral ventricle choroid plexus of human by immunoblotting and immunohistochemistry. Functional studies are presently the strongest evidence for a basolateral NHE in choroid epithelia. An AE is the other main acid –base transporter functionally studied in the choroid epithelium. Johanson et al. (1985) used radiolabeled DMO to perform in vitro measurements of pHi in adult rat choroid plexus where they found that epithelium 2 [HCO2 3 ] decreased in proportion to synthetic CSF [HCO3 ]. The presence of AE was 2 reflected by a decrease in [HCO3 ]i elicited by applying SITS, and an increase in 2 [HCO2 from the synthetic CSF. These results confirm the 3 ]i caused by removing Cl suggestion by others that a Cl –HCO3 exchange mechanism is responsible for decreases in CSF [Cl2] elicited by applying systemic DIDS (Frankel and Kazemi, 1983; Deng and Johanson, 1989).

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Molecular work on AE expression in the choroid plexus complements the aforementioned functional studies. Lindsey et al. (1990) used in situ hybridization and immunocytochemistry to reveal AE2 expression in the rat choroid plexus epithelia, and more specifically on the basolateral membrane (see Fig. 4). Similar expression in the lateral and fourth ventricle choroid plexus of rat was later shown by both Alper et al. (1994) and Wu et al. (1998). In support of earlier proposals (Johanson, 1984; Johanson and Murphy, 1990), there is evidence for the presence of an NBC. Based on short-circuit current measurements of frog chororid plexus, HCO2 3 movement from blood to CSF across the epithelium is dependent on Naþ and sensitive to ouabain, acetazolamide, furosemide, and stilbene derivatives 2 (Saito and Wright, 1983). Transepithelial HCO2 3 movement could involve a HCO3 þ conductance on the apical membrane (as suggested by the authors), and/or Na -coupled HCO2 3 transporters on either the apical or basolateral membranes. Using both in situ hybridization and immunohistochemical techniques, Schmitt et al. (2000) demonstrated the expression of NBCe1 in epithelial cells of choroid plexus, ependyma, and meninges of rat. The probes cannot distinguish between the three similar NBCe1 variants (see above). Labeling may therefore reflect the presence of any one (or more) of the variants. As shown in Fig. 4, an acid-loading NBC on the apical membrane may secrete HCO2 3 into the CSF. Based on reported transmembrane Naþ and HCO2 3 gradients on the apical membrane of the epithelium, we estimate that an NBC with a 1:3 Naþ:HCO2 3 stoichiometry would have a reversal potential of , 2 50 mV. Because the cells have approximately the same Vm ; it is not unreasonable to suggest that this NBC could mediate HCO2 3 secretion into the CSF. On the other hand, an acid-extruding NBC with a 1:2 stoichiometry—or a 1:1 stoichiometry for that matter—on the basolateral membrane could also contribute to transepithelial HCO2 3 movement into the CSF. On the basolateral membrane, such an 2 NBC would transport HCO2 3 into the epithelial cell. Subsequently, the HCO3 could exit 2 on the apical membrane through HCO3 -conducting anion channels (Saito and Wright, 1984; also see Speake et al., 2001). Clearly, further functional and molecular studies are required to determine the stoichiometry and location of NBCs and related proteins in these cells. The following is one model of pHCSF regulation by the choroid epithelium (see Speake et al., 2001; Nattie, 1998; Johanson, 1984; Johanson et al., 1985). The pH of the secreted CSF is regulated by the combined activities of NHE and AE on the basolateral membrane and possibly NBC on the apical membrane (Fig. 4). CA catalyzes the hydration of CO2 to Hþ and HCO2 3 . NHE on the basolateral membrane exchanges the newly formed intracellular Hþ for blood Naþ. The Naþ gradient that drives NHE is established by the Naþ pump on the apical membrane. AE on the basolateral membrane exchanges the newly 2 2 formed intracellular HCO2 3 for blood Cl . However, HCO3 might have another fate: secretion into the CSF by an NBC on the apical membrane that cotransports Naþ and 2 HCO2 3 . Alternatively, HCO3 -conducting anion channels on the apical membrane could 2 allow HCO3 entry into the CSF. Working in concert therefore, NHE and AE on the basolateral membrane move NaCl from blood to cell. Subsequently, the Na/K/Cl cotransporter, NBC, and probably Cl2 channels on the apical membrane move the NaCl from cell to CSF. The transepithelial movement of ions into the CSF drives the osmotic flow of water, probably through aquaporin 1 in the apical and maybe basolateral

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membranes (see Speake et al., 2001). The direction of transport by the Na/K/Cl cotransporter can vary. Keep et al. (1994) reported transport out of cells bathed in CO2/ HCO2 3 -buffered solutions, whereas Wu et al. (1998) reported opposite transport for cells bathed in the nominal absence of the physiological buffer. HCO2 3 appears to reduce one or more of the ion gradients (e.g., Cl2 or Naþ) necessary for inward transport (Speake et al., 2001). According to development studies, the choroid plexus of the neonatal rat (1 week old) displays poor secretory ability compared to that of the adult rat. In addition, the CSF pH and [HCO2 3 ] is more susceptible to changes in arterial pH elicited by acid –base disturbances (Johanson et al., 1976, 1988). As described above, acetazolamide elicits an increase in the pH and [HCO2 3 ] of choroid epithelial cells from adult rats. In contrast, the CA inhibitor has no effect on the pH of the cells from neonatal animals (Johanson et al., 1992). The neonatal data could be explained by low levels of either CA activity or specific acid – base transport mechanisms necessary for HCO2 3 secretion into the CSF. Examining the developmental profiles of acid –base transporters in the choroid plexus at both the functional and molecular levels will contribute to our identification and understanding of the specific processes involved in the regulation of CSF pH and [HCO2 3 ].

5.3. pH regulation by endothelial cells of brain capillaries Similar to the choroid plexus epithelia, capillary endothelial cells are polarized and express acid –base transporters on their basolateral (blood) and apical (ECF) membranes. The predominant acid – base transporter identified in these endothelial cells is the NHE. There is also some evidence for the presence of an AE. An NHE appears responsible for saturable 22Naþ uptake into rat brain following an intracarotid bolus injection, as shown by Betz (1983b). The uptake is amiloride-sensitive. In an accompanying study, the same investigator studied isolated microvessels from rat brain and reported amiloride-sensitive 22Naþ uptake that was stimulated by low pHi, and inhibited by extracellular Naþ, Hþ, Liþ and NHþ 4 (Betz, 1983a). The author concluded that 22 þ Na uptake by NHE probably occurs across the apical membrane because the collapsed lumen of the microvessels restricts access to the basolateral membrane. In an in vivo study documenting NHE activity, 22Naþ uptake across rat cerebral capillaries into the parietal cortex, pons-medulla, and CSF was reduced by either systemic acidosis or amiloride (Murphy and Johanson, 1989a). The combined results from these three studies are consistent with the presence of NHE on both the apical and basolateral membranes. Cultured cerebral endothelial cells from pig cells, with a steady-state pHi of , 7.2 in CO2/ HCO2 3 , also displayed amiloride-sensitive NHE activity as measured with BCECF (Hsu et al., 1996). According to amiloride-binding and RT-PCR studies, the Na –H exchanger in cerebral microvessels of the rat, pig and human is NHE-1 (Kalaria et al., 1998). A Cl –HCO3 exchanger may also contribute to the pHi physiology of cerebrovascular endothelial cells. For example, Smith and Rapoport (1984) performed in vivo studies on rats to examine 36Cl2 movement into the cerebrovascular endothelium following

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intravenous injection. The authors found that 36Cl2 uptake displays Michaelis– Menten saturation kinetics, consistent with a carrier-mediated process. A model of pHECF regulation by capillary endothelial cells is similar in some respects to that described above for the choroid plexus epithelium (see Nattie, 1998). For example, NHE and possibly AE are present on the basolateral membrane. The NHE functions as an acid extruder and the AE functions as an acid loader. The Naþ gradient that fuels the NHE is established by a Naþ pump. An NHE is also present on the apical membrane where it probably also functions as an acid extruder. As of yet, there is no evidence for an NBC in capillary endothelial cells.

6. Concluding remarks As we have reviewed in this chapter, the regulation of pHi and pHECF is paramount for proper brain function. Glial cells express an array of acid – base transporters that maintain pHi and contribute to pHECF regulation. In addition, cells of the choroid plexus epithelium and capillary endothelium use some of these same transporters to regulate both their own pHi and subsequently pHCSF and pHECF. As detailed above, there is a rich literature that highlights many of the functional and molecular properties of non-neuronal acid –base transporters. Additional functional and molecular studies will undoubtedly contribute to our understanding of the importance of individual acid –base transporters in the brain. Future functional studies include further characterizing the pHi physiology of microglia and NBC activity in oligodendrocytes, as well as identifying NBCs in the choroid plexus where they may mediate HCO2 3 secretion into the CSF. Future molecular studies include examining the expression profiles of NBCs and NDCBEs throughout the CNS. As found with the NHEs and AEs, NBC and NDCBE isoforms probably exhibit different expression profiles that depend on cell type, brain region, and stage of animal development. Cationcoupled HCO2 3 transporters can markedly influence neuronal physiology, as exemplified by the link between NBC activity and neuronal activity. The molecular information obtained on cation-coupled HCO2 3 transporters provides us the opportunity to use genetic approaches to characterize the function of these transporters in the CNS further. It would be particularly exciting, for example, to examine cellular pH physiology and neuronal firing patterns in an NBC or NDCBE knock-out mouse. The combination of cellular, molecular, and genetic approaches will be necessary to understand the complete physiology of individual acid –base transporters in the context of a functioning nervous system.

Acknowledgements We thank Dr Carmel M. McNicholas-Bevensee and Ms Jennifer B. Williams for reading the manuscript carefully and making suggestions. In addition, we thank Drs Mitchell Chesler and Conrad E. Johanson for helpful information and valuable comments. Dr Carmel M. McNicholas-Bevensee kindly obtained the neuronal action potential shown in Fig. 3.

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AVP effects and water channels in non-neuronal CNS cells Ye Chenp and Maria Spatza p

Correspondence address: Department of Operational and Undersea Medicine, Naval Medical Research Center, 503 Robert Grant Ave., Silver Spring, MD 20910, USA E-mail: [email protected] a Stroke Branch, NINDS, National Institutes of Health, 36 Convent Drive, MSC 4128, Bethesda, MD 20892-4128, USA

Contents 1. 2.

3.

4.

Introduction Ion and water homeostasis in non-neuronal parenchymal cells 2.1. Cerebrocortical astrocytes 2.2. The hypothalamo-hypophysial system Brain barrier functions in ion and water homeostasis 3.1. Capillary endothelium 3.2. Choroid plexus 3.3. Ependyma and tanycytes Concluding remarks

Arginine vasopressin (AVP) is a nona-peptide, which is generally considered as an antidiuretic and vasoconstrictive hormone. It is mainly synthesized and released from the hypothalamo-neurohypophysial system, but it has also been recognized to play a crucial role in the regulation of water and ion homeostasis in the brain. The responses to AVP by cerebral non-neuronal cells have been characterized as follows, according to cell type, function and mechanism. (1) Astrocytes and specialized astrocytes: In cerebrocortical astrocytes AVP regulates water content and cell volume changes induced by hydroosmotic challenges through V1b/V3 receptors by increasing water permeability. Circumventricular astrocytes and pituicytes regulate AVP secretion and release by altering the spatial relationship between neurons and their adjacent astrocytes. In addition, attention has been drawn to the role of putative modulators (specially endothelins), which could be involved in modulation of AVP functions in neuronal and non-neuronal cells. (2) Capillary endothelium and choroid plexus epithelium: These cells are the main cellular components of the blood – brain barrier (BBB) and blood – cerebrospinal fluid (CSF) barrier. AVP regulates ion movements between blood and brain across BBB to control Advances in Molecular and Cell Biology, Vol. 31, pages 747–771 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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extracellular potassium concentration and water content in brain. This process includes activation of Naþ,Kþ,2Cl2 cotransporter activity at the luminal membrane of capillary endothelial cells and opening of Kþ channels at their abluminal membrane, secondary to an AVP-induced stimulation of endothelin (ET) release. AVP also regulates ion movement across the blood – CSF barrier to control of CSF formation by decreasing Cl2 secretion in the epithelial cells of the choroid plexus. Moreover, AVP regulates water movement between CSF and brain by altering the water permeability in ependymal cells. Although it is known that water channel proteins, aquaporins, are widely distributed in non-neuronal cells, the capacity of AVP to affect aquaporins is yet unknown.

1. Introduction Water and ion homeostasis of the central nervous system (CNS) is of profound importance, both physiologically and under pathological conditions, when brain edema rapidly may become life-threatening, because the brain is encaged within the rigid skull with no possibility for expansion. The presence of a vasopressinergic neuroendocrine system, regulating water transport in the CNS was first proposed by Raichle (1981), and over the past two decades it has become evident that vasopressin (AVP) does have such a role in addition to its well-known regulation of whole-body water homeostasis (Chen et al., 1992; Hertz et al., 2000a; Niermann et al., 2001). AVP is synthesized in the hypothalamus, primarily by magnocellular neurons (see chapters by Mercier and Hatton and by Salm et al.), located in the supraoptic nucleus (SON) and paraventricular nuclei (PVN). It is released into the systemic circulation, where it exerts multiple hormonal effects, most notably regulating water reabsorption by the kidney. AVP released into the systemic circulation does not easily cross the blood –brain barrier, but physiologically significant amounts of AVP are also released into the brain and into the cerebrospinal fluid (CSF) (Ludwig, 1995; de Vries and Miller, 1998). Centrally released AVP is involved in control of brain water homeostasis (Raichle and Grubb, 1978), brain edema (Doczi et al., 1988), CSF formation (Davson and Segal, 1970), and secretion of pituitary peptides (Segal and Zlokovic, 1990). Among non-neuronal cells, astrocytes, capillary endothelium, and epithelial cells of the choroid plexus, as primary targets of AVP, play important roles in maintenance of physiological brain volume by changing water permeability and by modifying ion transport (Hertz et al., 2000a). Moreover, the specialized astrocytes in circumventricular organs (CVOs), including pituicytes play important roles in regulating AVP release into the general circulation. In this chapter, the current status of knowledge regarding effects of AVP on cellular water content and ion transport system in non-neuronal cells of the CNS will be discussed together with their response to changes of extracellular tonicity. Attention will be drawn to the role of some neuromodulators and/or neurotransmitters in the regulation of AVP release by specialized astrocytes. The possibility that AVP acts on water channels, aquaporins (AQP), in non-neuronal cells will also be considered, although the existence of such a functional relationship is still uncertain.

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2. Ion and water homeostasis in non-neuronal parenchymal cells 2.1. Cerebrocortical astrocytes 2.1.1. Ion uptake Neuronal activity triggers net ion fluxes between different cellular and extracellular compartments. An increase of extracellular Kþ concentration ([Kþ]e) occurs in the brain physiologically as a response to neuronal activity and to an even larger degree pathologically during seizures, brain ischemia, and other insults (Sykova, 1983; Walz and Hertz, 1983; Hertz et al., 2000b—see also chapter by Walz). To ensure an appropriate neuronal environment, increased [Kþ]e must be removed by redistribution between cells or by uptake into cells. Astrocytes play a crucial role in clearing [Kþ]e by passive and/or active mechanisms (Hertz, 1990). Except over short distances, or long times, the diffusion through the extracellular space is of minor importance. The so-called ‘spatial buffering’, a rapid redistribution of a local increase in [Kþ]e through an astrocytic syncytium or along individual Mu¨ller cells in the retina, taking up Kþ through Kþ channels in regions of high Kþ and liberating it in low Kþ regions, has been considered important for Kþ redistribution in the brain. It exerts such a function in the retina (see chapter by Bringmann et al.), but it probably plays a limited role in redistributing elevated [Kþ]e in the CNS under normal conditions (see chapter by Walz), and it cannot increase Kþ inside the cells, as equal amounts of Kþ enter and leave the syncytium (Amedee et al., 1997). Nevertheless, it can contribute to changes in the magnitude of the extracellular space secondary to ion redistribution (Dietzel et al., 1980; Witte et al., 2001). Two mechanisms exist by which the intracellular Kþ concentration in astrocytes increases. One is passive uptake of KCl through ion channels. The driving force for such an uptake is Donnan forces, and the uptake is crucially dependent upon a high membrane permeability for not only Kþ but also chloride ions (Cl2). This mechanism plays probably also at most a minor role in the normal CNS, whereas it is important in reactive astrocytes (Walz, 2002—see also chapter by Walz). In addition, astrocytes actively take up Kþ by activation of the Naþ,Kþ-ATPase and the Naþ,Kþ,2Cl2 cotransporter. The Naþ,Kþ-ATPase is known to be stimulated by Kþ at its extracellular site and by þ Na at its intracellular site (Sweadner, 1995). The affinity of the astrocytic Naþ,KþATPase is low (high Km ), so that its activity is stimulated by increases in [Kþ]e within the physiological and pathological range (Grisar et al., 1979; Mercado and Hernandez, 1992; Hajek et al., 1996). In contrast, the neuronal Naþ –Kþ-ATPase has a high affinity for extracellular Kþ, so that the Kþ-sensitive site is saturated at normal values of [Kþ]e and not affected by an increase (Grisar et al., 1979; Sweadner, 1995; Hajek et al., 1996). However, Naþ,Kþ-ATPase-mediated Kþ uptake in neurons can be activated by intraneuronal increase in sodium ions (Naþ) resulting from neuronal excitation. Moreover, at low [Kþ]e, e.g., during the so-called undershoot following neuronal excitation (Heinemann and Lux, 1975), the neuronal Naþ,Kþ-ATPase might also be stimulated at its Kþ-sensitive site, and due to its higher affinity it may favor neuronal uptake at lower [Kþ]e, enabling a gradual return to neurons of Kþ that initially was accumulated by astrocytes and subsequently released to the extracellular space (Hajek et al., 1996; Hertz et al., 2000a).

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A substantial part of the Kþ clearance from the extracellular space is not directly dependent upon the Naþ,Kþ-ATPase, but occurs via a Naþ,Kþ,2Cl2 cotransporter system (Walz and Hertz, 1984; Tas et al., 1987; Su et al., 2002a,b). In vivo expression of the Naþ,Kþ,2Cl2 cotransporter protein in rat astrocytes has recently been reported (Yan et al., 2001). The Naþ,Kþ,2Cl2 cotransporter, which operates in the inward direction in astrocytes, provides not only Kþ uptake, but also uptake of Naþ, which in turn stimulates the Naþ,Kþ-ATPase at its intracellular Naþ-sensitive site, triggering efflux of accumulated Naþ and stimulation of energy metabolism (see chapter by Hertz, Peng et al.) The joint operation of the Naþ,Kþ-ATPase and the cotransporter makes the Kþ uptake very effective and essentially mediates combined uptake of Kþ and of Cl2. The cotransporter is inhibited by loop diuretics, such as furosemide and ethacrynic acid, and more selectively by bumetanide, and it is stimulated by elevated [Kþ]e (Walz and Hertz, 1984; Su et al., 2002b). Joint stimulation of the Naþ,Kþ-ATPase/cotransporter in astrocytes and of the Naþ,Kþ-ATPase in neurons following neuronal excitation may contribute to the undershoot in [Kþ]e following neuronal excitation (Hertz et al., 2000a). Kþ uptake into astrocytes is not stimulated by AVP (Chen et al., 1992). A stimulation of Cl2 uptake by AVP is inhibited by bumetanide (Katay et al., 1998), indicating that it is secondary to uptake by the cotransporter. However, this stimulation does not become apparent immediately, suggesting that it is not a direct effect on cotransporter activity but rather secondary to osmotically induced cell swelling. 2.1.2. Fluid spaces Uptake of Naþ, Kþ, and Cl2 in cells leads to an osmotically driven uptake of water, i.e., swelling of the cells. Astrocytes exposed to high [Kþ]e with concomitant reduction of the extracellular Naþ concentration showed a significant increase in water content (Del Bigio and Fedoroff, 1990; Chen et al., 1992; Su et al., 2002b), which can be abolished either by the cotransporter inhibitor bumetanide or by cotransporter deletion (Naþ,Kþ,2Cl2 cotransporter 2 /2 mice) (Su et al., 2002a,b). The increase in water content reflects uptake of Naþ, Kþ, and Cl2 together with osmotically obliged water. In astrocytes, but not in neurons, the Kþ-induced increase in water content is greatly enhanced (50% of nonstimulation water content) by exposure to AVP (Fig. 1). Since the AVP-induced swelling is not caused by further stimulation of Kþ uptake (see above), it was concluded that it was due to a facilitation of water uptake (Chen et al., 1992), as has been directly demonstrated by Latzkovits et al. (1993). No corresponding effect was found at normal [Kþ]e, indicating that AVP facilitates rapid vectorial water movement driven by osmotic gradients (Fig. 2). The effect of AVP is potent, with a distinct enhancement of the Kþ-stimulated swelling at 10212 M AVP. AVP also increases free cytosolic Ca2þ concentration ([Ca2þ]i) in primary cultures of cerebrocortical astrocytes (Chen et al., 1992; Torday et al., 1997), identifying the receptor as a phospholipase C-linked V1 receptor. This effect is independent of extracellular Ca2þ (Chen et al., 2000), suggesting that it may be evoked by stimulation of a V1b/V3 AVP receptor, rather than of the V1a receptor that depends upon Ca2þ influx (Thibonnier et al., 1997). An AVP-mediated increase in [Ca2þ]i in cortical astrocytes was ‘rediscovered’ by Zhao and Brinton (2002), stating that theirs was the first description of this phenomenon,

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Fig. 1. Water content in primary cultures of mouse astrocytes, measured by aid of [14C]urea as described by Walz (1987), after 30 min of incubation in tissue culture medium at 37 8C under control conditions (black column), exposure to 60 mM Kþ in the incubation medium (hatched column) and exposure to vasopressin (AVP) under control conditions (white column) and in the presence of the elevated Kþ concentration (gray column). The water content of 4 mL/mg protein under control conditions (exposure to 5 mM Kþ in the medium) is unaltered in the presence of 10212 M vasopressin. After incubation at the elevated Kþ concentration (with concomitant reduction in the Naþ concentration) the water content is slightly, but significantly, increased. Incubation in the joint presence of elevated Kþ and 10212 M AVP leads to a large and significant additional increase in the water content, which reaches almost 7 mL/mg protein. No corresponding effect of AVP was seen in neuronal cultures (Chen et al., 1992).

and adding the new information that nuclear Ca2þ is also increased by AVP. However, these authors concluded that the receptor was a V1a receptor, a difference from the results by Chen et al. (2000), which may reflect that the astrocytes they studied were relatively immature. The effect of AVP on water permeability in astrocytes has been further studied during exposure of cultured astrocytes to severe hypotonic conditions, a non-physiological condition, which leads to rapid swelling, followed by a regulatory volume decrease (Sarfaraz and Fraser, 1999). In the presence of AVP, astrocytes exposed to hypotonic medium increase water space more than 50% at 5 min, at a time when cells in the absence of AVP had achieved complete regulatory volume decrease. This effect was inhibited by a subtype non-specific V1 antagonist, whereas a V2 agonist had no effect. Accordingly, although the effect of AVP on water permeability in the kidney is mediated by a V2induced increase in cAMP, all authors agree that AVP’s volume-regulatory effect in cortical astrocytes is triggered by one of the two V1 receptors (V1a and V1b/V3) that are linked to the phosphatidyl inositide second messenger system, increasing [Ca2þ]i (Chen et al., 1992; Sarfaraz and Fraser, 1999; Zhao and Brinton, 2002). Moreover, the AVP mediated increase in Cl2 uptake mentioned above is obviously secondary to AVP-induced swelling, and it was also inhibited by a V1 antagonist (Katay et al., 1998). Recently the effect of AVP on activity-dependent water flux has been studied in cerebrocortical brain slices (Niermann et al., 2001). The slices were incubated in lowchloride incubation medium in order to prevent participation of the Naþ,Kþ,Cl2

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Fig. 2. Effects of AVP on Naþ,Kþ,2Cl2 cotransporter activity and/or vectorial H2O movement created by the ion transport (as osmotically obliged H2O) in astrocytes, brain capillary endothelial cells, and choroid plexus epithelial cells. In astrocytes, Kþ-stimulated cotransporter-mediated ion uptake is independent of AVP, but movement of obliged water is enhanced by AVP; Kþ-induced cell swelling is accordingly increased by AVP. In endothelial cells, H2O permeability is unaffected by AVP, but cotransporter-mediated ion uptake at the luminal surface is stimulated by AVP, creating a demand for movement of osmotically obliged water; ion and water release across the abluminal surface is mediated by opening of ion channels, as indicated for Kþ, which in order to maintain electroneutrality is likely to travel with an anion [probably Cl2]; accordingly uptake of water and ions from the systemic circulation into brain is enhanced by AVP. In choroid plexus epithelial cells, H2O permeability and cotransporter activity are unaffected by AVP, but channel-mediated uptake of Cl2 (in exchange with HCO2 3) from the systemic circulation at the basal surface is decreased, reducing intracellular availability of Cl2 for transport by the cotransporter at the apical surface, which in these cells normally functions in the outward direction; accordingly secretion of Cl2 and of H2O is reduced. If uptake of H2O (and ions) in all three cell types is stimulated simultaneously, there will be an increase in water and ion contents in brain parenchyma, which is largely intracellular (although the accumulated ions and water originate from the systemic circulation), and this increase will be compensated for by a decrease of ion contents and amount of fluid in CSF, secondary to a decreased secretion by the choroids plexus.

cotransporter, and under these conditions evoked neuronal activity generates a rapid radial water flux in the slices, which at least partly may be due to the operation of the spatial buffer (Witte et al., 2001). AVP and V1a receptor agonists facilitated the water flux, whereas it was reduced by a V1 antagonists even in the absence of added agonist, suggesting a tonic vasopressinergic input. Due to the rapidity and high capacity of the flux it was concluded that it in all probably occurred as a result of modulation by vasopressin of aquaporin-mediated water flux through astrocytic membranes. Astrocytes express the water channels, AQP4 and 9 (Nielsen et al., 1997; Elkjaer et al., 2000; Baduat et al., 2001). Highly polarized AQP4 expression is found in astrocytic foot processes near, or in direct contact with capillaries. AQP4 expression is also very pronounced in glia limitans (Nielsen et al., 1997). Although AQP4 deletion has no affect on general behavior, neuromuscular co-ordination, or response to sensory stimulation in AQP-null mice (Ma et al., 1997), the deletion is associated with greatly reduced cerebral edema in response to water intoxication and stroke (Manley et al., 2000), implying a key role of AQP4 in the development of brain edema. It has been shown that AQP4 is not AVP-sensitive in the kidney collecting duct (Dibas et al., 1998), but since the AVP receptor activating water flux in the kidney is an adenylate cyclase-associated V2, and

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AVP effects on astrocytes are exerted on the phospholipase C-associated V1 receptor, this does not necessarily mean that the astrocytic AQP4 could not be stimulated by AVP. It is consistent with this point of view that the channel is regulated by protein kinase C (PKC) (Han et al., 1998; Nakahama et al., 1999). Because the promoter activity of AQP4 has not been analyzed, the precise regulatory mechanisms remain unknown (Venero et al., 2001). However, AQP4 is known to contain three putative phosphorylation sites, and it was demonstrated by Niermann et al. (2001), that AVP lost its ability to facilitate water flux in brain cortex slices after treatment with either an inhibitor of PKC or thapsigargin, which by inhibiting refill of intracellular Ca2þ stores (Norup et al., 1986) prevents the increase in [Ca2þ]i evoked by stimulation of phospholipase-C associated receptors such as the V1 receptor. It was concluded that both PKC and Ca2þ are involved in the facilitation of water redistribution, and that PKC may act either by phosphorylation of the channel itself or by phosphorylating other components of the signal transduction pathway (Niermann et al., 2001). The latter possibility may appear more likely, since phosphorylation of AQP4 may lead to a decrease in channel activity (Han et al., 1998). AQP4 is tethered to perivascular astrocytic endfeet by binding of its C terminus to the PDZ domain of synthrophin, a component of the dystrophin complex, and its expression in astrocytic membranes facing the neuropil is considerably lower (Neely et al., 2001). Its total expression in brain is normal in dystrophin null mice, but the normal cellular polarity is disturbed, with AQP expression being markedly reduced in perivascular astrocyte endfeet, but present at higher than normal levels in astrocyte membranes facing the neuropil. Another aquaporin, AQP9, is found on astrocytic processes and cell bodies. Similarities of distribution pattern within the brain between AQP4 and 9 in rodents suggest that the two proteins mediate common functions, and that they can act in synergy (Badaut et al., 2002). Recent studies, determining gene expression by reverse transcription polymerase chain reaction, have demonstrated that astrocytes also express mRNA for AQP3, 5, and 8 (Yamamoto et al., 2001). The physiologic role of these proteins remains to be determined.

2.2. The hypothalamo-hypophysial system 2.2.1. Astrocytes in the hypothalamo-hypophysial system As described elsewhere (see chapters by Mercier and Hatton and by Salm et al.) AVP and oxytocin are produced in magnocellular and parvocellular neuronal somata, respectively, of hypothalamic nuclei like the SON, the PVN, both of which contain mainly AVP-secreting neurons, and the arcuate nucleus, containing oxytocin-secreting neurons. The fibers terminate in the posterior, neural lobe of the pituitary (the neurohypophysis). In response to appropriate stimuli, astrocytes undergo significant morphological changes, possibly triggered by stimulation of b-adrenergic receptors expressed by SON astrocytes (Lafarga et al., 1992), as will be discussed below. Astrocytic membranes covering neuronal somata and dendrites withdraw and neuronal surfaces become directly apposed to each other (Tweedle and Hatton, 1977; Theodosis et al., 1981; Beagley and Hatton, 1994). The resulting cessation of astrocytic removal of neuronally released Kþ and glutamate as well as the absence of astrocytical release of the inhibitory

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transmitter taurine enhance neuronal stimulation (van den Pol et al., 1990; Mecker et al., 1993; see also chapters by Mercier and Hatton and by Salm et al.). When hormone demand returns to basal levels, astrocytic processes are once again observed between the neuronal elements (Hatton et al., 1984). In the neurohypophysis, specialized astrocytes, the pituicytes, envelop axons and terminals of vasopressin- and oxytocin-secreting neurons and their processes occupy portions of the basal lamina, constituting a barrier between neuronal terminals and capillaries (Wittkowski, 1986). During stimulation of hormonal secretion, release of engulfed axon and retraction of pituicyte endfeet from the vascular surface favor release of AVP and of oxytocin into the general circulation. AVP is not only released from axonal terminals but also from the dendrites of magnocellular neurons in the SON during its activation (Ludwig, 1995). Cultured astrocytes from SON express AVP receptors, which appear to be of V1b/V3 subtype (Hatton, 1997). Pituicytes respond to AVP with an increase in [Ca2þ]i in the absence of extracellular Ca2þ (Hatton et al., 1992), which identifies also this receptor as being of the V1b subtype. This is consistent with an immunocytochemical study, which showed that pituicytes express V1b receptors (Hernando et al., 2001). AVP induced [Ca2þ]i elevation in pituicytes may serve as an inhibitory mechanism of peptide release in neural lobe terminals (Hatton, 1999) by spread of Ca2þ waves from glia to neurons (see chapter by Cornell-Bell et al.). Induction of increased [Ca2þ]i in the terminals by the closely apposed astrocytes might inactivate Ca2þ channels and inhibit further release. In contrast to most brain areas, where astrocytic AQP4 protein localization is highly polarized, AQP4 in SON and PVN is evenly distributed over the membrane of astrocytes, notably in lamellae in direct apposition with neuronal elements (Nielsen et al., 1997). This distribution suggests a particular functional significance of regulation of water movement through astrocytes. Considering that SON astrocytes reside at an interface between extraparenchymal fluid (CSF and blood) and magnocellular neurons, and that astrocytes undergo morphological changes in response to osmotic stimulations, it is possible that astrocytes in SON may sense the surrounding extracellular tonicity and act as ‘vesicular osmometers’ (Wells, 1998). If AVP increases water permeability in these cells as in other astrocytes, this might facilitate such a function.

2.2.2. Astrocytes in afferent nuclei Two CVOs, the subfornical organ (SFO) and the organum vasculosum lamina terminalis (OVLT) send direct projections to SON and PVN. Since CVOs are devoid of a blood –brain barrier and are in direct contact with both plasma and CSF, cells in CVOs are ideally placed to readily sense any change in extracellular fluid composition. Astrocytes in SFO and OVLT express AVP receptors and the receptor subtype proved to be V1b, because AVP induced [Ca2þ]i increase in cultured astrocytes from SFO and OVLT is Ca2þ-independent (Jurzak et al., 1995). The presence of functional AVP receptors in CVOs may reflect CNS control of peripheral AVP level. AQP4 is densely expresses in SFO (Venero et al., 1999). Astrocytes from SFO and OVLT also express receptors for ETs, and binding sites for both the ETA and the ETB receptor were demonstrated by autoradiography. In addition ET-3 induced an intracellular Ca2þ transient through ETA

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receptors, whereas ET-1-stimulated Ca2þ mobilization was mediated by ETA and ETBreceptors (Gebke et al., 2000). ETs, whether produced locally or circulating, may participate with AVP to maintain extracellular fluid balance, as will be discussed below. 2.2.3. Putative modulators The likelihood of neuronal – glial interaction has been substantiated by the observed formation of synaptic contact between axonal terminals containing various neurohormones/neurotransmitters (i.e., AVP, oxytocin, enkephalin, adrenaline, serotonin, and GABA) and their respective receptors expressed on the pituicytes in the neurohypophysis. Some of these substances are co-localized or localized in adjacent neuronal and non-neuronal tissues. Moreover, circulating substances derived from peripheral organs may reach the AVP and oxytocin releasing brain areas due to the absence of blood – brain barrier (pituitary) or localization in close proximity to a large vascular bed (SON glia), as illustrated in the chapter by Mercier and Hatton. Based on recent in vivo and in vitro studies, evidence has been accumulated indicating that catecholamines, ATP (endogenous and exogenous) and its metabolite adenosine, as well as cardiovascular peptides may play a role in regulating the secretion of AVP and oxytocin and their control of water homeostasis (Ritz et al., 1992; Wall and Ferguson, 1992; Yamamoto et al., 1992; Lange et al., 1994; Rossi et al., 1997). Catecholamines. SON receives a rich catecholaminergic innervation (McNiel and Sladek, 1980) and (as mentioned above) the SON astrocytes express b-adrenergic receptors (Lafarga et al., 1992). The involvement of a catecholaminergic system in the modulation of SON and pituitary astrocytes was suggested by osmotically stimulated increase in number of these receptors that was associated with a change in astrocyte morphology (Beagley and Hatton, 1994). The absence of osmotically induced astrocytic retraction in adrenalectomized animals indicated that catecholaminergic signals from the adrenal gland could also directly or indirectly modulate the function of the hypophysiohypothalamic system. In support of this contention, morphological changes were observed in cultured pituicytes exposed to adrenergic agonist, which altered their shape from flat polygonal to stellate. This event was mimicked with 8-bromo cAMP, a permeable cAMP analog or forskolin (an activator of adenylate cyclase). The effect of this treatment of the pituicytes suggests an involvement of cAMP-mediated process in modulation of pituicyte morphology. Thus, cAMP signal transduction induced by catecholamine or other neurotransmitters/neurohormones in pituicytes may influence the neuronal release of AVP and/or oxytocin from the neurohypophysis (Miyata et al., 1999). ATP and adenosine. ATP is co-stored with neuropeptides in the secretory granules of the neurohypophysial nerve endings, and an ectoATPase terminating the extracellular action of ATP is localized in the same organ. The ATP, which is released along with AVP and oxytocin by electrical stimulation of the neurohypophysis, may have a role in regulation of AVP secretion via P2X2-purine receptor. The effects of ATP on AVP secretion have been shown to be both stimulatory and inhibitory (Troadec et al., 1998; Sperlagh et al., 1999). Lemos and Wang have suggested that endogenous and exogenous ATP exert opposite effects on AVP secretion in neurohypophysial terminals (Lemos and Wang, 2000). Alternatively, the conflicting results of ATP could be explained by assuming

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that ATP, co-released with AVP, initially stimulates further release of AVP, whereas adenosine, which is rapidly formed by hydrolysis of ATP, could then act through A1 receptor to decrease the release of AVP (Wang et al., 2002). As mentioned above, the morphologic plasticity of astrocytes and pituicytes, demonstrated in vitro studies, mirror the in vivo depicted changes in these cells during hypothalmamo-hypophysial hormonal secretion. Therefore, the latest reported studies focused on the responses and mechanisms responsible for the morphologic changes in the pituicytes induced by either ATP or adenosine (Rosso et al., 2002a,b). ATP was demonstrated to elicit stellation of pituicytes (Fig. 3, lower left part). This effect is due to the ATP metabolite, adenosine, and it is mediated by A1-receptors and independent of intracellular Ca2þ and mitogen protein kinase pathway. It involves the adenosine-induced down-regulation of Rho A activity associated with F-actin depolymerization and reorganization of microtubular filaments in

Fig. 3. Upper part: Schematic illustration of proposed synergistic and antagonistic effects between AVP and ETA and ETB receptor stimulation on AVP secretion from the neurohypophysis and on astrocyte stellation. AVP secretion from magnocellular neurons in the SON is inhibited by stimulation of ETA receptors on these cells, but it is enhanced by stimulation of ETB receptors on AV3V cells, leading to efferent glutamatergic stimulation of NMDA receptors on the magnocellular neurons. In addition, AVP release from the neurohypophysis is enhanced by stimulation of pituitary ETA receptors. Lower left part: Mechanisms involved in adenosine- and cAMPmediated stellation of astrocytes (stress fiber (actin) depolymerization and decreased tyrosine phosphorylation of the focal adhesion associated proteins FAK and paxillin). Oppositely directed reactions (enhanced polymerization of actin and inhibited tyrosine dephosphorylation) are presumably involved in the conversion of stellate astrocytes to flat, polygonal astrocytes by endothelins (ETs) and AVP.

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the pituicytes. Rho, a member of small GTPase proteins, was shown to control the basal (flat—nonstellate) state of cultured pituicytes and glial cells. In addition, it was found that AVP and oxytocin prevented the adenosine-induced stellation of pituicytes (Fig. 3, lower left part). This effect was mediated by a V1a-receptor mechanism and entailed Ca2þdependent activation of Cdc42, another small GTPase protein linked to alterations of the cytoskeleton (Rosso et al., 2002b). It appears therefore that the signal transduction pathway taking part in altering the adenosine-elicited structural features of pituicytes differs from that described to prevent it. The AVP and oxytocin prevention of adenosine-induced stellation of pituicytes is indicative of an existing potential negative feedback mechanism provided by these substances. It is still unknown whether morphologic responses of hypothalamic astrocytes to adenosine alone or in the presence of AVP and oxytocin is similar to those of pituicytes. Nevertheless, the morphologic observations in vitro support the concept of purinergic regulation of functional plasticity in pituicytes, which may be involved in modulation of hormonal (AVP and oxytocin) release occurring during physiological stimulation. Endothelin. The ET family of peptides is known to exert various and diverse biological effects on many organs, tissues and cell types through activation of ETA and ETB receptors (Rubanyi and Polokoff, 1994). Therefore it is conceivable that ET-elicited action and interaction with other peptides may play a crucial regulatory role in the function of a given organ. In the brain, ETs were shown to affect neuronal AVP secretion irrespective of whether they had been produced centrally or locally. However, little is known about the effects of ETs on hypothalamo-hypophysial astrocytes. Therefore the information presented below pertains to localization of ETs and their involvement in regulating neuronal release of AVP, which may have ramifications for the functional participation of astrocytes and pituicytes in these endeavors. Endothelin-1 (ET-1), the most potent vasoconstrictor, is a member of 21-amino acid family (ET-1,2,3) originally isolated from porcine aortic endothelium. Subsequently, ET-1 and ET-3 were localized in various organs including CNS (Takahashi et al., 1991; Nakamura et al., 1993; Rubanyi and Polokoff, 1994; Gajkowska and Viron, 1997; Lange et al., 2002). In the brain, ET peptides, ET mRNA, ET1-converting enzymes, and ET-1 receptors have also been identified in non-vascular tissues, and they may be derived from neurons as well as non-neuronal cells (astrocytes, endothelial cells). Particularly, ET-1 and ET-3 were localized within the region involved in the secretion of AVP and in the control of body fluid homeostasis (i.e., neurohypophysis, SON and anteroventrical (AV3V) neurons, projecting to the SON (Wall and Ferguson, 1992; Yamamoto et al., 1995). An increased AVP plasma content was reported after intravenous or intracerebroventricular administration of ET-1 or ET-3 (Beagley and Hatton, 1994). These observations suggested activation of SON and paraventricular neurons by ETs, reaching these areas and/or AV3V neurons from the systemic circulation. A reported reversible ET-3 induced inhibition of water reabsorption in rats and the attenuation of the ET-3 induced increase in AVP plasma levels by brain natriuretic peptides is indicative of peptidergic interactive effects (Makino et al., 1992; Yamamoto et al., 1995). These findings also suggest that endogenously released ETs may play a role in the central control of fluid and electrolyte homeostasis. A potentiating effect of ET-1 and ET-3 on AVP secretion, observed in Kþ-depolarized isolated nerve endings, implicated involvement of the ETs as an autocrine regulator.

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However the reports concerned with mechanism and mediation involved in the AVP release are somewhat contradictory, dependent on the specific region studied in the compartmentalized hypothalamo-neurohypophysial explants. Thus, the accumulated experimental data indicate that the ET-1 inducible modulation of AVP secretion depends on the region and the subtype of ET-receptor activation. The ET-stimulated AVP release in neurohypophysis is mediated by ETA receptor and depolarization (Rossi, 1995), but the role of Ca2þ is still debatable. The demonstrated ET-effects on the hypothalamohypophysial region are rather complex (Rossi and Chen, 2002), as indicated in Fig. 3. Previous electrophysiologic studies demonstrated that ET directly inhibited the phasic firing of AVP neurons within SON. However, it stimulated the neuronal activity within the AV3V region, which contains projections to SON, and therefore excited the magnocellular neurons (Yamamoto et al., 1993). In hypothalamo-neurohypophysial explants, which included the AV3V region, Rossi and Chen (2002) lately demonstrated an increased AVP secretion from the neurohypophysis through pharmacological activation of ETB receptors in the hypothalamic area, probably in the AV3V region, whereas the stimulation of ETAreceptors on the vasopressinergic neurons of the SON inhibited the AVP release. Moreover, the stimulatory effect on AVP release induced by activation of the ETB receptor was shown to be mediated by hypothalamic NMDA-receptors, mediating the afferent stimulation from AV3V (Fig. 3). In addition, it was suggested that NMDA-activation may be associated with local release of GABA which in turn could decrease the AVP secretory response to ETB activation. A recent report by Miyata et al. (1999) indicated that ET-1 or ET-3 reversed the adenosine or cAMP-induced stellation of pituicytes in vitro (lower left part of Fig. 3). This observation implies that ETs known to be directly involved in the regulation of neuronal AVP release may also influence the AVP release through pituicyte activities. As far as the mechanism is concerned, the studies on pituicytes suggested participation of tyrosine phosphorylation in the pituicyte shape conversion, as it has been described in astrocytes. Until now, no other reports exist regarding the mechanism of ETs partaking in reversal of pituicyte stellation. Therefore, it is noteworthy to mention some of the factors entailed in altering astrocytic shape that might be relevant to those in pituicytes. In general, it appears that the mechanism involved in this event depends to a certain extent on the substances used for induction of cellular stellation (Goldman and Abramson, 1990; Koyama and Baba, 1994; Koyama and Baba, 1996; Padmanabhan et al., 1999). The observed common denominator for stellation is the reorganization of actin filaments due to its depolymerization, caused by various agents (i.e., cytochalasin, a disruptor of actin fibers, cAMP- or cAMP-mediated hormones). The association of tyrosine phosphorylation with focal adhesion molecules and involvement of tyrosine kinase was shown to be responsible for cAMP-induced changes in astrocytes. The stellation was manifested by decreased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin (focal adhesion associated proteins). This process seems to be downstream to Rho protein activity. On the other hand, ET-evoked prevention of astrocytic process formation was linked to induction of stress fibers (actin), ETB receptor-mediated tyrosine phosphorylation, and activation of Rho proteins (Miyata et al., 1999). It is therefore possible the same mechanism is involved in the reported ET-induced reversion of pituicyte stellation by ETs. Provided this is the case, and the same scenario occurs in vivo during a given physiological stimulus, it can be

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envisioned that retraction and recovery of pituicytes as well as of astrocytes, induced by AVP release involves a co-ordination of AVP and ET signals between neurons and glia in the hypothalamo-hypophysial region of the brain. 2.2.4. Adrenocorticotrops The anterior pituitary releases several hormones in response to hypohysiotropic hormones produced in the hypothalamus. Among these, the release of adrenocorticotropic hormone (ACTH) has repeatedly been found to be stimulated by application of AVP to preparations of isolated hormone-secreting cells (Jard et al., 1986; Tse and Lee, 1998; Livesey et al., 2000). The stimulation is associated with the evoked increase in [Ca2þ]i, which is temporally correlated with a burst of exocytosis and can be replaced by release of Ca2þ via flash photolysis of caged IP3 (Tse and Lee, 1998). The stimulatory effect is independent of extracellular Ca2þ, identifying the receptor as being of the V1b subtype, and the V1b subtype of the AVP receptor was first identified in pituitary corticotrops (Jard et al., 1986). In addition, systemic ET-1-induced stimulation of the hypothalamohypophysial – adrenal axis is mediated at the early stages by AVP and subsequently by AVP and corticotropin-releasing hormone, supporting the notion of interaction between ET-1 and AVP in a variety of endocrine systems (Malendowicz et al., 1998). 3. Brain barrier functions in ion and water homeostasis 3.1. Capillary endothelium Cerebral capillaries and microvessels have a very limited permeability to most plasma constituents, including Naþ and Kþ, due to tight junctions between the endothelial cells and specialized membrane properties (see chapter by Couraud et al.). This ‘blood– brain barrier’ is extremely important for neuronal function by preventing systemic changes in Kþ concentration from altering [Kþ]e in the brain. Thus, during hypo- and hyperkalemia, despite the change in plasma, Kþ concentration in brain is maintained constant by the regulation of flux of Kþ between blood and brain (Bradbury, 1979; Jones and Keep, 1988—see also chapter by Walz). A recent cytochemical study reveals localization of Naþ,Kþ-ATPase on both luminal and abluminal membranes (Manoonkitiwongsa et al., 1998). Consistent with this observation, a biochemical study of luminal and abluminal membrane vesicles derived from bovine brain endothelial cells after fractionation in a discontinuous Ficoll gradient led to the conclusions that although Naþ,Kþ-ATPase activity is primarily located on the abluminal membrane, approximately 25% of the activity is of luminal origin, and that different isoforms of the enzyme may be found at the two surfaces (Sanchez del Pino et al., 1995). During chronic dietary hyperkalemia, a significant decrease in the amount of the a3 subunit of Naþ,Kþ-ATPase may reflect a down-regulation of the luminal enzyme to reduce transportation of Kþ from blood to brain (Keep et al., 1999). In contrast to this evidence suggesting Naþ,Kþ-ATPase activity both luminally and abluminally, several studies have shown a distinct abluminal polarity of the enzyme, which possibly may be explained by a decrease of luminal, but not of abluminal, activity when certain fixation procedures are used (Manoonkitiwongsa et al., 2000).

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Similar to what has been found in astrocytes, increases in [Kþ]e above the normal resting level stimulate Naþ,Kþ-ATPase activity in microvessels (Schielke et al., 1990). Cultured brain microvessels and capillary endothelial cells express Naþ,Kþ,2Cl2 cotransporter proteins (Yerby et al., 1997) and exhibit Naþ,Kþ,2Cl2 cotransporter activity (Keep et al., 1994; Vigne et al., 1994; Sun et al., 1995; Kawai et al., 1995a,b). Although there is substantial evidence suggesting a luminal localization of Naþ,Kþ,2Cl2 cotransporters (Keep et al., 1993; Kawai et al., 1995b), in vivo direct cytochemistry for its existence on the luminal membrane is still lacking. The cotransporter mediates uptake of all three ions, and Ca2þ-dependent Kþ channels on the abluminal surface may mediate Kþ entry into the CNS (Fig. 2). Brain vascular cells express mRNA for the V1a receptor. AVP induced increase in [Ca2þ]i is dependent upon extracellular Ca2þ, and therefore by definition acting on a V1a subtype (Hess et al., 1991). AVP receptors have been identified at the luminal membrane of brain endothelial cells, and systemic administration of AVP antagonists of V1 subtype reduce vasogenic brain edema and Naþ accumulation (Rosenberg et al., 1990; Nagao et al., 1994). However, a reduction in blood-to-brain Naþ flux in AVP-deficient Brattleboro rat during cerebral ischemia is abolished by intraventricularly administrated AVP, but not by systemic treatment with AVP, suggesting that AVP receptors are also expressed abluminally (Dickinson and Betz, 1992). Along similar lines, intracereboventricular injection of AVP exacerbates acute ischemic brain edema (Liu et al., 1991). AVP enhances release of ET-1 in cultured endothelial cells (Spatz et al., 1993; Kawai et al., 1995a, 1997), and ET-1, in turn, stimulates Naþ,Kþ,2Cl2 cotransporter activity (Vigne et al., 1994; Spatz et al., 1994, 1997, 1998) and Naþ,Kþ-ATPase activity (Kawai et al., 1995b), which may enhance uptake of ions and osmotically obliged water into endothelial cells, as illustrated in Fig. 2. The effects of ET-1 on the ionic transport systems occur through activation of ETA receptors and phospholipase C (PLC), linked to intracellular Ca2þ and PKC. This may also be the reason why AVP-induced increase of Naþ,Kþ,2Cl2 cotransporter activity has been found to be secondary to a Ca2þ- and calmodulin-dependent phosphorylation of the cotransporter protein (O’Donnell et al., 1995). On the other hand, an ET-1 stimulated Naþ/Hþ exchange, coupled to ETA and PLC activation, is PKC independent and is partially mediated by tyrosine kinase and Ca2þ calmodulin (Kawai et al., 1995b). At the abluminal side, AVP and ET separately or possibly together mediates opening of Ca2þ-dependent Kþ channels (Keep et al., 1993; Van Renterghem et al., 1995). It is quite interesting that AVP has been found to induce the ET-1 gene in endothelium, although this has not yet been demonstrated in brain capillaries (Imai et al., 1992), and that astrocyte-conditioned medium increases cotransporter activity in endothelial cells (O’Donnell et al., 1995). It is therefore possible that an AVP/ET circuit takes part in a co-ordination of astrocytic and capillary event in order to maintain the intracellular and extracellular volume and ionic composition in the brain. AVP-activated Naþ,Kþ,2Cl2 cotransporter activity in endothelial cells may also be functionally co-ordinated with AVP-facilitated water uptake in astrocytes in general during normalization of resting [Kþ]e in brain after physiological activity. In the absence of AVP, astrocytic removal of Kþ and Cl2 without osmotically obliged water uptake, together with Naþ-stimulated neuronal Naþ/Kþ exchange after neuronal activity, lead to a post-excitatory decrease in [Kþ]e, i.e., an undershoot. AVP-mediated uptake of

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blood-borne Naþ, Kþ, and Cl2 in endothelial cells, together with the opening of Ca2þdependent Kþ channel on the abluminal membrane, may respond to this decrease by transporting Kþ from blood to brain and thereby normalize [Kþ]e (Hertz et al., 2000a,b). Thanks to the concomitant AVP-induced increase in water flux across the astrocytic cell membrane, such a transendothelial uptake of Kþ may cause [Kþ]e to rise above its normal level. In turn, the increase in [Kþ]e may stimulate the abluminally located Naþ,KþATPase, mediating uptake of Kþ across the abluminal endothelial membrane to normalize [Kþ]e. There are no indications that AVP directly affects water permeability in cerebral endothelial cells, possibly reflecting an absence of AQP protein in endothelial cells in the brain in situ (Nielsen et al., 1997). However, as discussed above, AQP4 is abundant in perivascular astrocytic endfeet, and astrocytic processes cover . 99% of the capillary surface in brain (see chapter by Wolff and Chao). This arrangement opens the possibility that at least part of the exchange of H2O between the systemic circulation and the brain may involve both transendothelial and transastrocytic processes. Moreover, it might explain the detection of mRNA and proteins of AQP4 and 9 in isolated rat cerebral microvessels and in cultured cerebral microvascular endothelial cells (Sobue et al., 1999; Kobayashi et al., 2001, 2002). In any case there is substantial reduction in brain edema following water intoxication by intraperitoneal water infusion in AQP null mice (Manley et al., 2000). This infusion leads to serum hyponatremia, which creates an osmotic gradient driving water into the brain as a ‘vasogenic brain edema’ (Klatzo, 1987). Nevertheless, the loss of pronounced polarity of AQP expression to the astrocytic endfeet in dystrophin null mice is not accompanied by a lack of ability to develop brain edema or a reduced mortality after intraperitoneal injection of distilled water and AVP, but the development of the edema is slightly delayed (Vajda et al., 2002). As summarized in legend of Fig. 2, simultaneous stimulation of astrocytes and of capillary endothelial cells in the brain will cause an increase in water and ion contents in brain parenchyma, which is largely intracellular, although the accumulated ions and water originate from the systemic circulation.

3.2. Choroid plexus The choroid plexuses secrete CSF and consist of villi containing a connective tissue stroma, which is a richly vascularized extension of the subarachnoid space, protruding into the ventricles and covered by epithelial cells connected by tight junctions (see chapters by Mercier and Hatton and by Weaver et al.). The epithelial cells are the site of the blood – CSF barrier (see chapter by Couraud et al.). In isolated choroid plexuses, Naþ,Kþ-ATPase and Naþ,Kþ,2Cl2 cotransporter are located on apical membrane of the choroid plexus epithelial cells (Masuzawa et al., 1984; Ernst et al., 1986; Keep et al., 1994; Klarr et al., 1997; Plotkin et al., 1997), as indicated in Fig. 2. The direction of the fluxes mediated by the cotransporter has been uncertain. Keep et al. (1994) suggested that they are involved in secretion of CSF, but Wu et al. (1998) found in vitro experiments that inhibition of this transporter by bumetanide caused cell shrinkage and therefore concluded that the cotransporter normally mediates inwards fluxes of the three ions. Recently, the pendulum

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has swung back to the concept that the cotransporter in vivo functions in the outward direction, partly accounted for by high inward concentrations of Naþ and Cl2, established by the operation on the basolateral membrane of Hþ/Naþ and bicarbonate/Cl2 exchangers, respectively (Murphy and Johanson, 1990; Garner and Brown, 1992), and driven by metabolic activity of the cells, producing CO2, which is dissociated to Hþ and HCO2 3 (Speake et al., 2001). Whereas the cotransporter and the Naþ,Kþ-ATPase both mediate Kþ uptake in astrocytes and in endothelial cells, the outward direction of Naþ, Kþ and Cl2 transport by the cotransporter is contrasted by a ‘normal’ inwardly directed Naþ,Kþ-ATPase, which may serve the purpose of regulating CSF Kþ concentration. In isolated preparations from adult animals this uptake has been found to increase in a stepwise fashion with each 2 mM increase in [Kþ]e, up to a 90% increase over control (3 mM [Kþ]e) with 9 mM [Kþ]e (Parmelee et al., 1991). In contrast, choroid plexus from 3-day-old animals increased uptake in 5 mM [Kþ]e, but could not increase Kþ uptake further with higher [Kþ]e, and kinetic analysis of the ouabain-inhibitable component suggested that the immature tissues may express a different isoform of the a-subunit of the Naþ,Kþ-ATPase. These data may explain an inability of neonatal rats to regulate CSF [Kþ]e, when serum [Kþ]e is elevated, and they indicate that active Kþ transport by the choroid plexus epithelial cells plays an integral role in the regulation of [Kþ]e in CSF. In contrast, in intact adult animals with increasing plasma [Kþ]e during dietary hyperkalemia, the a1 and b1 subunits of the Naþ,Kþ-ATPase were up-regulated as an indication of enhanced Kþ extrusion, whereas during hypokalemia, Naþ,Kþ-ATPase activity was decreased (Klarr et al., 1997). In addition to ion channels and carriers discussed above the choroid plexus epithelial cells are likely to express Kþ and HCO2 3 channels on their apical membrane (Speake et al., 2001), and on the basolateral membrane, a Naþ –Cl2 cotransporter has been identified, which may participate in Kþ exit into blood (Pearson et al., 2001). The concentration of AVP is several times higher in CSF than in plasma (Vorherr et al., 1968; Luerssen and Robertson, 1980; Reppert et al., 1982). Synthesis of AVP by choroidal epithelium close to the apical membrane has been demonstrated in situ (Chodobski et al., 1997, 1998b), and AVP release can be regulated by cAMP-dependent signaling, although other second messenger systems may also be involved (Chodobski and Szmydynger-Chobodska, 2001). Therefore, it is conceivable that changes in blood-borne AVP do not affect CSF formation under normal physiological conditions (Chodobski et al., 1998a), although morphological changes in choroid plexus epithelial cells mimicking those observed after intraventricular administration of AVP (see below) have been reported after systemic administration of AVP (Schultz et al., 1977), and circulating AVP is a potent vasoconstrictor of choroidal arterioles (Segal et al., 1992), which might secondarily decrease CSF production. Intracerebroventricular injection of AVP reduces blood flow in the choroid plexus and decreases CSF secretion (Faraci et al., 1988, 1990; Maktabi et al., 1993; Chodobski et al., 1998a) as well as transfer of 22Na from blood to CSF (Davson and Segal, 1970). AVP binding sites are abundant in choroid plexus epithelial cells (van Leeuwen et al., 1987; Tribollet et al., 1999), and they are both of the V1a and V1b subtype (Ostrowski et al., 1994; Burbach et al., 1995). In addition, V2 receptor mRNA was detected in the choroid plexus of newborn rats, but it was not detectable in the adult (Kato et al., 1995).

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Hypernatremia in rats increases the expression of both AVP and V1bR mRNA expression in choroid plexus, indicating the involvement of the V1b receptor in the regulation of ion and water homeostasis (Zemo and McCabe, 2001). A study of the effect of AVP on Cl2 efflux from acutely isolated choroid plexuses after previous labeling with 36Cl have shown that AVP potently reduces Cl2 efflux (Fig. 2) and increases the number of dehydrated epithelial cells by a V1 receptor-mediated effect (Johanson et al., 1999). The inhibitory effect of AVP on Cl2 efflux is probably due to þ þ interference with Cl2/HCO2 3 and Na /H exchange at the basolateral membrane. As a 2 þ result of slower uptake of Cl and Na , efflux of Cl2 efflux and thus of osmotically obliged fluid across the apical membrane into CSF decreases and many choroid plexus epithelial cells become dehydrated, which is consistent with the observed V1-dependent inhibition of CSF formation. An alternative explanation, i.e., that cotransporter-mediated efflux of Cl2 together with Naþ and Kþ should be inhibited is less likely, since it would lead to cell swelling rather than shrinkage. Immunocytochemical studies have shown that the aquaporin 1 (AQP1) protein is expressed in the apical membrane of the rat choroid plexus epithelium (Nielsen et al., 1997; Wu et al., 1998; Speake et al., 2003), and it is likely that it plays an important role in mediating water transport across this membrane during CSF secretion. A recent study has shown that AQP4 protein is also expressed in the choroid plexus of the fourth and the lateral ventricles of rat brain (Speake et al., 2003), but is diffusely distributed throughout the cytoplasma. The route by which water crosses the basolateral membrane remains to be established. The application of AVP to Xenopus oocytes injected with AQP1 cRNA increased the membrane permeability to water, implying that AQP1 may be an AVPregulated water channel (Patil et al., 1997). If this is the case, the AVP-mediated cell dehydration could reflect not only a reduced influx of Cl2 and Naþ, but perhaps also a more rapid flux of osmotically obliged water across the apical membrane together with the cotransporter-induced efflux of Naþ, Kþ, and Cl2.

3.3. Ependyma and tanycytes Ependymal cells line the cerebral ventricles of the brain and form the interface that separates the CSF and the brain (see chapter by Wolff and Chao). They may have functional roles in regulating the transport of ions, small molecules, and water between cerebrospinal and interstitial fluids (Bruni, 1998). Although the morphology is similar to that of most epithelial membranes, ependyma possess electrophysiological characteristics of glia cells (Bruni, 1998). In hypothalamus, ependyma may take up and spatially buffer Kþ released from adjacent endocrine neurons, and tanycytes, a related cell type lining the ventricles at certain locations (see chapter by Wolff and Chao), shunt Kþ to the CSF or to capillaries, thereby influencing neuronal excitability (Jarvis and Andrew, 1988). AVP increases water permeability of ependymal cells as shown by an augmentation in water movement from CSF to blood across the ependymal and capillary interfaces in the presence of AVP (Rosenberg et al., 1986). AQP4 is abundant in ependymal cells (Jung et al., 1994; Nielsen et al., 1997).

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As summarized in the legend of Fig. 2, the reduction of CSF formation during exposure to AVP is purposeful, because it will provide a partial compensation for the increase in water and ions within brain parenchyma, which will be produced if astrocytes and brain capillaries are exposed to AVP at the same time.

4. Concluding remarks The role of AVP in the regulation of water and ion balances in brain is explored, and in particular, its important influences on non-neuronal brain cells. AVP affects non-neuronal cells in diverse manners, an effect which may be modulated by other neuronal hormone and/or neurotransmitter systems. The modulation by ETs may be specially important and takes place both in astrocytes and in endothelial cells. In cerebral astrocytes, AVP facilitates water permeability to regulate water content at the cellular level of brain parenchyma. This effect is marked in astrocytic endfeet covering microvessels, which may lead to an enhanced fluid uptake in brain, facilitating the development of brain edema. In endothelial cells, AVP enhances cotransporter activity and channel opening to control [Kþ]e in brain and further enhance edema formation under pathological conditions. In ependymal cells, AVP increases water permeability to facilitate transport between CSF and CSF extracellular fluid. In choroid plexus, AVP decreases Cl2 efflux into CSF to reduce formation of CSF. Circumventricular astrocytes and pituicytes regulate systemic AVP secretion by morphological changes in their relationship with neurons, and they may react to local AVP release by reducing further AVP secretion. In the anterior lobe of the pituitary gland AVP facilitates release of ACTH. Many of these effects are associated with aquaporins, either by directly increasing water permeability or by mediating ion transport that drives vectorial water movement.

References Amedee, T., Robert, A., Coles, J.A., 1997. Potassium homeostasis and glial energy metabolism. Glia 21, 46 –55. Baduat, J., Hirt, L., Granziera, C., Bogousslavsky, J., Magistretti, P.J., Regli, L., 2001. Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia. J. Cereb. Blood Flow Metab. 21, 477– 482. Badaut, J., Lasbennes, F., Magistretti, P.J., Regli, L., 2002. Aquaporins in brain: distribution, physiology, and pathophysiology. J. Cereb. Blood Flow Metab. 22, 367–378. Beagley, G.H., Hatton, G.I., 1994. Systemic signals contribute to induced morphological changes in the hypothalamo-neurohypophysial system. Brain Res. Bull. 33, 211– 218. Bradbury, M.W.B., 1979. The Concept of a Blood– Brain Barrier. Wiley, Chichester. Bruni, J.E., 1998. Ependymal development, proliferation, and functions: a review. Microsc. Res. Tech. 41, 2 –13. Burbach, J.P.H., Adan, R.A., Lolait, S.J., van Leeuwen, F.W., Nezey, E., Palkovits, M., Barberis, C., 1995. Molecular neurobiology and pharmacology of the vasopressin/oxytocin receptor family. Cell Mol. Neurobiol. 15, 573–595. Chen, Y., McNeill, J.R., Hajek, I., Hertz, L., 1992. Effect of vasopressin on brain swelling at the cellular level— do astrocytes exhibit a furosemide– vasopressin-sensitive mechanism for volume regulation? Can. J. Physiol. Pharmacol. 70, S367– S373.

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Alexander disease: a primary disease of astrocytes Lawrence F. Enga,b,* and Yuen Ling Leeb a

Department of Pathology, School of Medicine, Stanford University, Stanford, CA 94305, USA b Department of Veterans Affairs Medical Center, Palo Alto, CA 94304, USA p Correspondence address: Pathology Research (151B), PAVA Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304, USA E-mail: [email protected]

Contents 1. 2. 3. 4. 5.

6. 7.

Introduction GFAP and astrogliosis Function of GFAP Alexander disease Increased GFAP production of the human GFAP gene in a transgenic mouse 5.1. Transgenic mice carrying the human GFAP gene 5.2. Astrocytes cultured from a low GFAP overexpressor mouse, Tg73.2 Role of GFAP in Alexander disease Consequences of GFAP mutations

In the central nervous system (CNS) of higher vertebrates, astrocytes become reactive and respond in a typical manner following injury, termed astrogliosis, either as a result of aging, trauma, disease, neurodegenerative disorders, genetic disorders, mechanical insult or chemical insult. Reactive astrogliosis is characterized by rapid synthesis of glial fibrillary acid protein (GFAP), the principal intermediate filament of mature astrocytes. The GFAP astrocytic response serves as a microsensor of the injured microenvironment at any location in the CNS. While GFAP is involved with almost any insult to the CNS, no convincing evidence of a primary astrocyte disease had been demonstrated until the development of the transgenic mouse model expressing a human GFAP transgene. The discovery that these mice formed abundant Rosenthal fibers (RFs) suggested that mutations in the GFAP gene were a cause of Alexander disease. Only in rare cases and in Alexander disease do reactive astrocytes contain RFs. The finding that GFAP mutation is found in many cases of Alexander disease offers the possibility of diagnosing Alexander disease through analysis of patient DNA samples. This non-invasive assay will eliminate morphological autopsy and brain biopsy analyses. Advances in Molecular and Cell Biology, Vol. 31, pages 773–785 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction The pathogenic role of astrocytes is debated hotly in the case of many neurological diseases (see chapters by Barger, by Brown and Sassoon, and by Przedborski and Goldman). As yet, however, there is only one disease, Alexander disease, which is unequivocally a primary disorder of astrocytes and of glial fibrillary protein (GFAP). 2. GFAP and astrogliosis Astrocytes account for 25% of the cells and 35% of the mass in the CNS. They have intimate contact with the pia of the brain, neurons, oligodendrocytes, endothelial cells, pericytes, myelin membrane internodes, synapses, and microglia. Glial fibrillary acidic protein (GFAP) in the mature CNS is found in protoplasmic astrocytes in gray matter, fibrous astrocytes in white matter, radial glia in the cerebellum (Bergmann glia), and subependymal astrocytes adjacent to the cerebral ventricles. At the surface of the brain, GFAP is especially concentrated in astrocytes, which form the outer limiting membrane, the glia limitans. Astrocytes in the CNS react to injury by hypertrophy, and in some cases proliferation. The functions of reactive astroglia are still not well understood (see chapter by Kalman). A common feature of these cells is enhanced expression of GFAP (Eng and Ghirnikar, 1994; Eng and Lee, 1995). In the CNS of higher vertebrates, astrocytes become reactive and respond in a typical manner, termed astrogliosis following injury either as a result of aging, trauma, disease, neurodegenerative disorders, genetic disorders, or chemical insult. Numerous in vitro and in vivo studies on the molecular profiles of substances, which are upregulated during astrocyte activation, document the complex and varied responses of astrocytes to injury (Eddleston and Mucke, 1993). Reactive astrogliosis is characterized by rapid synthesis of GFAP intermediate filaments, and increased protein content or immunostaining of GFAP has been found in experimental models involving gliosis. These include the cryogenic lesion in the brain, stab wounds, experimental allergic encephalomyelitis (EAE), hyperthermia, electrically induced seizures, and toxic lesions. The GFAP astrocytic response serves as a microsensor of the injured microenvironment at any location in the CNS. The precise mechanism of this response is still unknown. Growth factors, hormones, cytokines, and chemokines have been implicated, but no single common factor has been identified (Eng et al., 2000). In the above conditions, the GFAP content of astrocytes at the site of injury or activation increases until the astrocyte cell body and its processes become completely filled. Only in rare cases and in Alexander disease do reactive astrocytes also contain RFs. 3. Function of GFAP The rapid advances in molecular biology and newer techniques such as knockout mice, the use of the gfa-2 promoter to prepare transgenes, antisense RNA methodology, and DNA sequencing have greatly increased our knowledge of GFAP function in CNS development, injury, and disease. The technique of gene knockout (KO) has been used to

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examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. A putative function for GFAP is as a structural protein in association with the other cytoskeletal proteins—microfilaments (mainly actin) and microtubules (mainly tubulins) (Schliwa, 1986). Studies to determine other possible functions for GFAP have analyzed GFAP null mice (Gomi et al., 1995; Pekny et al., 1995; McCall et al., 1996; Liedtke et al., 1996). While GFAP null mice exhibit some abnormalities, they survive, reproduce, and live a normal life span. There is an intricate relationship between expression of GFAP and of vimentin, another intermediate filament protein, which is widely expressed in embryonic development. Homozygous vimentin knockout (vim 2 ) mice develop and reproduce without an obvious phenotype (Colucci-Guyon et al., 1994) but show a cerebellar defect and impaired motor coordination (Colucci-Guyon et al., 1999). Fibroblasts derived from these mice are also mechanically weak and severely disabled in their capacity to migrate and to contact a 3-D collagen network. Wounds in the vim 2 adult animal showed delayed migration of fibroblasts into the wound site (Eckes et al., 2000). The GFAP network in the vim 2 mice has disrupted GFAP and fails to assemble into a filamentous network in astrocytes that normally co-express GFAP and vimentin, i.e., corpus callosum astrocytes and Bergmann glia (Galou et al., 1996). Based on GFAP, vimentin, and double knockout mice studies, Pekny (2001) has provided a concise review on the possible functions of glial filaments. Besides providing structural support (Nawashiro et al., 1998), reactive astrogliosis, and scar formation, there is now evidence that GFAP is involved with long-term depression (Shibuki et al., 1996), longterm potentiation (Tanaka et al., 2002), control of astrocytic glutamine level (Pekny et al., 1999), circadian rhythm (Fernandez-Galaz et al., 1999), and regulation of cell volume and cell motility (Lepekhin et al., 1999; Anderova et al., 2001), and that it promotes normal blood – brain barrier formation (Pekny et al., 1998). These topics will not be discussed in this chapter; instead an illness, which in most cases is caused by a mutation in GFAP, i.e., Alexander disease, will be described.

4. Alexander disease In 1949 W. Steward Alexander described the pathologic condition which was given his name by Friede (1964). This illness is characterized clinically by megalencephaly accompanied by progressive spasticity, psychomotor retardation, and dementia. Pathological features include astrocytosis of white matter of the CNS, extensive demyelination, and less pronounced axonal loss. Histologically, it is characterized by the presence of large numbers of homogeneous eosinophilic masses forming elongated tapered rods scattered throughout the cortex and white matter, which are most numerous in the subpial, perivascular, and subependymal regions. Morphologically, the eosinophilic deposits are identical, by both light and electron microscopy, with the so-called RFs. Rosenthal fibers probably result from degenerative changes, which have taken place in the cytoplasm and cytoplasmic processes of differentiated astrocytes. They are inclusions within astrocytes, which are present in various situations where reactive gliosis has been in progress for a long time, e.g., in pilocytic astrocytomas,

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in optic nerve gliomas, in astrocytic scars, in multiple sclerosis plaques, in chronic infarcts, in ovarian teratomas, and most prominently in Alexander disease (Herndon et al., 1970; Grcevic and Yates, 1957; Alexander, 1949; Borrett and Becker, 1985). The RFs appear to be similar among the different disorders. RFs vary in size from round, focal deposits of a few microns to elongated, cigar-shaped bodies, one hundred microns or more in length for those that reside in astrocyte processes. At the ultrastructural level, RFs appear as dense, osmophilic masses lying on a meshwork of intermediate filaments (Herndon et al., 1970). The inclusion is composed of two low-molecular-weight heat shock proteins, aB-crystallin and HSP27 (Iwaki et al., 1989; Tomokane et al., 1991). Some of the aB-crystallin is conjugated to ubiquitin (Goldman and Corbin, 1991). Levels of aB-crystallin and HSP27 mRNA are elevated in Alexander disease (Head et al., 1993; 1994). It has been suggested that a variety of ‘stresses’ might induce the accumulation of RFs in astrocytes (Chin and Goldman, 1996). aB-crystallin is a lowmolecular-weight (22 kDa) protein which has a wide distribution of lens and nonlenticular tissues. It is a stress-related protein, which can be induced in culture by heat or hypertonicity, is associated with intermediate filaments, and is thought to stabilize cells in culture (Wisniewski and Goldman, 1998). It is generally soluble in aqueous solutions but exists in an aggregated form in RFs. We reported that only the periphery of RFs is immunostained with GFAP antibodies (Eng and Bigbee, 1978). Paraffin sections of an infant brain with Alexander disease immunostained with antibody to GFAP by the Sternberger PAP technique is shown in Fig. 1. A vessel surrounded by reactive astrocytes filled with RFs is shown in Fig. 1a. At higher magnification of an area with abundant RFs (Fig. 1b) one can clearly see the unstained fibers surrounded by intense GFAP immunostaining of the glial filaments (Ramsay et al., 1979).

Fig. 1. (a) A vessel surrounded by reactive astrocytes filled with Rosenthal fibers. (b) An area with abundant unstained Rosenthal fibers surrounded by intense immunostaining of the glial filaments.

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Russo et al. (1976) identified three clinical subgroups of Alexander disease. The first consists of the infantile group, which includes Alexander’s original patient. Onset of symptoms is from birth to early childhood, and the course is one of neurological deterioration with psychomotor retardation, seizures, quadriparesis, and megalencephaly. The average duration of the illness is two-and-a-half years. The second is the juvenile group, which has an onset from 7 to 14 years of age. The course is characterized by progressive bulbar symptoms and plasticity. Seizures and cognitive deterioration are less prominent and the average duration of the illness is 8 years. The adult cases comprise the third group. Onset of symptoms may occur between the second and seventh decade. Clinically, these adults, whose pathological picture is similar to that of the infantile and juvenile groups, may follow a course consistent with classical multiple sclerosis or they may be asymptomatic. The pathological hallmark of all groups is the diffuse accumulation of RFs, particularly in the subependymal, subpial, and perivascular regions. Demyelination is extensive in the infantile cases, less severe in the juvenile group, and variable in the adults. Magnetic resonance (MR) imaging studies in three patients with an autopsy-based diagnosis of Alexander disease were analyzed to define MR criteria for diagnosis. Five MR imaging criteria were defined: (i) extensive cerebral white matter changes with frontal predominance; (ii) a periventricular rim with high signal T1-weighted images and low signal on T2-weighted images; (iii) abnormality of basal ganglia and thalami; (iv) brain stem abnormalities; and (v) contrast enhancement of particular gray and white matter structures. Four of the five criteria had to be met for an MR image-based diagnosis. In a retrospective analysis of 217 children with leukoencephalopathy of unknown origin, 19 were found who fulfilled these criteria. In four of the 19 patients, subsequent histologic confirmation was obtained. The clinical symptomatology was the same in the patients with and without histologic confirmation, correlated well with MR abnormalities, and was in close agreement with the known histopathological findings in Alexander disease. The authors conclude that the defined MR imaging criteria are sufficient to diagnose Alexander disease (van der Knaap et al., 2001). Becker and Teixeira (1988) have stated that pathologic features strongly indicate that Alexander disease represents a non-neoplastic disease of astrocytes. The infant cases may represent a single disease entity. However, questions have been raised as to whether the juvenile and adult cases that have RFs, but different pathologic features, have the same etiology. As recently as 1999, one of the early workers on this subject suggested that while Alexander disease in infants is a disease entity, the boundaries of the entity remain illdefined and much of what is called Alexander disease is not that entity (Herndon, 1999). Recent GFAP transgene studies may now help clarify this conclusion (Messing et al., 1998; Brenner et al., 2001). 5. Increased GFAP production of the human GFAP gene in a transgenic mouse 5.1. Transgenic mice carrying the human GFAP gene In order to determine the properties of astrocytes containing increased amounts of GFAP without external stimulation and activation, six lines of transgenic mice were generated which carry added copies of the normal human GFAP (hGFAP) gene

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and express the human transgene at different levels (Messing et al., 1996; 1998). Mice in lines that expressed high levels of the hGFAP gene (Tg73.1 and 73.8) died young, while mice in lines that expressed lower levels of the transgene (Tg73.2, 73.4, and 73.5) attained adulthood and survived for more than a year. At the light microscopic level, astrocytes in the high-expressing lines were distended by aggregates of globular eosinophilic material. Ultrastructural examination of a transverse section of optic nerve from a 13-day-old high-expressing mouse showed that astrocytes contained abundant cytoplasmic filaments in association with irregular osmophilic deposits resembling RFs.

5.2. Astrocytes cultured from a low GFAP overexpressor mouse, Tg73.2 Astrocytes in primary cultures generally contain larger amounts of GFAP than astrocytes in vivo. Consistent with this, astrocyte cultures prepared from a low overexpressor (Tg73.2) exhibited abnormal cytoplasmic inclusions identical to those seen in vivo in the high overexpressors (Eng et al., 1998). Astrocytes in the Tg73.2 cultures appear odd-shaped and enlarged, express increased levels of GFAP (both human and mouse, as will be discussed below), express aB crystallin protein, HSP27 protein, and vimentin protein. At the light microscopic level, many but not all astrocytes in 18-day Tg73.2 cultures exhibited large odd-shaped cells (Fig. 2a) that immunostained with antibody specific for hGFAP (SMI-21). Tg73.2, but not wild-type astrocytes, in culture for 18 days, immunostained for aB-crystallin (Fig. 2b). Vimentin staining was seen in the Tg73.2 cultures (Fig. 2c), but was also seen in the wild-type cultures. At 14 days in culture, both types of cultures immunostained sparsely for Hsp27, but at 18 days the heat shock protein staining became more evident in the Tg73.2 cultures (Fig.2d). Staining for hGFAP was already intense by 6 days in culture whereas staining for aB-crystallin continued to increase, suggesting that the overexpression of hGFAP may contribute to the induction of aB-crystallin. Immunostaining of Tg73.2 astrocytes with SMI-21 (hGFAP only) and counterstaining with eosin showed irregular-shaped eosin positive bodies surrounded by SMI-21 immunostain. Conventional ultrastructural examination of Tg73.2 astrocytes showed numerous osmophilic deposits in a bed of intermediate filaments (Fig. 3a) identical to that seen in a case of Alexander disease (Fig. 3b). Double immunogold staining with SMI -21 (hGFAP only) and 18 nm gold second antibody followed by R-68 (human and mouse GFAP) and 12 nm gold second antibody showed that the GFAP in the wild-type astrocytes bound only to the 12 nm gold particles (Fig. 4a), while the GFAP in the Tg73.2 astrocytes bound to both 18 and 12 nm gold particles (Fig. 4b). This provided additional evidence demonstrating that both mouse and human GFAP are present in some of the Tg73.2 astrocytes. It appears that Tg73.2 mouse astrocytes in culture do not require additional stress from external sources or contact with other neuroectodermal cells to produce RFs. This suggests that the added hGFAP gene is sufficient to induce RFs and that excess of GFAP in astrocytes and/or the presence of GFAP that differs from species-specific GFAP may be detrimental to normal function.

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Fig. 2. Astrocytes in culture for 18 days from a Tg73.2 mouse were immunostained for hGFAP (a); aB-crystallin (b); Vimentin (c); and Hsp-27 (d).

6. Role of GFAP in Alexander disease The overexpressing GFAP mouse studies suggested that a primary alteration in GFAP may be responsible for Alexander disease (Messing et al., 1998; Eng et al., 1998). Genomic DNA samples from 11 unrelated patients in whom the diagnosis of Alexander disease had been confirmed by autopsy were analysed by Brenner et al., (2001). Each exon with some adjoining intron segments and 1717 bp of the 50 flanking region of GFAP were amplified by PCR and sequenced. Of the 11 DNAs from the Alexander disease patients, 10 contained novel heterozygous mutations of GFAP predicting nonconservative amino acid

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Fig. 3. (a) Astrocytes in culture for 20 days from a Tg73.2 mouse were analyzed at the ultrastructural level. Note the dense Rosenthal fibers among the glial filaments. (b) Astrocytes from a 17-month-old infant brain with Alexander’s disease were examined at the ultrastructural level. Note the dense deposits in the astrocyte cell body, which are identical to those seen in the Tg73.2 astrocyte culture.

changes, all involving arginine. Amino acid changes were: arginine 79 to cysteine or to histidine (one case of each); arginine 239 to cysteine (1 case); arginine 258 to proline (4 cases) or to histidine (1 case); and arginine 416 to tryptophan (2 cases). None of these mutations was seen in the two non-Alexander disease leukodystrophy control DNAs that were fully sequenced, or in 53 control DNA samples from individuals without neurologic disease that were specifically analyzed for these mutations by restriction digestion. In each case, the Alexander disease patient was heterozygous for the mutation, suggesting a dominant mode of action. Since all the parents were phenotypically normal, the authors predicted that these mutations arose de novo. To test this hypothesis, DNA samples from five patients were analyzed by restriction digestion. None of the parental samples contained the mutations found in the affected children. Additional studies showed that of 14 parental DNAs tested, none had the non-conservative mutation found in the affected children. These initial studies showed that most cases of pathologically proven Alexander

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Fig. 4. Ultrastructural analysis of wild-type (a) and Tg73.2 (b) astrocytes double immunogold staining with SMI-21 (human GFAP) with 18 nm gold particle followed by R-68 (Bovine GFAP) with 12 nm gold particle antisera. The wild-type astrocytes bind only to the 12 nm gold bound antiserum while the Tg73.2 astrocytes bind to the 12 and 18 nm gold bound antisera.

disease are associated with de novo mutations (occurring in the embryo or in the parental gametes) in the coding region of GFAP. A review (Messing et al., 2001) and several additional studies by other groups have confirmed the original observations. The DNA of a series of additional infancy-onset patients, who had heterogeneous clinical symptoms but were candidates for Alexander disease on the basis of neuroimaging abnormalities (see above), were analyzed for GFAP mutations by Rodriguez et al. (2001). Missense, heterozygous, de novo GFAP mutations were found in exon 1 or 4 for 14 of the

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15 patients analyzed, including patients without macrocephaly. Nine patients carried one of the previously established mutations (arginine 79 to histidine [4 cases]; arginine 239 to cysteine [4 cases] or to histidine [1 case]). The other five had one of four novel mutations, of which two affected arginine (arginine 88 to cysteine [2 cases] or to serine [1 case]) and two affected non-arginine residues (leucine 76 to phenylalanine and asparagine 77 to tyrosine [each in one case]). All were located in the central rod domain of GFAP, and there was a correlation between clinical severity and affected amino acid. Another study on patients suspected of having Alexander disease was conducted by Gorospe et al. (2002) to determine the extent to which clinical and MRI criteria could accurately diagnose affected individuals, using GFAP gene sequencing as the confirmatory assay. Patients showing MRI white matter abnormalities consistent with Alexander disease, unremarkable family history, normal karyotype, and normal metabolic screening, were included in the study. Genomic DNA from patients was screened for mutations in the entire coding region, including the exon –intron boundaries, of the GFAP gene. Twelve of the 13 patients were found to have mutations in GFAP. Seven of the 12 patients presented in infancy with seizure and megalencephaly. Five were juvenile-onset patients with more variable symptoms. Two patients in the latter group were asymptomatic or minimally affected at the time of their initial MRI scan. The mutations were distributed throughout the gene, and all involved sporadic single amino acid heterozygous changes that altered the charge of the mutant protein. Most of the GFAP mutations were sporadic single amino acid heterozygous missense changes: methionine 73 to arginine (1 case), arginine 79 to cysteine (1 case) or to histidine (2 cases); arginine 88 to cysteine (2 cases), arginine 239 to cysteine (1 case); tyrosine 242 to aspartic acid (1 case); glutamic acid 373 to lysine (1 case), arginine 416 to tryptophan (2 cases). Moreover, two Japanese juvenile-onset cases have been demonstrated, one in which alanine 244 was mutated to valine, and another in which arginine 239 was changed to cysteine (Aoki et al., 2001; Shiroma et al., 2001). At this time, 41 mutations have been confirmed by GFAP sequencing (Li et al., 2002). The majority of the described mutations involve a change of the positively charged (at pH 7) amino acid arginine to a non-charged residue and there is one change of the noncharged methionine to arginine. In one patient the missense amino acid changed a negative charge on glutamic acid to a positive charge on lysine and in another patient, non-charged tyrosine was altered to negatively charged aspartic acid.

7. Consequences of GFAP mutations In the review by Messing et al. (2001), several mechanisms have been discussed by which GFAP mutations might lead to Alexander disease. Jaeken (2001) has proposed that Alexander disease is a conformational disease, arising when a constituent protein undergoes a change in size or shape with resultant self-association and intra- or extracellular deposition. The frequent changes in charged amino acids in the mutations linked to Alexander disease are consistent with this notion. GFAP mutations might cause disease by raising levels of the protein and increasing its stability. The mutation might lead to an accumulation of a particular form of GFAP, which compromises astrocyte function.

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The mutation can lead to accumulation of a toxic form of GFAP, which interferes with its polymerization into normal intermediate filaments. The emerging evidence that GFAP deletion alters functional characteristics of astrocytes is in agreement with such a concept, and a reason for the more severe form in infants may be interference with neuronal – astrocytic and endothelial – astrocytic interactions during development. We hypothesized that the normal mechanism for GFAP turnover may be insufficient to handle the excess GFAP, thus inducing an accumulation of stress proteins (Eng et al., 1998). Alexander disease astrocytes do display properties of physiological stress as evidenced by the elevation of the stress proteins aB-crystallin and HSP27. Brenner and co-workers suggest that Alexander disease likely results from a dominant gain of function that in turn partially blocks filament assembly, which leads to accumulation of an intermediate that participates in toxic interactions (Li et al., 2002). In the past, diagnosis of Alexander disease was based on a combination of clinical, morphological, and MRI imaging examinations. The recent finding that GFAP mutation is found in many cases of Alexander disease offers the possibility of diagnosing most cases of Alexander disease through analysis of patient DNA samples. This non-invasive assay will eliminate morphological autopsy and brain biopsy analyses. The belief that Alexander disease is a primary disease entity of astrocytes (Becker and Teixeira, 1988; Eng et al., 1998) has now been confirmed.

References Alexander, W.S., 1949. Progressive fibrinoid degeneration of fibrillary astrocytes associated with mental retardation in a hydrocephalic infant. Brain 72, 373– 381. Anderova, M., Kubinova, S., Mazel, T., Chvatal, A., Eliasson, C., Pekny, M., Sykova, E., 2001. Effect of elevated K(þ ), hypotonic stress, and cortical spreading depression on astrocyte swelling in GFAP-deficient mice. Glia 35, 189– 203. Aoki, Y., Haginoya, K., Munakata, M., Yokoyama, H., Nishio, T., Togashi, N., Ito, T., Suzuki, Y., Kure, S., Linuma, K., Brenner, M., Matsubara, Y., 2001. A novel mutation in glial fibrillary acidic protein gene in a patient with Alexander disease. Neurosci. Lett. 312, 71–74. Becker, L.E., Teixeira, F., 1988. Alexander’s disease. In: Norenberg, M.D., Hertz, L., Schousboe, A. (Eds.). The Biochemical Pathology of Astrocytes. Alan R. Liss Inc, New York, pp. 179–190. Borrett, G., Becker, L.E., 1985. Alexander’s disease: a disease of astrocytes. Brain 108, 367 –385. Brenner, M., Johnson, A.B., Boespflug-Tanguy, O., Rodriguez, D., Goldman, J.E., Messing, A., 2001. Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nature Genet. 27, 117– 120. Chin, S.M., Goldman, J.E., 1996. Glial inclusion in CNS degenerative diseases. J. Neuropath. Expt. Neurol. 55, 499– 508. Colucci-Guyon, E., Portier, M.-M., Dunia, I., Paulin, D., Pournin, S., Babinet, C., 1994. Mice lacking vimentin develop and reproduce without obvious phenotype. Cell 79, 679–694. Colucci-Guyon, E., Gimenez, Y., Ribotta, M., Maurice, T., Babinet, C., Privat, A., 1999. Cerebellar defect and impaired motor coordination in mice lacking vimentin. Glia 25, 33 –43. Eckes, B., Colucci-Guyon, E., Smola, H., Nodder, S., Babinet, C., Krieg, T., Martin, P., 2000. Impaired wound healing in embryonic and adult mice lacking vimentin. J. Cell. Sci. 113, 2455–2462. Eddleston, M., Mucke, L., 1993. Molecular profile of reactive astrocytes; implications for their role in neurologic diseases. Neuroscience 54, 15–36. Eng, L.F., Bigbee, J.W., 1978. Immunohistochemistry of nervous system-specific antigens. In: Agranoff, B.W., Aprison, M.H. (Eds.), Advances in Neurochemistry, vol. 3. Plenum Press, New York, pp. 43–98.

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Eng, L.F., Ghirnikar, R.S., 1994. GFAP and astrogliosis. Brain Pathol. 4, 229–237. Eng, L.F., Lee, Y.L., 1995. Intermediate filaments in astrocytes. In: Kettenmann, H., Ransom, B.R., Schousboe, A. (Eds.), Neuroglial. Oxford University Press, New York, pp. 650–667. Eng, L.F., Lee, Y.L., Kwan, H., Brenner, M., Messing, A., 1998. Astrocytes cultured from transgenic mice carrying the added human glial fibrillary acidic protein gene contains Rosenthal fibers. J. Neurosci. Res. 53, 353–360. Eng, L.F., Ghirnikar, R.S., Lee, Y.L., 2000. Glial fibrilllary acidic protein: GFAP-thirty-one years (1969–2000). Neurochem Res. 25, 1439–1451. Fernandez-Galaz, M.C., Martinez Munoz, R., Villanua, M.A., Garcia-Segura, L.M., 1999. Diurnal oscillation in glial fibrillary acidic protein in a perisuprachiasmatic area and its relationship to the luteinizing hormone surge in the female rat. Neuroendocrinology 70, 368–376. Friede, R.L., 1964. Alexander’s disease. Arch. Neurol. 11, 414 –422. Galou, M., Colucci-Guyon, E., Ensergueix, D., Ridet, J.L., Gimenez y Ribotta, M., Privat, A., Babinet, C., Dupouey, P., 1996. Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice. J. Cell Biol. 133, 853–863. Goldman, J.E., Corbin, E., 1991. Rosenthal fibers contain ubiquitinated aB-crystallin. Am. J. Pathol. 139, 933 –938. Gomi, H., Yokoyama, T., Fujimoto, K., Ikeda, T., Katoh, A., Itoh, T., Itohara, S., 1995. Properties of astrocytes from mice devoid of the glial fibrillary acidic protein develop normally and are susceptible to scrapie prions. Neuron 14, 29–41. Gorospe, J.R., Naidu, S., Johnson, A.B., Puri, V., Raymond, G.V., Jenkins, S.D., Pedersen, R.C., Lewis, D., Knowles, P., Fernandez, R., De Vivo, D., van der Knaap, M.S., Messing, A., Brenner, M., Hoffman, E.P., 2002. Molecular findings in symptomatic and pre-symptomatic Alexander disease patients. Neurology 58, 1494–1500. Grcevic, N., Yates, P.O., 1957. Rosenthal fibres in tumours of the central nervous system. J. Pathol. Bact. 73, 467 –472. Head, M., Corbin, E., Goldman, J.E., 1993. Overexpression and abnormal modification of the stress proteins aB-crystallin and HSP27 in Alexander disease. Am. J. Pathol. 143, 1743–1753. Head, M.W., Corbin, E., Goldman, J.E., 1994. Coordinate and independent expression of aB-crystallin and hsp27. J. Cell Physiol. 159, 41–50. Herndon, R.M., 1999. Is Alexander’s disease a nosologic entity or a common pathologic pattern of diverse etiology? J. Child Neurol. 14, 275 –276. Herndon, R.M., Rubinstein, L.J., Freeman, J.M., Mathieson, G., 1970. Light and electron microscopic observations on Rosenthal fibres in Alexander disease and in multiple sclerosis. J. Neuropathol. Exp. Neurol. 29, 524–551. Iwaki, T., Kume-Iwaki, A., Liem, R.K.H., Goldman, J., 1989. aB-crystallin is expressed in non-lenticular tissues and accumulates in Alexander disease. Cell 57, 71–78. Jaeken, J., 2001. Alexander disease and intermediate filaments in astrocytes: a fatal gain of function. Eur. J. Pediatr. Neurol. 5, 151–153. van der Knaap, M.S., Naidu, S., Breiter, S.N., Blaser, S., Stroink, H., Springer, S., Begeer, J.C., van Coster, R., Barth, P.G., Thomas, N.H., Valk, J., Power, J.M., 2001. Alexander disease: diagnosis with MR imaging. Am. J. Neuroradiol. 22, 541–552. Lepekhin, E.A., Eliasson, C., Berthold, C.H., Berezin, V., Bock, E., Pekny, M., 1999. Intermediate filaments regulate astrocyte motility. J. Neurochem. 79, 617–625. Li, R., Messing, A., Goldman, J.E., Brenner, M., 2002. GFAP mutations in Alexander disease. Int. J. Develop. Neurosci. 720, 259–268. Liedtke, W., Edelmann, W., Bieri, P., Chiu, F.-C., Cowan, N.J., Kucheriapa, R., Raine, C.S., 1996. GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination. Neuron 17, 607–615. McCall, M.A., Gregg, R.G., Behringer, R.R., Brenner, M., Delaney, C.J., Galbreath, E.J., Zhang, C.L., Pearce, R.A., Chiu, S.Y., Messing, A., 1996. Targeted deletion in astrocyte intermediate filament (GFAP) alters neuronal physiology. Proc. Natl. Acad. Sci., USA 93, 6361–6366. Messing, A., Galbreath, E.J., Sijapati, K.K., Brenner, M., 1996. Overexpression of GFAP in transgenic mice. J. Neuropath. Expt. Neurol. 55, 620 (Abstract).

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Messing, A., Head, M.W., Galles, K., Galbreath, E.J., Goldman, J.E., Brenner, M., 1998. Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice. Am. J. Pathol. 152, 391–398. Messing, A., Goldman, J.E., Johnson, A.B., Brenner, M., 2001. Alexander disease: new insights from genetics. J. Neuropath. Expt. Neurol. 60, 563 –573. Nawashiro, H., Messing, A., Azzam, N., Brenner, M., 1998. Mice lacking glial fibrillary acidic protein are hypersensitive to traumatic cerebrospinal injury. Neuroreport 9, 1691–1696. Pekny, M., 2001. Astrocytic intermediate filaments: lessons from GFAP and vimentin knock-out mice. Prog. Brain Res. 132, 23 –30. Pekny, M., Leveen, P., Pekna, M., Eliasson, C., Berthold, C.-H., Westermark, B., Betsholtz, C., 1995. Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally. EMBO J. 14, 1590–1598. Pekny, M., Stanness, K.A., Eliasson, C., Betsholtz, C., Janigro, D., 1998. Impaired induction of blood–brain barrier properties in aortic endothelial cells by astrocytes from GFAP-deficient mice. Glia 22, 390 –400. Pekny, M., Eliasson, C., Siushansian, R., Ding, M., Dixon, S.J., Pekna, M., Wilson, J.X., Hamberger, A., 1999. The impact of genetic removal of GFAP and/or vimentin on glutamine levels and transport of glucose and ascorbate in astrocytes. Neurochem. Res. 24, 1357–1362. Ramsay, P., Norman, M., Eng, L.F., 1979. Chemical study of an Alexander brain. Trans. Am. Soc. Neurochem. 10, 125 (Abstract). Rodriguez, D., Gauthier, F., Bertini, E., Bugiani, M., Brenner, M., N’guyen, S., Goizet, C., Gelot, A., Surtees, R., Pedespan, J.-M., Hernandorena, X., Troncoso, M., Uziel, G., Messing, A., Ponsot, G., Pham-Dinh, D., Dautigny, A., Boespflug-Tanguy, O., 2001. Infantile Alexander disease: spectrum of GFAP mutations and genotype–phenotype correlation. Am. J. Hum. Genet. 69, 1134–1140. Russo, L.S., Aron, A., Anderson, P.J., 1976. Alexander’s disease. A report and reappraisal. Neurology 26, 607–614. Schliwa, M., 1986. The cytoskeleton: an introductory survey, Cell Biol. Monogr., Vol. 13. Springer-Verlag, New York. Shibuki, K., Gomi, H., Chen, L., Bao, S., Kim, J.J., Wakatsuki, H., Fujisaki, T., Fujimoto, K., Katoh, A., Ikeda, T., Chen, C., Thompson, R.F., Itohara, S., 1996. Deficient cerebellar long-term depression, impaired eyeblink conditioning, and normal motor coordination in GFAP mutant mice. Neuron 16, 587–599. Shiroma, N., Kanazawa, N., Izumi, M., Sugai, K., Fukumizu, M., Sasaki, M., Hanaoka, S., Kaga, M., Tsujino, S., 2001. Diagnosis of Alexander disease in a Japanese patient by molecular genetic analysis. J. Hum. Genet. 46, 579–582. Tanaka, H., Katoh, A., Oguro, K., Shimazaki, K., Gomi, H., Itohara, S., Masuzawa, T., Kawai, N., 2002. Disturbance of hippocampal long-term potentiation after transient ischemia in GFAP deficient mice. J. Neurosci. Res. 67, 11–20. Tomokane, N., Iwaki, T., Tateishi, J., Iwaki, A., Goldman, J.E., 1991. Rosenthal fibers share epitopes with aB-crystallin, glial fibrillary acidic protein and ubiquitin, but not with vimentin: immunoelectron microscopy with colloidal gold. Am. J. Pathol. 138, 875–885. Wisniewski, T., Goldman, J.E., 1998. aB-crystallin is associated with intermediate filaments in astrocytoma cells. Neurochem. Res. 23, 385 –392.

Glial reaction and reactive glia M. Ka´lma´n Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary E-mail: [email protected](M.K.)

Contents 1. 2. 3. 4. 5. 6. 7. 8.

9.

10.

11.

12.

Definition and general characteristics Physiological and clinical importance The main types of glial reactions and reactive astrocytes: anisomorphic and isomorphic Structure and cell junctions of reactive astrocytes The cytoskeleton of reactive astrocytes Noncytoskeletal protein markers of reactive astrocytes Recruiting astrocytes for the reaction The sequence of events and the phases of glial reaction 8.1. Inflammatory phase 8.2. Astroglial phases: isomorphic and anisomorphic 8.3. Scarring Regulation of the glial reaction 9.1. General comments 9.2. Tissue destruction and microglial activity 9.3. Activation of astroglia 9.4. Astroglial activity 9.5. Scarring 9.6. Feedback effects and balancing 9.7. Remarks to the humoral regulation 9.8. Intracellular regulation Functions of the glial reaction 10.1. Introductory remarks 10.2. Debris elimination and secondary tissue damage 10.3. Neuroprotection 10.4. Demarcation CNS regeneration and the glial reaction 11.1. In the mature mammalian CNS 11.2. Other inhibitors of regeneration 11.3. In the immature mammalian CNS 11.4. In the nonmammalian CNS 11.5. Attempts to inhibit the inhibitors 11.6. Is there a ‘biological rationale’ for regeneration failure? Concluding remarks

Advances in Molecular and Cell Biology, Vol. 31, pages 787–835 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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Abbreviations ATP: adenosine triphosphate; BBB: blood – brain barrier; bFGF: basic fibroblast growth factor; BHN: brain hyaluronectin (also called GHAP, HBGP); BrDU: bromodeoxyuridine; cAMP: cyclic adenosine monophosphate (cyclic AMP); CNS: central nervous system; CNTF: ciliary neurotrophic factor; dBcAMP: dibutyryl cyclic adenosine monophosphate; E: embryonic day; ECM: extracellular matrix; EGF: epidermal growth factor; Erk: extrinsic receptor signalling kinase (see also MAPK); FGFR: fibroblast growth factor receptor; GFAP: glial fibrillary acidic protein; GHAP: glial hyaluronate associated protein (called also BHN, HBGP); HBGP: hyaluronate-binding glial protein (also called BHN, GHAP); HSP: heat-shock protein; ICAM: intercellular adhesion molecule; IFAP: intermediate filament associated protein; IL: interleukin; IL-1bRa: interleukin-1b receptor antagonist; MAP: microtubule associated protein; MAPK: mitogen-activated protein kinase (see also Erk); MIP: macrophage inflammatory protein; NGF: nerve growth factor; P: postnatal day; PCNA: proliferative cell nuclear antigen; PDGF: platelet derived growth factor; PECAM: plasma endothelial cell adhesion molecule; PNS: peripheral nervous system; PSA-NCAM: polysialic acid-containing nerve cell adhesion molecule; PTHrP: parathyroid hormone related peptide; TGF: transforming growth factor; TNF: tumor necrosis factor; trk, tyrosine kinase; VCAM: vasoactive cell adhesion molecule; VEGF: vasoactive endothelial growth factor. This chapter describes both morphological and functional features of the glial response following lesions of central nervous tissue. The sequence of stages, and a combination of different cell activities are emphasized. Especially, the effect on neural regeneration is described in detail, including data on immature and nonmammalian brains. The main point is that the glial reaction is the CNS-type of wound healing, and it has combined multilateral, beneficial and adverse effects, depending on the circumstances. The glial reaction to lesion is a result of evolution and neurohistogenesis, and it cannot be considered simply as a ‘misfortune’. 1. Definition and general characteristics Glia more or less ‘react’ to any alterations of neurons, either functional or dysfunctional. The terms ‘glial reaction’ and ‘reactive glia’, however, refer to the characteristic response, which follows different injuries (physical, chemical, ischemic, etc.) of the CNS. The predominant process is the vigorous reaction of astrocytes, and the hallmarks are the increased number of glial cells, the hypertrophy of astrocytes and the accumulation of cytoplasmic fibrillary material: the intermediate filaments. The reactive astrocytes exhibit signs of intense metabolism, and several factors characteristic of the immature astroglia are re-expressed (for details, see later; for reviews, see e.g., Eddleston and Mucke, 1993; Ridet et al., 1997). Although the morphologic phenomena are attributed mainly to astroglia, the contribution of macrophages/microglia seems to be essential in the early phase (Fernaud-Espinosa et al., 1993). Some oligodendritic reaction has also been mentioned (Ludwin, 1985; Xie et al., 1995).

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The first description of ‘fibrous gliosis’ was made by Virchow (according to Bignami and Dahl, 1976), on a case of tabes dorsalis. Following a series of studies by aid of the classical impregnation techniques (see, e.g., Rio Hortega and Penfield, 1927; Ramon y Cajal, 1928; cited by Reier, 1986), the major breakthrough came with the discovery of GFAP (Eng et al., 1971), the intermediate filament protein and immunohistochemical marker of astroglia. The increased GFAP expression proved to be a hallmark of the glial reaction (Bignami and Dahl, 1976), and its immunohistochemical demonstration has gained a general acceptance in the investigations of the glial reaction. The observations on the glial reactions have been summarized in a series of reviews (see, e.g., Bignami et al., 1980; Lindsay, 1986; Reier, 1986; Malhotra et al., 1990; Hatten et al., 1991; Norton et al., 1992; Brodkey et al., 1993; Eddleston and Mucke, 1993; Fernaud-Espinosa et al., 1993; Eng and Ghirnikar, 1994; Norenberg, 1994; Ridet et al., 1997; Wu and Schwartz, 1998; Fawcett and Asher, 1999; Eng et al., 2000).

2. Physiological and clinical importance The glial reaction corresponds to the wound healing in the other parts of the organism. The main difference is that in the CNS, the access of extrinsic cells (i.e., hematogenous cells, immune cells, etc.) and large proteins (i.e., antibodies) is restricted by the BBB (Brodkey et al., 1993; Eddleston and Mucke, 1993; Lawson and Perry, 1995; Merrill and Benveniste, 1996). Therefore, the astrocytes have to take on some tasks performed otherwise by connective tissue cells (including hematogenous cells). These features have been acquired during the evolution (see Abbott, 1995) and during the maturation of the CNS (Lawson and Perry, 1995). The predominant view is that the reactive glia is the main obstacle to neural regeneration (for details, see later). According to the reviews of Stichel and Mu¨ller (1998a) and Ho¨ke and Silver (1994), it was Ramon y Cajal (1928), who first described the abortive effect of reactive gliosis on the growing axons. In the CNS, glial reaction follows vascular, traumatic and degenerative damages, which accounts for its clinical importance. In Alzheimer’s disease it has even been assigned a pathogenetic role (Ho¨ke and Silver, 1994; Ho¨ke et al., 1994; Shaffer et al., 1995; Xu et al., 1999; Martins et al., 2001; for reviews see Unger, 1998; Schubert et al., 2000; Abraham, 2001), as it also has in multiple sclerosis and experimental allergic encephalitis (EAE) (Luo et al., 2000). Glial reaction can interact with the integration of CNS transplants (Azmitia and Whitaker, 1983; Kruger et al., 1986; Malhotra et al., 1990; for a review, see Reier et al., 1992), and it may act as an epileptogenic focus (see e.g., Lee et al., 1995; Aronica et al., 2001a). It is to be noted that the former consensus that the glial reaction has a negative effect is about to be modulated, pointing to its neuroprotective and regenerative effects; therefore, efforts are being made to find ways to make use of the glial reaction rather than to suppress it (see chapter by Dezawa).

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3. The main types of glial reactions and reactive astrocytes: anisomorphic and isomorphic The glial reaction has two main forms: isomorphic and anisomorphic (Hatten et al., 1991; Fernaud-Espinosa et al., 1993; Ridet et al., 1997). In the former case, the astrocyte processes have no special orientation, in the latter the processes are oriented in a main direction, and form a ‘palisade-like’ arrangement. The anisomorphic reaction occurs when there is a confined area to be demarcated, i.e., mainly in the so-called ‘open’ lesions, when the glio –meningeal and/or glio – vascular barrier (glia limitans, glial endfeet and basement membrane towards the connective tissue) is penetrated, by mechanical lesions, severe strokes (bleeding or emollition), invading tumours, or abscess formation. It is noteworthy that the ‘anisomorphic’ arrangement of the glial processes is formed only in an advanced stage of the reaction, and that it is confined to the vicinity of the damage. So even the ‘anisomorphic’ reactions have an early ‘isomorphic phase’, and later a collateral ‘isomorphic zone’, after the formation of the demarcating anisomorphic core. ‘Proximal’ and ‘distal’ astrocytes can be distinguished by their localization in the adjacent or distant zone around the lesion. The ‘open’ lesions usually result in intrusions of hematogenous cells and connnective tissue (meningeal, perivascular) cells. In these cases a new glia limitans (so-called ‘secondary’ or ‘accessory’), and new basement membrane are formed (Carbonell and Boya, 1988; Maxwell et al., 1990a), restoring the sequestered position of the CNS tissue. Fully developed, it results in a permanent glial scar, which is formed by the proximal astrocytes, with a contribution of meningeal and/or perivascular connective tissue, and neo-vascularization. The barrier (i.e., permanent) function is obvious in the anisomorph zone, where the glia demarcate the surviving nervous tissue from the decaying one and from the invading connective tissue. The isomorphic, collateral zone is supposed to have a neuroprotective (i.e., temporary) function by aid of its metabolic, antitoxic and antioxidant activities and its immune effects (Ridet et al., 1997). It is also believed that the activation mechanisms for the different reaction types and zones are different (Ho¨ke and Silver, 1994). Both the proximal and the distal astrocytes display signs of hypertrophy, similar ultrastructural features and GFAP-immunopositivity. Proximal astrocytes exhibit a different molecular profile from the distal ones (Eddleston and Mucke, 1993; Fernaud-Espinosa et al., 1993; Ho¨ke and Silver, 1994; Ridet et al., 1997) in accordance with their different functions. The modifications characteristic of the ‘reactive glia’ are shown mainly by the proximal astrocytes. However, no data seem to be available on possible biochemical differences between astrocytes of the entirely isomorphic reaction and of the distal, isomorphic astrocytes of the anisomorphic reaction. The nonpenetrating, so-called ‘closed’ injuries (toxic, metabolic, and smaller ischemic injuries) result only in an isomorphic lesion. Isomorphic gliosis also follows every type of CNS degeneration, independently of its etiopathogenesis. Most reviews list a number of examples (see, e.g., Eng and Ghirnikar, 1994; Montgomery, 1994). Without any attempt of completeness, let us look at several examples: retrograde degenerations (Vaughn et al., 1970; Graeber and Kreutzberg, 1986; Tetzlaff et al., 1988); anterograde degenerations (Hajo´s et al., 1990a,b; Murray et al., 1990), cryogenic lesions (Amaducci et al., 1981);

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ischemic, hypoxic injuries (Miyazaki et al., 2001); toxic effects (e.g., Block and Schwarz, 1984; Eriksdotter-Nilsson et al., 1986), multiple sclerosis (e.g., Luo et al., 2000), experimental allergic encephalitis (EAE) (Smith et al., 1983; Goldmuntz et al., 1986; Aquino et al., 1988), tumor-invaded areas (e.g., Lee et al., 1995; Aronica et al., 2001a), amyotrophic lateral sclerosis (e.g., Aronica et al., 2001b), tabes dorsalis, Huntington’s disease (Vacca and Nelson, 1984; Vacca-Galloway, 1986), as well as those, which are in focus today: prion diseases (Forloni et al., 1994; Gomi et al., 1995) and Alzheimer’s disease (Ho¨ke and Silver, 1994; Ho¨ke et al., 1994; Shaffer et al., 1995; Xu et al., 1999; Martins et al., 2001; for reviews, see Unger, 1998; Schubert et al., 2000; Abraham, 2001), in which the glial reaction is supposed to be not only a consequence of neurodegeneration, but a pathogenic factor as well (see chapters by Brown and Sassoon and by Barger). According to Mirza et al. (2000) Parkinson’s disease seems to be an exception by not creating morphologically obvious glial reactions, but this does not mean that the glial cells do not respond to the neuronal degeneration (see chapter by Predzborski and Goldman). Following lesions astrocytes can be activated in patches far away and discontinuous from the reactive glia localized to the lesion. This phenomenon is known as ‘remote glial reaction’ (Hajo´s et al., 1990a). Similar observations have been reported by Barrett et al. (1981), Isacson et al. (1987), Anders and Johnson (1990) and Block and Schwarz (1984). ‘Remote’ glial reaction is induced by the degeneration of synapses belonging to the lesioned neurons, as revealed by electron microscopy. Aside from the anisomorphic – isomorphic classification, the glial reactions seem to display similar morphology, independent of the provoking effect. According to Norton et al. (1992), and Ridet et al. (1997), however, in diverse forms of CNS injuries the astrocytes can develop biochemical differences, exhibiting a plasticity to the microenvironment. The isomorphic gliosis is usually reversible upon cessation of its stimulus (see, e.g., Eriksdotter-Nilsson et al., 1986 for toxic gliosis; Ka´lma´n et al., 1993 for ‘remote’ gliosis), as is the collateral, ‘isomorphic’ zone (distal astrocytes) of the ‘anisomorphic’ glial reactions (see, e.g., Mathewson and Berry, 1985).

4. Structure and cell junctions of reactive astrocytes By conventional light microscopic examination, the glial reaction is revealed by an increased number of glial cells, mainly astrocytes, and also microglia. The astrocytes hypertrophy, the nuclear contours become irregular, nucleoli are conspicuous, processes thicken upon being visible, and eosinophilic crystalline (Rosenthal-fibers) appears in the cytoplasm (see chapter by Eng). Mitotic figures are frequent (see e.g., Cavanagh, 1970; Norenberg, 1994). Most characteristics of glial reactivity are exhibited in both gray and white matter. The gray matter astrocytes, which mainly belong to the ‘protoplasmic’ type, become ‘fibrous’ during the reaction. For the ultrastructural details, see e.g., Reier (1986), Maxwell et al. (1990a), Eddleston and Mucke (1993), and Norenberg (1994). Astrocytes exhibit cytoplasmic swelling, enlargement in nuclear size, increases in cell organelles (mainly mitochondria) and in microtubule and microfilament content, accumulation of glycogen and several lipid

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droplets, and apparent additions of more cisternae to the Golgi complex and rough endoplasmic reticulum. Organelles either assemble into aggregates or increase to such an extent that they fill the cytoplasm. Astrocytes replace the neural elements with their enlarged, prolonged, convoluted, interdigitating processes. The cell-junction structures are also modified in the reactive glia. The number of the intramembranous ‘orthogonally arranged particles’ increases (Anders and Brightman, 1979). In the intact brain, these particles characterize mainly the perivascular astrocytes (Landis and Reese, 1974), and they are supposed to represent connections between the intermediate filaments inside the cells and the ECM on its outside. In the reactive astroglia, the distribution of the gap junctions becomes uneven. High numbers of gap junctions, tight junctions and puncta adherentes (Vaughn and Pease, 1970; Alonso and Privat, 1993) were found, especially in the proximal astrocytes and mainly in the glia limitans secondaria. The immunopositivity to connexin43, the major protein of the astrocytic-gap junctions, decreased following lesion with kainic acid (Vukelic et al., 1991; Sawchuk et al., 1995). However, in some other injuries, e.g., retrograde degeneration in the motor nucleus of facial nerve (Rohlmann et al., 1993), spinal cord compression (Theriault et al., 1997), and cerebral ischemia (Hossain et al., 1994), the immunopositivity became more intense. High expression of connexin in peritumoral and other reactive astrocytes suggests that they may contribute to tumor-related seizures (Lee et al., 1995; Aronica et al., 2001a – see also chapter by Cornell-Bell et al.). Bordey et al. (2001) reported, however, a reduced electrophysiological coupling in the reactive glia, suggesting a compromised ion distribution within the astrocytic syncytium. For recent reviews on gap junctions in astroglia, see Giaume and McCarthy (1996) and Dermietzel (1998) as well as chapter by Scemes and Spray. N-cadherins have consistently been found to be upregulated in astrocytes of the glial scar (Vazquez-Chona and Geisert, 1999). Integrins (for a review, see Jones, 1996) are essential for the connection to the ECM. A new member of the adhesion molecules, gicerin, a membrane glycoproteid mediating cell –cell and cell –ECM interactions in the CNS, was observed in reactive astrocytes around retrograde degeneration of hypoglossus neurons (Li et al., 1999).

5. The cytoskeleton of reactive astrocytes As first described by Bignami and Dahl (1976), the GFAP-immunopositivity increases dramatically following lesions. GFAP-immunopositivity appears even in those areas, where it is not detectable by light microscopical methods in the intact brain. For an account of the ‘GFAP-free’ areas of the rat brain see Ka´lma´n and Hajo´s (1989) and Hajo´s and Ka´lma´n (1989), and for a discussion of the role of GFAP, see the recent reviews by Rutka et al. (1997) and Eng et al. (2000), and the research report by Menet et al. (2001). Three mechanisms should be considered as underlying the appearance of GFAPimmunopositivity: (i) the appearance of new, GFAP-expressing astrocytic populations by proliferation and/or migration; (ii) the increase of immunopositivity without increase of GFAP; (iii) and increase in GFAP-expression of the resident astroglia, which is by far the predominant factor.

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Several studies have demonstrated an increased amount of GFAP and its mRNA (Mathewson and Berry, 1985; Condorelli et al., 1990, 1999; Hozumi et al., 1990a,b; Steward et al., 1991; Norton et al., 1992; Eng and Ghirnikar, 1994) in the reactive glia, an increase that could be blocked by protein synthesis inhibitors (see, e.g., Steward et al., 1997). Our results on the chicken Bergmann-glia, which is free of GFAP in the intact cerebellum, clearly demonstrated that injury provokes GFAP expression in this resident astroglial population (Ajtai and Ka´lma´n, 1998). The same signaling mechanisms are involved in increasing GFAP expression as in other intracellular processes in response to extracellular stimuli, e.g., activation of MAP-kinase and of receptor tyrosine kinase (see e.g., Norenberg, 1994; Kahn et al., 1997; Steward et al., 1997; Condorelli et al., 1999). However, in some cases the increase of the GFAP-immunopositivity occurred so rapidly and was so intense, that the authors assumed that it reflected an early disassembly of filaments, which make more epitopes available (Amaducci et al., 1981; Eng et al., 1989; Du et al., 1999; Lee et al., 2000). Moreover, tissue loss may result in a relative increase of astrocyte number and GFAP-content (Norenberg, 1994). The first studies on GFAP null-mutants did not establish that GFAP-expression could be a conditio sine qua non for the reactivity of astrocytes (Gomi et al., 1995; Pekny et al., 1995), although the lack of GFAP intermediate filaments was not compensated for by any increase in vimentin (Pekny et al., 1995). However, double null-mutants (i.e., GFAP plus vimentin) (Pekny et al., 1999; Menet et al., 2001) as well as the use of antisense mRNAs (Weinstein et al., 1991) proved to be more effective for demonstrating the importance of these proteins. The obtained data suggest that GFAP is necessary for the formation of stable glial processes, e.g., in the glial reaction models in vitro (Yu et al., 1994; Ghirnikar et al., 1994; Xu et al., 1999), and for the motility of the astrocytes (Lepekhin et al., 2001). The surface and adhesion characteristics of the cells correlate with the presence or absence of GFAP (Menet et al., 2001); therefore GFAP affects cell – cell and cell – ECM connections. In general, the enhanced expression of GFAP seems to be the main indicator of the activation of astroglia (Eddleston and Mucke, 1993; Norenberg, 1994). However, an increase of GFAP-expression does not always indicate reactive gliosis, but occurs also as a consequence of different neural activities (Kraig et al., 1991; Steward et al., 1991, 1997; Canady et al., 1994) or hormonal effects (see e.g., Garcia-Segura et al., 1999; Hajo´s et al., 2000 as well as Section 9.7). As GFAP-expression during evolution seemed to decrease to the lowest possible level (Ka´lma´n, 2002), it is not surprising that microenvironmental alterations are usually followed by upregulation of GFAP, whereas downregulation is infrequent (for an example, see Missler et al., 1994). Several authors reported the expression in reactive astrocytes of the intermediate filament proteins of the immature astroglia, vimentin (e.g., Dahl et al., 1982; Schiffer et al., 1986; Janeczko, 1993) and nestin (Clarke et al., 1994; Sahin Kaya et al., 1999; Krum and Rosenstein, 1999; Ko¨rnyei et al., 2000). Vimentin also occurs in some mature, but nonreactive types of astrocytes, e.g., Bergmann-glia (Pixley and de Vellis, 1984). It becomes expressed in the proximal astrocytes, and nestin is only found at the enlarged glial processes oriented towards the lesion (Schiffer et al., 1986; Ho¨ke et al., 1994; Ko¨rnyei et al., 2000). They are coexpressed with GFAP, and vimentin and GFAP form copolymers (Janeczko, 1993). The altered composition of the intermediate filaments

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changes their characteristics, including their effects on cell morphology, motility and adhesion (Abd-el-Basset et al., 1989; Rutka et al., 1997; Menet et al., 2001; Lepekhin et al., 2001). An increased level of actin was also found in the gliotic tissue in jimpy and shiverer mutant mice (Chen et al., 1993). Despite some in vitro data (e.g., Gagelin et al., 1995), an increased expression of the actin-anchoring proteins: vinculin, talin, and paxillin could, however, not be demonstrated, at least at the light microscopic level (Ka´lma´n and Szabo´, 2001a, see also for a review). No conspicuous immunopositivity was detected of plectin (Ka´lma´n and Szabo´, 2000), which is important in the organization of the cytoskeleton (as an intermediate filament associated protein (IFAP), see, e.g., Seifert et al., 1992; Rutka et al., 1997). IFAPs are minor quantity proteins that regulate the organization of intermediate filaments. The IFAP 70/280, which is characteristic of immature astroglia, is re-expressed in the glial reaction, in the proximal astrocytes (Yang et al., 1997). Reactive astrocytes also express the protein IFAP48, a protein related to ‘stellation’ in vitro (transition from epitheloid into stellate, process-bearing form), which is due to the cross-linking of GFAP into bundles (Abd-el-Basset et al., 1989). They also express high levels of microtubule associated protein 2 (MAP2) (Geisert et al., 1990) as well as of the Tau protein, also an MAP, otherwise known as a neuronal marker (Schinstine and Iacovitti, 1996).

6. Noncytoskeletal protein markers of reactive astrocytes Glutamine synthetase and glutamate transporters, EAAT1 and EAAT2 (see chapter by Schousboe and Waagepetersen) are highly expressed in the mature ‘resting’ astroglia, but their expression is further increased during the astrocyte reaction (Condorelli et al., 1990; Norenberg, 1994; Lievens et al., 2000). Glutamine synthetase is glia-specific, but it may also be expressed in oligodendroglia (Tansey et al., 1991) (see, however, also chapter by Derouiche). The expression of the calcium-binding S-100b protein, which is not strictly astrocytespecific (Boyes et al., 1986), also increases in reactive astroglia (reviewed by Norenberg, 1994), and at least part of the astrocytes become immunopositive to annexin-II, even at the light microscopical level (Ka´lma´n and Szabo´, 2001b). Beside other effects (see later), these substances decrease the disassembly of GFAP filaments to subunits (see, e.g., Ziegler et al., 1998; Bianchi et al., 1995; Garbuglia et al., 1995). A few other factors were also reported to be associated with reactive glia during the last decade. SCI occurs in mature astrocytes, but following lesion it is induced mainly in the proximal astrocytes (McKinnon and Margolskee, 1996; Mendis et al., 1996). J-31 is found in tanycytes and area postrema glia in the intact CNS (Malhotra et al., 1993; Ho¨ke and Silver, 1994). Following lesion, it associates with the intermediate filaments, similarly to the antibody 6.17, which was first used as a marker at the neuromuscular lesions, and later proved its usefulness on the white-matter (‘fibrous’) astrocytes as well as on the glial scar (Ridet et al., 1996). Another antibody, M22, applied at first on the AIDS virus (HIV)-induced reactive glia has also proved to be an effective marker of a fraction of the reactive astrocytes (Eddleston et al., 1996).

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7. Recruiting astrocytes for the reaction Various sources of reactive astrocytes should be considered: (i) the mature, local resident population; (ii) its mitotic derivatives; (iii) immigrating mature astrocytes; and (iv) newly generated cells from the persisting glioblasts or stem cells, mainly from the subventricular zone. Another question is, whether any astrocyte can participate in the response, or whether it is the privilege of a subpopulation. In a quantitative study an increase of mitoses around brain lesions was found by Cavanagh (1970), while the mitotic capability of mature, GFAP-positive astrocytes was evaluated by simultaneous immunocytochemistry and autoradiography as described by Hajo´s et al. (1981). Double-labeling studies with GFAP and autoradiography, incorporation of BrDU, or use of proliferative cell nuclear antigen PCNA (Latov et al., 1979; Janeczko, 1988, 1989; Miyake et al., 1992; Schiffer et al., 1993) have provided undeniable evidence of postlesional astrocyte proliferation. It is to be noted that cumulative labeling experiments suggested more intense mitotic activity than pulselabeling. No amitosis was found (Cavanagh, 1970). Astrocytes, however, accounted only for up to one-fifth of the dividing cells, and the mitotic astrocytes represented only 2% of the total astrocytic population (Cavanagh, 1970; Latov et al., 1979; Ludwin, 1985; Reier, 1986). Similar proportions were estimated by Janeczko (1988, 1989) and Schiffer et al. (1986, 1993). The general opinion is that this proliferation contributes only a small number of cells (Vaughn et al., 1970; Lindsay, 1986; Reier, 1986; Hatten et al., 1991; Fernaud-Espinosa et al., 1993; Hajo´s et al., 1993; Ridet et al., 1997), and carefully conducted quantitative studies often have shown no evidence of cell proliferation (Norenberg, 1994). The microglia accounted for the majority of mitotic cells following lesion by kainic acid (Murabe et al., 1982), whereas oligodendrocytes were the least responsive cells to injury. Following lesion, Ludwin (1985) and Xie et al. (1995) reported mitoses of mature oligodendrocytes alongside with astrocytes and microglia, but Maxwell et al. (1990a) reported that oligodendroglia exhibited neither qualitative nor quantitative alterations. Mitoses are more frequent around the ‘open’ lesions (Miyake et al., 1992), and mainly the proximal astrocytes proliferate, whereas the distal ones rather react with hypertrophy (Schiffer et al., 1993; Ho¨ke and Silver, 1994). Inhibition of cell proliferation by cytostatics did not inhibit the glial reaction (Billingsley and Mandel, 1982; Politis and Houle, 1985; Ka´lma´n and Fu¨lo¨p, 1991). Migration of mature astrocytes to the lesion is another possibility to recruit cells. Astrocytes do migrate, e.g., from the brain to the retina (Watenabe and Raff, 1988; Ling et al., 1989; Huxlin et al., 1992), and their velocity was estimated by Huxlin et al. (1992) to be 300 mm/day. Several authors reported astrocytic migration evoked in vitro by factors promoting glial reaction (e.g., Faber-Elman et al., 1996). Janeczko (1989) and Nieto-Sampedro (1998) suggested astrocyte migration towards the wound, in the latter paper including migration of BrDU-labeled astrocytes. Hatton et al. (1993), however, labeled astrocytes with fluorescent granules and found no migration to near the lesion in vivo. Hajo´s et al. (1993) also concluded that the migration could not be meaningful. In his review, Norenberg (1994) stated: “astrocyte migration has occasionally been cited…but not documented”. Eddleston and Mucke (1993) in their review ‘were unable to

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find convincing in vivo evidence that mature GFAP-positive astrocytes are able to migrate effectively’ (i.e., toward the lesion), and they suggested that the glial reaction “…reflects predominantly phenotypic changes of resident astroglia rather than migration or proliferation of such cells.” Some ‘immature’ features are displayed by the reactive astrocytes (Ridet et al., 1997). They express substances characteristic of immature astroglia, including the aforementioned nestin and vimentin (see above), IFAP 70/280, PSA-NCAM (Yang et al., 1997), and GD3 ganglioside (Amat et al., 1996; Lekman and Fredman, 1998). Several authors attributed these features to a dedifferentiation of the mature astrocytes (e.g., Malhotra et al., 1990; Yang et al., 1997; Hatten et al., 1991), which is necessary to re-enter the cell cycle, although they did not rule out the possibility that these cells originated from glioblasts and/or stem cells. In his review, however, Norenberg (1994) pointed out that “while a derivation from a progenitor cell cannot be excluded, little evidence supports this view”. Nevertheless, Frise´n et al. (1995) and Holmin et al. (1997) reported the proliferation of immature cells in the subventricular zone and their migration during maturation (GFAPexpression) towards the injury, although the authors did not rule out the capability of the resident glia to react. Schinstine and Iacovitti (1996) found markers of immature neurons (Tau, MAP2) in the reactive glia in vitro, and attributed it to their origin from a common neuron –glia progenitor. Ridet et al. (1997) also emphasized the role of stem cells. Our studies of the lesions of the molecular layer of cerebellum (Ajtai and Ka´lma´n, 1998) showed that the appearance of extrinsic astrocytes (from any source) around the lesion was negligible (although this could have been the reason that no demarcation developed). Another important piece of information is that vimentin and GFAP appear together in the reactive glia (e.g., Janeczko, 1993) and not sequentially, as during development (e.g., Pixley and de Vellis, 1984). These observations suggest that at least the major part of reactive astrocytes are neither newly formed nor ‘dedifferentiated’ to immaturity but mature, resident cells which express a characteristic spectrum of molecules. Studies based on the detection of the A2B5 marker (Miller et al., 1986, see also Ho¨ke and Silver, 1994) raised the possibility that reactive astrocytes may be a subpopulation (the so-called type-2 astrocytes). Recent results have, however, demonstrated that the classification of astrocytes into type-1 and type-2 according to the absence or presence of A2B5 has importance only in vitro (see, e.g., Franklin and Blakemore, 1995).

8. The sequence of events and the phases of glial reaction Already Del Rio-Hortega and Penfield (1927, cited by Reier, 1986) pointed out that the glial reaction proceeds in phases, in which different phenomena predominate: (i) an inflammatory phase with microglial reaction, (ii) an astroglial reaction with hypertrophy; (iii) a palisade-like arrangement of glial processes; (iv) the appearance of a connective tissue core; and (v) the final formation of definite glial scar (the latter three phases, of course, only in the case of the anisomorphic reaction to open lesions). This sequence of events will be followed in this review, except that the two last phases will be treated together. Several investigators have carried out time-course studies (Bignami and Dahl, 1976; Barrett et al., 1981, 1984; Berry et al., 1983; Miyake et al., 1988; Takamiya et al., 1988;

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Moumdijan et al., 1991; Fernaud-Espinosa et al., 1993; Norenberg, 1994). The most precisely timed descriptions are those of Mathewson and Berry (1985) and Maxwell et al. (1990a) for GFAP-immunohistochemistry and the ultrastructure, respectively, and that of Hozumi et al. (1990a,b) for the GFAP-synthesis and content. Considering that their descriptions do not diverge from each other and from the authors mentioned above, I do not cite them separately in the following description, which refers to rat cortex, and mainly to anisomorphic gliosis.

8.1. Inflammatory phase In the case of ‘closed’ injuries the inflammation is usually weak, and confined almost exclusively to the resident microglia, leading Norton et al. (1992) to state that ‘clearly some forms of injury do not involve inflammation and microglia’. Following ‘open’ injuries the inflammation is strong, committed by bleeding, and it recruits a number of blood-borne cells. In this case the inflammatory phase (peaking about 2 days postlesion) is characterized by tissue destruction (edema, cell swelling, tissue debris). Macrophages appear already during the first day. Their number peaks at day 4, then decreases until day 16, and they have almost disappeared by day 30, i.e., their presence, although gradually decreasing, extends into the following phases. The macrophages are of dual origin: bloodborne monocytes, and activated resident microglia. At first the resident microglia is activated (e.g., Amat et al., 1996). The reduction in blood-borne macrophages coincides with the increase in microglia between days 4 and 16, which corresponds to the suggested transformation from one to the other. According to Schnell et al. (1999), the inflammatory response to mechanical injury of comparable magnitude is more intense and lasts longer in the spinal cord than in the brain. The earliest expression of altered GFAP-immunopositivity can already occur during the inflammatory phase. Transitory (6 –24 h) decreases in GFAP-content and astrocyte number have been observed (Bjo¨rklund et al., 1986; Miyake et al., 1988; Hozumi et al., 1990a). Norenberg (1994) attributed these results to a damage of astrocytes, or to posttraumatic edema which could ‘dilute’ the astroglia. Ultrastructural studies also revealed the transitory disappearance of intermediate filaments and glycogen from the astrocytes (Reier, 1986). Very early increases of GFAP (in 0.5 –6 h postlesion) were also reported (Amaducci et al., 1981; Okimura et al., 1996; Hadley and Goshgarian, 1997), which could not be explained easily by ‘de novo’ synthesis (see Section 5). The GFAP mRNA level increases within 6 – 24 h (Hozumi et al., 1990b; Steward et al., 1991). By an extremely sensitive method (i.e., the induction of the GFAP-lacZ hybrid gene in transgenic animals), the increase could be detected within 1 h following the lesion (Mucke et al., 1991). The cell proliferation is most intense in the inflammatory phase and at the beginning of the astroglial phase. The mitoses appear on the first day (Cavanagh, 1970; Reier, 1986), even already at 2 h postlesion, according to Janeczko (1989). The peak of proliferation is during the first 2 –3 days (Cavanagh, 1970; Ludwin, 1985; Janeczko, 1989, 1993; Schiffer et al., 1993). The nearer to the lesion, the later the mitoses continue, until day 4– 8 (Cavanagh, 1970; Janeczko, 1989). As mentioned before, only a small fraction of the

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proliferating cells are astrocytes, the others are macrophages (blood-borne or resident), or even oligodendrocytes. The microglial proliferation (day 2 –3) precedes that of bloodborne macrophages and astrocytes (day 3 –4: Amat et al., 1996). 8.2. Astroglial phases: isomorphic and anisomorphic The astroglial phase lasts usually from the 3rd to the 14th postlesional days, i.e., when hypertrophic astrocytes dominate the landscape. This phase is indicated by a dramatical increase (or appearance) of GFAP-immunopositivity, within 36 –48 h. At first, there is an isomorphic phase (for about one day). By day 4 following open lesions, however, the proximal astrocytes orient their processes toward the lesion, to form a palisade-like pattern (anisomorphic zone). Electron microscopic investigations revealed a large number of gap junctions in these processes, which form a compact ‘cytoplasmic sheet’ between days 4 and 8. By days 7 or 8, the territory of reactive astrocytes is at its maximum. At cortical injuries, it almost occupies the whole ipsilateral hemisphere in the cross-section (see also Okimura et al., 1996). It extends even contralaterally along the corpus callosum (Amaducci et al., 1981; Ludwin, 1985; Moumdijan et al., 1991), although this was not seen by Mathewson and Berry (1985). The wide reach of the gliotic reaction may be due to signals spreading along extracellular pathways and/or through gap junctions (Moumdijan et al., 1991; Norenberg, 1994). After the second week the collateral (distal, isomorphic) reaction gradually disappears, the more peripheral the sooner. After 3 weeks the reaction is confined to the glial scar, which is formed by then (see below). Quantitative measurements of the GFAP-content reflect the immunohistochemical phenomena, with a rapid increase on the second postlesional day (Miyake et al., 1988; Hozumi et al., 1990a). The expressions of vimentin (Schiffer et al., 1986; Janeczko, 1993), nestin (Krum and Rosenstein, 1999), and IFAP 70/280 (Yang et al., 1997) have a similar time-course as that of GFAP. 8.3. Scarring The last phase is the formation of the glial scar (Berry et al., 1983; Maxwell et al., 1984, 1990a; Carbonell and Boya, 1988). Preliminary features of scarring appear already in the astroglial phase. The intruding meningeal fibroblasts infiltrate the core of the lesion between 4 and 8 days postlesion, and peak between days 8 and 10. Laminin is produced after 3– 5 days by the astrocytes for their basal lamina (see also Bernstein et al., 1985). This process is promoted by the meningeal cells and collagen fibers invading the site of the lesion (see also chapter by Mercier and Hatton). In a recent publication, Liesi and Kauppila (2002) found that the formation of a complete basement membrane is extended beyond one month, and that the astrocytes expressed type IV collagen, beside the laminin. Collagen fibers and ECM appear by days 8. They fuse with the astrocytic basement lamina, so that between days 8 and 12, a glia limitans and basement membrane develop. In parallel with the disappearance of the major part of distal reactive astrocytes, by day 16 or 20 a complete glia limitans membrane is established, and the fibroblasts become less numerous. Unlike the original one, the reconstituted glia limitans is thicker, consists of a

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complex system of multiple layers of flattened interwoven glial processes which are frequently joined by gap junctions, and the surface is uneven, covered by basal lamina along a (sometimes very thin) connective tissue core of fibroblasts and collagen fibers (Carbonell and Boya, 1988). Schwann-cells in some cases share a common basal lamina with astrocytes, and thus a PNS – CNS transition zone is formed. Between days 20 and 30, the scar contracts and persists at least for months. New blood vessels appear already after the first week. In some instances, revascularization is related to the basal lamina formation (Lawrence et al., 1984). It should be noted that the microglial and astroglial reaction and the scar formation overlap each other temporally. Since they are not strictly confined to one phase, the different phases can be distinguished only by the predominance of one or more characteristic phenomena. Let me take the liberty for a personal unpublished observation: the phases, mainly the astroglial one, seemed to be delayed when the lesion was severe, with a wide necrotic zone. It should also be emphasized that the above-mentioned description referred to the anisomorphic reactions following the ‘open’, penetrating injuries. In some isomorphic cases of gliosis following ‘closed’ injuries, e.g., in EAE (Aquino et al., 1988; Goldmuntz et al., 1986; Smith et al., 1983) or toxic necrosis (Brock and O’Callaghan, 1987) the appearance and disappearance of GFAP were reported to be slower. 9. Regulation of the glial reaction 9.1. General comments As mentioned before, the glial reaction corresponds to inflammation and wound healing, therefore, the regulating factors (interleukins [ILs], transforming growth factor [TGF-]-b1, parathyroid hormone-related peptide [PTHrP], etc.) are similar (Brodkey et al., 1993; Eddleston and Mucke, 1993; Lawson and Perry, 1995; Merrill and Benveniste, 1996; Funk, 2001). However, there are considerable differences from the other regions of the organism. According to Eddleston and Mucke (1993), these were evolutionary responses of CNS ‘to two different types of selective pressures: (i) to restrict the access of hematogenous cells to the brain by the BBB; (ii) the need of the factors secreted by hematogenous cells in the case of other wounds.’ Merrill and Benveniste (1996) also emphasized that ‘BBB excludes cells and large proteins, and in the CNS the expression of MHC I and II molecules, which are critical for antigen presentation, is extremely low, as well as the antigen-capturing effect of lymphatic system’. Lawson and Perry (1995) also mentioned “…scarcity of neutrophils and the delay of macrophages in the CNS inflammation…”, due to the following factors: (i) CNS endothelium does not express adhesion molecules for the blood cells; (ii) deficit in cytokine/ chemokine cascade to promote the activation of leukocytes; (iii) the microenvironment of CNS is anti-inflammatory (ECM, astrocyte products). Considering the ‘open’ injuries with bleeding, the ‘low access’ sounds peculiar, but it is supported by the glial demarcation by day 4 (see Sections 8.2, 8.3 and 10.4). Present data, which mainly have been obtained in in vitro experiments, suggest that the damage of neurons has less direct effect on astroglia, although direct effects of neuronal

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injury have also been reported (Forloni et al., 1994; Ho¨ke et al., 1994; Guenard et al., 1996; Ko¨rnyei et al., 2000). The effect is mediated by the cells of the inflammatory phase, i.e., mainly the macrophages (including microglia); however, lymphocytes, thrombocytes, endothelial cells, as well as meningeal and perivascular fibroblasts, participate when the injury has destroyed the gliomeningeal and gliovascular barriers. The primary trauma is, therefore, followed by sequences of events, including secondary injuries of neurons (Ridet et al., 1997; Wu and Schwartz, 1998; Schubert et al., 2000; Schwab et al., 2001). The mediating factors are mainly cytokines (ILs, etc.) forming a very complex system of cascades and loops, with auto- and paracrine mechanisms and positive and negative feedback and feed-forward controls, which have not yet been completely understood. For general reviews, see Ridet et al. (1997) and Schubert et al. (2000); for cytokines in inflammatory CNS reactions, see Merrill and Benveniste (1996); for the cooperation with the immune system, see Aloisi et al. (2000), for the effects on BBB, see Abbott (2000); for the factors produced by the astroglia the most comprehensive review even today is Eddleston and Mucke (1993).

9.2. Tissue destruction and microglial activity The tissue injury produces several factors activating the glial reaction: potassium ions (Kþ), bFGF, CNTF, IL-6, PDGF, TNF-a, TGF-b1, PTHrP (from destroyed neurons and/or astrocytes – see chapter by Nakagawa and Schwartz), MBP (myelin basic protein), endothelin (from vessels), thrombin, PDGF, bFGF (from blood), T-factors, TNF-a, IL-1b, TGF-b1 (from lymphocytes); components of the complement system, and vasogenic edema and invading blood serum may also act as triggers (for review, see e.g., Brodkey et al., 1993; Eddleston and Mucke, 1993; Norenberg, 1994; Merrill and Benveniste, 1996; Compston et al., 1997; Ridet et al., 1997; Acarin et al., 2000; Streit, 2000; Funk, 2001). These factors activate the microglia (e.g., Brodkey et al., 1993; Levison et al., 1996; Ridet et al., 1997; Luo et al., 2000; Schubert et al., 2000), and/or astroglia and fibroblasts (see later). Platelet activating factor (PAF) mediate the effect of injury, among others, to the lymphatic system (Brodie, 1994). Apoptosis, however, does not elicit a local inflammatory response (e.g., Compston et al., 1997). Other humoral factors of inflammation, e.g., bradykinin, histamine, serotonin (Abbott, 2000) and substance P (Liu, 1995) are rapidly produced and have role in the vasoregulation and opening of BBB. In vitro data suggest that neurotransmitters, such as b-adrenergic agonists (e.g., Imura et al., 1999, see, however, opposite opinions: Wandosell et al., 1993; Schubert et al., 2000, in details in Section 9.8) and purine derivatives, including ATP (Brambilla et al., 2000; Brambilla and Abbracchio, 2001), and its degradation products, such as adenosine (Schubert et al., 2000) also promote glial reaction (for a review on the effect of purine derivatives on glia in general, see Neary et al., 1996). The macrophages produce a wide spectrum of factors, such as TNF-a, IL-1b, IL-6, bFGF, g-IFN, TGF-b1, MIPs (Giulian et al., 1986; Brodkey et al., 1993; Eddleston and Mucke, 1993; Norenberg, 1994; Merrill and Benveniste, 1996; Compston et al., 1997; Ridet et al., 1997; Acarin et al., 2000; Luo et al., 2000; Pousset et al., 2000; Schubert et al., 2000).

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Several of these factors (mainly TNF-a, IL-1b, MIPs), promote and/or prolong the inflammation directly or via their cytotoxic effects (secondary neuronal injuries). These factors also activate the astrocytes, and can even affect the fibroblasts, promoting their reaction and ability of scar formation. On the other hand, microglia can also produce neuroprotective and anti-inflammatory substances either directly (EGF, IL-1Ra), or by activating astrocytes. 9.3. Activation of astroglia Some of the factors activating the astrocytes following injury have also a role in the astrogliogenesis of the intact CNS. EGF, bFGF, PDGF are mitogens for the astrocytes as well as their different precursor cells (Cameron and Rakic, 1999; Compston et al., 1997), and promote astrocytic migration (mainly in vitro, Takamiya et al., 1986; Faber-Elman et al., 1996). CNTF is an activator of GFAP-expression and astrocytic maturation (Compston et al., 1997; Kahn et al., 1997). Some data, however, suggest that at least bFGF (Eclancher et al., 1990; Hou et al., 1995); and CNTF (Kahn et al., 1995) can provoke the astroglial reaction alone. Other factors mainly acquire a role following injuries, e.g., TGF-b1 (Lindholm et al., 1992; Logan et al., 1992, 1994; for review, see Bottner et al., 2000), TNF-a (Balasingam and Yong, 1996; Zhang et al., 2000; Schubert et al., 2000), IL-1b (Giulian et al., 1988, 1993; Sievers et al., 1993; Liu et al., 1994; Herx and Yong, 2001; Schubert et al., 2000), although the last two, according to Mizuno et al. (1994), also participate in the gliogenesis. These factors (mainly TGF-b1 and IL-1b) are considered to play a pivotal role in the glial reaction. They have complex effects, promoting astrocyte proliferation, migration, hypertrophy and GFAP expression. There are, however, differences: TGF-b1, in contrast to the other factors, stops astrocyte proliferation, and promotes maturation and GFAPsynthesis. These factors have effects on the inflammatory and scarring phases as well (see above), and are thus involved in every step of glial reaction. IL-6 (Fattori et al., 1995), and TGF-a (Rabchevsky et al., 1998) as well as MIPs (Luo et al., 2000) activate the astrocytes directly. Leukemia inhibitory factor (LIF) might also modulate the postlesional activity of astrocytes (Banner et al., 1997; Ridet et al., 1997). The lymphocyte-produced INF-g is mitogenic for astrocytes (Compston et al., 1997). The recently described stromal cell derived factor (SDF-1) promotes astrocyte proliferation in vitro (Bajetto et al., 2001). The wide progression of astroglial activation into the noninjured brain areas can be induced by diffusion of soluble activating factors along extracellular pathways and/or through gap junctions (see also Section 4; Schiffer et al., 1986; Moumdijan et al., 1991). It should be noted that the data on astrocyte proliferation and migration are mainly derived from in vitro observations, and their importance for the in vivo situation has been challenged (see Section 7). 9.4. Astroglial activity The most comprehensive list of astrocyte-derived factors is that of Eddleston and Mucke (1993); their data, however, have been verified and complemented by a series of other authors (see also chapter by Nakagawa and Schwartz). Astrocytes produce TGF-b1,

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bFGF, PDGF (McMillian et al., 1994; Compston et al., 1997; Ridet et al., 1997), PTHrP (Funk, 2001); GMF (glia maturation factor, Turriff and Lim, 1981; Wang et al., 1992; Norenberg, 1994), GDNF (glia derived nerve growth factor, Miyazaki et al., 2001), VEGF (Krum and Rosenstein, 1998, 1999; Proescholdt et al., 1999). NGF (Lindholm et al., 1992), CNTF (Stockli et al., 1991), TNF-a (Acarin et al., 2000), GIF (glia inhibitory factor, Hozumi et al., 1998); ILs (1b, 2, 3, 4, 6, 8,10) (Giulian et al., 1993; McMillian et al., 1994; Compston et al., 1997; Ridet et al., 1997; Acarin et al., 2000; Stanimirovic et al., 2001; Hulshof et al., 2002). These factors have effects in many directions. First, some of them affect the astrocytes themselves by autocrine activity (e.g., Stanimirovic et al., 2001) and thereby make the gliosis self-sustaining. Second, they provide a feedback to the microglia, e.g., by TGF-b1 (negatively, Schubert et al., 2000), by IL-3 and granule cell colony stimulating factor (G-CSF)-1 in response to IL-1b and TNF-a (Frei et al., 1986; Giulian et al., 1993; Compston et al., 1997), by stimulating microglial FGFR3 receptors with bFGF (Ballagriga et al., 1997), or by MIP-mediated induction of macrophage chemoattractants (Luo et al., 2000). Feedback mechanisms affect the BBB as well: TGFb1 limits (maybe due to its effect on the scarring, see below), whereas VEGF promotes BBB permeability and cell penetration (Eddleston and Mucke, 1993; Krum and Rosenstein, 1998, 1999; Proescholdt et al., 1999). Some factors are neuroprotective (see later), or communicate with the meningeal and perivascular tissue, and promote scar formation (see below).

9.5. Scarring The factors bFGF (Berry et al., 1983; Maxwell et al., 1990a; Mahler et al., 1996) and TGF-b1 (Logan et al., 1992, 1994; Eddleston and Mucke, 1993; Brodkey et al., 1993; Liesi and Kauppila, 2002) attract fibroblasts, regulate the deposition of ECM proteins (tenascin, laminin, fibronectin, collagen IV, produced by astrocytes) and the formation of the basement membrane. TGF-b1 is a key inducer of scarring. Its effect on ECM deposition is mediated by another cytokine, CTGF (connective tissue growth factor) (Schwab et al., 2001). EGF antagonizes this effect (Brodkey et al., 1993). IL-1b also induces laminin- and collagen IV expression in the astrocytes and it is a key factor in formation of the basement membrane (Scripter et al., 1997; Liesi and Kauppila, 2002). NGF stimulates process formation in meningeal cells, and may thus promote scarring (Frise´n et al., 1998). Meninges control glial scarring by the release of TGF-b1 and PTHrP, whereby they control the activity of dipeptidyl peptidase II, which degrades the ECM (Struckhoff, 1995a). Astrocytes have PTHrP-receptors, and a meningeal – astrocytic paracrine loop may operate (Struckhoff and Turzynski, 1995). In vitro meningeal cells stimulate the formation of astrocyte processes (Ness and David, 1997), most probably via PTHrP, and they induce a glia limitans-like structure (Struckhoff, 1995b). Brain injuries, mainly when followed by scarring, can induce the formation of new vessels. The growth factor VEGF (Krum and Rosenstein, 1998, 1999; Proescholdt et al., 1999) has important effects on angiogenesis by stimulating endothelial proliferation and upregulate the vasoactive adhesion molecules ICAM-1, VCAM-1, and PECAM

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(see also Schnell et al., 1999). TGF-b1 (Brodkey et al., 1993) also participates in the postlesional vascularization. 9.6. Feedback effects and balancing At every point of the cascade regulating the glial reaction, there are feedback effects and factors mitigating and/or balancing the tissue destruction. The cytotoxic effects of microglia, and the neuroprotective effects of astrocytes, which counter-balance each other (Giulian, 1993; Giulian et al., 1993) can also be regarded as positive and negative feedback, causing and preventing further secondary injuries. The feedback on microglia by astrocytes has already been mentioned. Some cytokines have anti-inflammatory effects. The same factors (e.g., IL-6, TGF-b1) may at first amplify, and then attenuate the reaction (Merrill and Benveniste, 1996). Production of IL-4, IL-6, IL-10 by reactive astrocytes (Balasingam and Yong, 1996; Schubert et al., 2000; Hulshof et al., 2002) attenuates reactive gliosis, e.g., by inhibition of the synthesis of TNF-a (a positive feedback factor, Schubert et al., 2000) and other pro-inflammatory cytokines synthesized by microglia. A so-called IL-1 receptor antagonist (IL-1Ra) is produced by both microglia and astroglia (Scripter et al., 1997; Pousset et al., 2000; Stanimirovic et al., 2001). A kinase inhibitor (p27) has a role in the contact-dependent inhibition of reactive gliosis in vitro and in vivo (Koguchi et al., 2002). TAPA (target of anti-proliferative antibody, CD –cluster determinant-81) is upregulated in the glial scar (Peduzzi et al., 1999), but not in the immature brain. Neurons suppress reactive astrocytes via the 5-HT5A serotonin receptor, which inhibits cAMP accumulation (Carson et al., 1996). The data listed here were mainly from in vitro experiments, and those obtained in vivo referred mainly to the glial reaction to an ‘open’ wound. However, those describing isomorph reactions to ‘closed’ wounds did not diverge from the others (see e.g., Murabe et al., 1982; Ballagriga et al., 1997; Acarin et al., 2000; Luo et al., 2000; Streit, 2000), except that in this case the contribution of the hematogenous cells is much less important, and that of connective tissue elements is negligible. 9.7. Remarks to the humoral regulation The general hormonal constitution of the organism has relatively little effect on the glial reaction. According to Vijayan and Cotman (1987) hydrocortisone decreases the inflammatory but increases the astroglial phase. Sex steroids (either male or female) mitigated the glial reaction after injury (Garcia-Estrada et al., 1993; Garcia-Segura et al., 1999). Thyroidectomy had no permanent effect (Miyake et al., 1989; Ka´lma´n et al., 1991a), and the same applied to adrenectomy in our preliminary experiments (Ka´lma´n et al., 1991a). Quercetin (vasopermeability factor, vitamin P) might possess an antigliotic effect. In vitro (i.e., independently from any effect on vessels) it reduced the increase of GFAP, and cell hypertrophy (Wu and Yu, 2000). At first the regulation of the glial reaction seems to be chaotic, a pell-mell, in which every cell produces every factor, and every factor affects every function of every cell. Some principles, however, should not be disregarded: (i) due to the limited access of CNS

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for hematogenous cells, astrocytes must contribute and complete an inefficient microglial production of the same pro-inflammatory and other factors, to keep them at their proper level (see the comments of Eddleston and Mucke, 1993; Lawson and Perry, 1995; Merrill and Benveniste, 1996, cited at the beginning of Section 9.1); (ii) although the effects are similar, they occur by/on different cells at different places and/or at different moments (Si duo faciunt idem, non est idem!: when two persons do the same thing, it is not the same thing!); (iii) several data refer to in vitro observations, whereas others were obtained in vivo; (iv) one factor can affect the same general process in multiple manners, at different points and via different effectors (in the same manner as an operation code to the different parts of an army), (v) the production intensity of the factors can change during the glial reaction (Acarin et al., 2000); (vi) the effects of a factor (e.g., FGF 1– 3) depend on the receptors expressed by the target cells (Ballagriga et al., 1997; Ridet et al., 1997), and when different factors act together, some effects can be enhanced by synergism or attenuated by antagonism: the spectrum of effects exerted by a single factor (e.g., IL-6, TGF-b1) may, therefore, be versatile (Merrill and Benveniste, 1996; Funk, 2001); (vii) simultaneous production of antagonistic factors (e.g., cytotoxic and cytoprotective interleukins, or IL-1b and IL-1bRa (Pousset et al., 2000; Stanimirovic et al., 2001) can balance an effect and keep it under control. Synergistic factors can in some cases affect parallel, but separate pathways leading to the same end result, i.e., they can replace each other (see Herx and Yong, 2001 for IL-1b null mutants, or Luo et al., 2000, for chemoattractant deficiency); in other cases they might act only when applied simultaneously (like the different keys of a safety box) to prevent adversive, unnecessary, ‘illegal’ effects.

9.8. Intracellular regulation The intracellular regulation of cell functions involved in the glial reaction occurs via similar mechanisms as in other tissues. In the astrocytes, the pro-inflammatory cytokines (e.g., IL-1b, TNF-a) induce the expression of the early genes, and these, in turn, recruit the acute phase genes. These genes code acute-phase proteins, such as complement C3 and antichymotrypsin. The transcription factors (CCAAT/enhancer binding protein beta and delta) are the key regulators in the activation of the genes of these proteins, i.e., these genes have roles as immediate-early genes (Cardinaux et al., 2000). Comprehensive studies of the activation and role of early genes (immediate genes), among them proto-oncogenes, were performed by Dragunow and Hughes (1993) and Wu and Yu (2000). As in other cells, the transduction pathways leading to gene activation operate via the MAPK (Erk), tyrosine kinase receptor systems (trkA: Lee et al., 1998, trkB: McKeon et al., 1997), forming a cascade (O’Callaghan et al., 1998). The role of cAMP has also been supposed for a long time (e.g., Fedoroff et al., 1984). Wandosell et al. (1993), however, has challenged this, considering dBcAMP-treated astrocytes as being much closer to untreated primary cultures of astrocytes than to reactive astrocytes. According to the review of Schubert et al. (2000), the role of the cAMP signaling is rather complex. It brings microglia into a less activated state and suppresses the cascade-stimulating effects, but not the negative feedback effects. It reduces the neurotoxic activity of astrocytes but

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promotes their neuroprotective activity. Participation of a system using cyclooxygenase-2 intermediates in reactive astrocytes, e.g., in the effect of purine derivatives, was reviewed already by Norenberg (1994) and Ridet et al. (1997). However, an increasing amount of data is becoming available, e.g., triggering of gene expression in reactive astroglia by inflammation-mediated activation of nuclear-factor kappaB (Gabriel et al., 1999; Stanimirovic et al., 2001) as well as involvement of target of anti-proliferative antibody (TAPA) (Peduzzi et al., 1999), the immediate early gene coding the connective tissue growth factor, CTGF (Schwab et al., 2001). An early activation of polyamine biosynthesis has been shown in various models of mechanic and ischemic injuries (Zini et al., 1990). The permeability of some ion channels and electrophysiological properties are also modified in the astroglia following brain lesions (see e.g., Ridet et al., 1997; Westenbroek et al., 1998; D’Ambrosio et al., 1999; Schro¨der et al., 1999– see also chapter by Walz). 10. Functions of the glial reaction 10.1. Introductory remarks The molecular profile reveals that reactive astrocytes may benefit the injured nervous tissue. Many of the molecules were already expressed during development and were downregulated when the development was complete (for a very comprehensive review see: Eddleston and Mucke, 1993; for recent ones: Ridet et al., 1997; Stichel and Mu¨ller, 1998a). Depending on the characteristics of the injury, and the stage of the glial reaction, different functions predominate. In the inflammatory phase, reactive astrocytes engage in elimination of necrotic tissue, debris, and blood, as well as in immune surveillance. In the astroglial phase they demarcate and ‘seal’, the decaying tissue, protect against toxic, ischemia-induced, and other pathological metabolic factors, and fill the space left over by neuronal destruction. In the scarring phase, they form a new glia limitans and basement membrane, and assist in the revascularization. With respect to neuronal regeneration, the reactive glia has in general been considered to be an impediment, although the beneficial effects are considerable. This contradictory phenomenon, which is of utmost clinical significance, will be discussed in the following sections. 10.2. Debris elimination and secondary tissue damage The elimination of the dead or dying neurons and the tissue debris is a precondition for healing. Both the resident microglia, and the blood-borne macrophages participate in this process, and also in the immune defense, e.g., by immune surveillance and antigen presentation, which indirectly promotes the elimination of unnecessary macromolecules and cell fragments (Aloisi et al., 2000; Hickey, 2001). In the case of nonpenetrating (toxic, ischemic, etc.) injuries the resident microglia undertake most of the task (Perry and Gordon, 1988; Graeber and Streit, 1990; Streit, 2001). The participation of astrocytes in these processes seems to be less extensive. Although the phagocytic activity of astrocytes is frequently mentioned in reviews (e.g., Norenberg, 1994), original reports demonstrating

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astrocytic phagocytosis are scarce. Most probably it does not match that by macrophages, and it may be confined to special cases and/or structures. Astroglial cells actively remove detritus (e.g., formed during exposure to toxic chemicals) from nerve cells (Cavanagh et al., 1990), and phagocytose degenerated nerve endings (Hajo´s et al., 1990a). In the very early stage of CNS development (E12 – E15 in the rat), neuroepithelial cells already phagocytose cell debris (Ka´lma´n, 1989). Astrocytes produce apolipoprotein E, which plays an important role in the removal of lipids (myelin debris) that are accumulated following injury (Eddleston and Mucke, 1993; Norenberg, 1994; Schubert et al., 2000)— see also chapter by Ito and Yokoyama). Astrocytes express MHC antigens in vitro, and present antigens to T-lymphocytes, but several studies argue against a major role of astrocytes in debris elimination in general (Frank et al., 1986; Eddleston and Mucke, 1993; Norenberg, 1994; Merrill and Benveniste, 1996). The protease systems (proteases and their inhibitors) also participate already in the elimination of cell and ECM remnants. They have, however, multiple functions during the remodeling of ECM, in blood coagulation, and in the inactivation of cytokines and other factors. By these activities, they have a role in neuroprotection, and influence axon growth. The members of these systems are, e.g., metalloproteases, cathepsin, a1-antichymotrypsin-like protein inhibitor, protease-nexin, plasminogen activator/plasmin (Gloor et al., 1986; Brodkey et al., 1993; Eddleston and Mucke, 1993; Norenberg, 1994; Ridet et al., 1997; Rivera et al., 2002; for a comprehensive review of the systems produced by reactive glia, see Eddleston and Mucke, 1993). Members of protease systems are manufactured by both astrocytes and microglia, and their expression is under control of cytokines (Norenberg, 1994; Ridet et al., 1997). The inflammatory processes and the blood coagulation affect each other mutually in the CNS as well as in the organism in general. Besides the aforementioned production of plasminogen activator and plasmin (Brodkey et al., 1993) the brain tissue coagulation factor, thromboplastin, is predominantly expressed by astrocytes (Eddleston and Mucke, 1993). In addition to its benefits, the inflammatory phase exerts seemingly adverse effects. As mentioned above, the neural destruction occurs in two subsequent phases: the primary, direct and the secondary, indirect phase. Following injuries, there is usually a collateral zone (penumbra in the case of ischemic damage [see chapter by Ha˚berg and Sonnewald]) of damaged but still viable neurons, which are subjected to secondary damage. The necrotic zone displays several metabolic abnormalities (ischemia, acidosis, increased intracellular calcium concentration, hyperammoniaemia, hyperkaliaemia), which are disadvantageous for the neurons. In addition, the microglia produce several neurotoxic agents during their elimination activity. The enzyme systems, which are involved in prostanoid and leucotriene synthesis and in the production of inflammatory mediators and second messengers, are very active, e.g., the membrane-bound and cytosolic phospholipase A2, cyclooxygenase 2, lipoxygenase. Their activity increases the levels of toxic intermediates and side products: free fatty acids, free radicals, lipid peroxidation products, as during inflammation in general. TNF-a enhances the neurotoxic effects. Astrocytes also contribute to the formation of toxic substances (mainly under influence of IL-1b), and a correlation is found between the level of these toxins and neuronal degeneration (Eddleston and Mucke, 1993; Ridet et al., 1997; Stephenson et al., 1999; Aloisi et al., 2000; Schubert et al., 2000).

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The intensity of the inflammation depends on the cause of the injury. The opinions are divided about the role of inflammation. On one hand, the more intense and prolonged the inflammatory phase and macrophage activity are, the more extended and intense the tissue destruction, and the more noticeable the gliomeningeal scar (e.g., Giulian, 1993; Giulian et al., 1993; Balasingam et al., 1996). According to Giulian (1993; see also Giulian et al., 1993), a neurodegenerative microglia and a neuroprotective astroglia are competing during the glial reaction. David et al. (1990), however, reported evidence that the axonal growth-promoting properties near the lesion (see below) may be produced by macrophages, at least in the optic nerve, and macrophages augment the production of NGF (Heumann et al., 1987), and fibronectin (Brodkey et al., 1993). Several authors also claim that the disadvantage of the CNS is the inappropriately poor access to tissue elimination by macrophages and lymphocytes, which cannot be perfectly compensated for by the activity of astrocytes (see also in Sections 11.2 and 11.4, and David et al., 1990; Norenberg, 1994; Abbott, 1995; Lawson and Perry, 1995; Merrill and Benveniste, 1996; Schwartz et al., 1999; Kipnis et al., 2001). Inappropriate astrocyte function may play a role in the pathogenesis of Alzheimer’s disease, by inhibition of macrophageal phagocytosis and destruction of b-amyloid protein (Shaffer et al., 1995; Schubert et al., 2000; Martins et al., 2001). To perform their tasks, the glial cells must survive, for which they have sufficient selfdefense systems. As in other part of the organism, the inflammatory effects are balanced by lipocortins, e.g., lipocortin-1 (annexin-1, calpactin II) (Johnson et al., 1989). These lipoproteins are induced by steroids and mediate their anti-inflammatory activity (Norenberg, 1994; Young et al., 1999). Annexin 1 has been demonstrated in both microglia and astrocytes (Eberhard et al., 1994; Johnson et al., 1989; Mullens et al., 1994). Annexin II was also detected in specimens from victims of human neurodegenerative diseases (Eberhard et al., 1994) and in reactive astrocytes (Ka´lma´n and Szabo´, 2001b). Heat-shock proteins are also induced during the glial reactions. They are chaperones, i.e., they correct the noncovalent assemblies of other polypeptides in vivo. They preserve the integrity of intermediate filaments, and prevent stress-induced unfolding of cellular proteins (for review, see, e.g., Head and Goldman, 2000). Among the several types of heatshock proteins, HSP27 stabilizes the cytoskeleton, and it is also an antioxidant in astrocytes; HSP32 is an antioxidant in macrophages; HSP47 contributes to the ECM remodeling (Salvador-Silva et al., 2001). APG-2, a member of the heat-shock protein 110 family, has been demonstrated in reactive astrocytes (Lee et al., 2002). The alphaB crystallin, which accumulates in reactive astrocytes as eosinophilic inclusions (see Section 4) is also a heat-shock protein, and it modulates intermediate filament organization (Sanz et al., 2001; Acarin et al., 2002).

10.3. Neuroprotection Several neuron-damaging effects menacing the injured but only partially damaged neurons are neutralized by the reactive glia. The neuroprotective role comprises several activities, including the production of appropriate molecules. For reviews, see Eddleston

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and Mucke (1993) and Muller et al. (1995). These functions are attributed mainly to the collateral, peripheral and transitory ‘isomorphic’ (distal) astrocytes. Since astrocytes can cover their energy demands by glycolysis (at least to a large degree), they generally survive transitory periods of oxygen-deprivation better than neurons. They supply neurons with anaplerotic metabolites, such as glutamate, which is crucial for normal glutamatergic and GABAergic transmission (see chapter by Schousboe and Waagepetersen), but during energy deprivation neuronal –astrocytic metabolic interactions become impaired (see chapter by Ha˚berg and Sonnewald). It has also been speculated that astrocytes may supply neurons with pyruvate and lactate, but this concept has been severely challenged (see chapter by Roberts and Chih). Accordingly the notion that astrocytes supply neurons with substrates for energy production (Eddleston and Mucke, 1993; Malhotra et al., 1993; Dringen et al., 1994) may have to be revised. Astrocytes express carboanhydrase activity (Cammer et al., 1989) as well as Hþ/Naþ and HCO32/Cl2 exchange channels, and they are important in pH regulation of the brain (see chapter by Bevensee). The astrocytes remove the toxic products of the metabolism of metals. Transferrin and its receptor on reactive astrocytes suggest that they may help diminish the excess of iron. The so-called ‘Go¨mo¨ri astrocytes’ have an important role in controlling metal toxicity. They express metallothionein, which binds heavy metals (Young et al., 1991; Eddleston and Mucke, 1993). Metallothionein was, however, not found in the remote (secondary) gliosis of thalamus (Acarin et al., 1999). Astrocytes provide protection against free radicals and oxidants (Norenberg, 1994). Antioxidant enzymes, e.g., heme oxygenase HO-1 exhibit high activity (see chapter by Schipper), and the apolipoproteins E and D are also antioxidants (Eddleston and Mucke, 1993). Astrocytes synthesize glutathione (Yudkoff et al., 1990), and re-reduction of ascorbic acid and oxidized glutathione are quite active. For the antioxidant defense of the brain by reactive astrocytes, see the review of Wilson (1997). One of the main tasks of astrocytes, be they normal or reactive, is to decrease the extracellular concentration of glutamate below toxic levels (e.g., Rosenberg and Aizenman, 1989; Condorelli et al., 1990; Norenberg, 1994; Lievens et al., 2000). They do so by intense glutamate uptake by the two astrocyte-specific glutamate-transporters EAAT1 (GLAST) and EAAT2 (GLT-1). Besides the destroyed neurons, microglia is also a source of glutamate (Schubert et al., 2000). However, reactive astrocytes in Alzheimer’s disease may show a decreased ability to accumulate glutamate (see chapter by Barger), and Krum et al. (2002) did not find increase of the glutamate transporter GLT-1 in reactive astrocytes around stab wounds. Glutamate is also used to detoxify ammonium ions by production of glutamine, catalyzed by the astrocyte-specific glutamine synthetase. Astrocytic enzymes metabolize quinolinic acid, an NMDA (N-methyl-D -aspartate) receptor agonist (Eddleston and Mucke, 1993). Metabotropic glutamate receptors (mGluR3 and 5) are upregulated in reactive astrocytes (see, e.g., Aronica et al., 2000, 2001b). Most probably, they affect the glia – neuron communication, e.g., in the epileptogenesis in the hippocampus (see chapter by Shuai et al.). The humoral factors most effective in provoking the inflammatory reaction and glial scar are frequently potent neuroprotective agents as well (CNTF, bFGF, PDGF) (Eddleston and Mucke, 1993; Krupinski et al., 1997). Reactive astrocytes provide neuroprotection via the production of GDNF (Miyazaki et al., 2001) and NGF (Goss et al., 1998;

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Lee et al., 1998), as well as insulin-like growth factor (IGF-1), which stimulates myelination, and possibly protects neurons from delayed postlesional neuronal death (Ridet et al., 1997). Other effects are indirect, e.g., those of TNF-a, IL-1b, TGF-b1, and bFGF, which induce NGF synthesis in astrocytes (Lindholm et al., 1992; Compston et al., 1997). Analogously, PTHrP and TNF-a induce astrocytic expression of IL-6, which has a neuroprotective effect (Funk, 2001). By decreasing further neuronal degeneration, the neuroprotection indirectly decreases the risk of future inflammatory events. 10.4. Demarcation The anisomorphic astrocytes with their palisade-like process system demarcate the decaying tissue, inhibit further inpouring of cells and humoral factors, and confine the cytotoxic, inflammatory and devastating processes, and in doing so defend the viable CNS tissue from secondary damage (e.g., Fitch and Silver, 1997a; Stichel and Mu¨ller, 1998a). The chondroitin sulfate proteoglycans, as inhibitors of cell-migration, contribute importantly to this separation (Fitch and Silver, 1997a,b), and their distribution correlates with the inflammation and BBB destruction. The effect of the progressive formation of a gliotic barrier is reflected by the observation that the level of diffusible factors decreases significantly in brain-implanted probes between day 3 and 4 (Osborne et al., 1991). After the formation of a barrier the collateral (distal, isomorphic) astrocytes, which probably mainly have a neuroprotective function, gradually revert towards their normal phenotype (i.e., their GFAP-immunopositivity decreases below the detectable level). As previously mentioned, the reactive glia form a new (‘secondary’ or ‘accessory’) glia limitans during the scar formation, restoring the sequestered localization of the CNS tissue. The immunologically competent cells, as well as antibodies, complement inflammationprovoking cells and factors are also excluded in order to defend the central nervous tissue. As a part of their barrier function, the reactive glia also prevents the invasion of Schwann cells, which have the capability to remyelinate demyelinated central axons (Fishman et al., 1983; Berry et al., 1992; Franklin and Blakemore, 1995 – see also chapter by Dezawa). 11. CNS regeneration and the glial reaction 11.1. In the mature mammalian CNS Since Ramo´n y Cajal (1928, cited by Reier, 1986; Ho¨ke and Silver, 1994; Stichel and Mu¨ller, 1998a) the predominant view is that the reactive glia is an obstacle to the growth of axons (Berry et al., 1983; Reier et al., 1983; Bernstein et al., 1985; Reier, 1986; Lindsay, 1986; Brodkey et al., 1993; for recent reviews, see Fitch and Silver, 1997a; Stichel and Mu¨ller, 1998a). This is in spite of the fact that during development, the astroglia promotes neuronal growth (see below). Following injury, the axons form the characteristic ‘growth cones’ at the site of the lesion, adjacent to the reactive glia, but considerable elongation of the axons does not occur (Ramon y Cajal, 1928, cited by Reier, 1986). Neurons in the mature mammalian CNS have the intrinsic ability to regenerate their axons for long distances in appropriate

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conditions, e.g., in implanted peripheral nerve segments (Aguayo et al., 1987; David et al., 1990), and in vitro astroglia promote or permit axon growth (see, e.g., Hatten et al., 1984; Noble et al., 1984). The failure of CNS regeneration appears to be a function of the glial environment (Aguayo et al., 1987; Brook et al., 1994; Kawaya and Gage, 1991; Schnell and Schwab, 1993; Schwab et al., 1993 – see also chapter by Dezawa). In a few areas the axons regenerate even in the mature CNS, despite glial reaction. These systems are: the ventral (but not dorsal) hypothalamus (Alonso and Privat, 1993); the hypothalamic monoaminergic fibers (except for mechanical lesions, Bjo¨rklund and Stenevi, 1979; Chauvet et al., 1998), the hypothalamo – hypophyseal axons (Kiernan, 1970), the olfactory fibers (Anders and Johnson, 1990; Burd, 1993; Ho¨ke and Silver, 1994), dentate gyrus (Gage et al., 1988) and hippocampus (Brodkey et al., 1993; Represa et al., 1995). In the telencephalic cholinergic system the reactive glia is permissive in the presence of NGF (Kawaya and Gage, 1991). The regrowth of primary sensory fibers through the PNS –CNS border is debated (Reier, 1986; Pindzola et al., 1993; Ho¨ke and Silver, 1994). In these areas, however, the astroglia usually exhibits unique features. A conventional glial scar did impede the reinnervation, when transplanted into a regenerative system, e.g., from the optic nerve into the olfactory bulb (Anders and Hurlock, 1996), indicating that it overrode the beneficial local effects. During development, the astroglia have an important role nursing and guiding axons (see also below). Dense populations of GFAP-immunopositive astrocytes in the presumptive position of the main neural pathways have been described (Bignami and Dahl, 1973, 1988; Bignami et al., 1980; Hatten et al., 1984; Ka´lma´n et al., 1991b), this phenomenon is sometimes called ‘natural gliosis’ (Bovolenta et al., 1987, 1994). The growth control involves adhesion molecules (N-cadherin, N-CAM, integrins), ECM components as well as growth factors and other soluble signals. For the actions of these factors in general, see Adams and Watt (1993), Aumailley and Gayraud (1998), and Boudreau and Jones (1999); for their role especially in the CNS, see Tomaselli et al. (1988), Fitch and Silver (1997a), and Powell et al. (1997); and for the biochemistry of the ECM components, see Dow and Wang (1998). The control of axon growth, however, also involves inhibitions, to prevent axon growing into unnecessary, disadvantageous directions. Therefore, so-called ‘restrictive astrocytes’ form cordons (or boundaries, Laywell and Steindler, 1991; Laywell et al., 1992 for recent reviews see Fitch and Silver, 1997a; Powell et al., 1997; Stichel and Mu¨ller, 1998a), and produce inhibitory molecules. Menet et al. (2001) emphasized the correlation between the structure of the cytoskeleton, and the adhesion features of the astrocyte membrane. Reactive astrocytes express materials (e.g., ECM components) in vivo which have proven to be growth inhibitors in vitro, e.g., isolated by a nitrocellulose filter first implanted into the brain and then applied in cell culture (Rudge et al., 1989; McKeon et al., 1991, 1995; Brodkey et al., 1993). These substances seem to be identical to the ‘cordon-forming’ substances of the developing brain. The growth inhibitory molecules are proteoglycans, such as keratansulfate and the different types of chondroitin sulfate (phosphacan, neurocan, brevican, versican), and glycoproteins, such as tenascin (Faissner and Kruse, 1990; McKeon et al., 1991, 1995; Brodkey et al., 1993; Eddleston and Mucke, 1993; Pindzola et al., 1993; Dow et al., 1994; Ho¨ke and Silver, 1994; Norenberg, 1994; Powell et al., 1997; Ridet et al., 1997; Stichel and Mu¨ller, 1998a).

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These materials form a surface that is unable to support axon growth. Proteoglycans, most of which are produced by astrocytes, influence cell attachment by interaction with receptors. The interactions of proteoglycans with growth factors may hinder axon growth by binding and functional removal of growth signals from the injury site. The structure, production and effects of tenascin were reviewed by Brodkey et al. (1993), and more recently by Faissner (1997) and Jones and Jones (2000). Tenascin seems to have adhesive properties opposite to those of fibronectin. The effect of tenascin, however, depends on the circumstances, such as its concentration and interaction with other molecules (Adams and Watt, 1993; Taylor et al., 1993). Several papers (e.g., Bignami et al., 1988, 1992a; Mansour et al., 1990) suggested the role of a hyaluronate binding glycoprotein (brain hyaluronectin, BHN, HBGP, GHAP), produced by white matter astrocytes, as axonal growth repellent. In the territory of the glial scar in vivo, the inhibition of axon growth exhibits a temporal and spatial correlation with the zone where tenascin and chondroitin sulfate are expressed (Pindzola et al., 1993; Ho¨ke and Silver, 1994; Davies et al., 1997; Fitch and Silver, 1997a, b; Moon et al., 2002), an observation which supports the inhibitory role of these molecules. The molecular changes, which mediate the expression of inhibitory molecules following injury, however, have not yet been completely understood. The meningeal invasion seems to be an essential factor in the failure of axonal regeneration (Berry et al., 1983; Carbonell and Boya, 1988; Struckhoff, 1995a; Stichel and Mu¨ller, 1998a; Klapka et al., 2002). Meningeal cells in vitro decrease the permissiveness of astrocytes to axon-growth (Ness and David, 1997). Alonso and Privat (1993) found that the nonpermissive scar in the dorsal hypothalamus contained more tight and gap junctions between the glial processes than were found between astrocytes in the ventral hypothalamus, where regeneration did occur. The physical obstacle suggested earlier (see Reier, 1986) might at least partly be due to these junctions. In the tri-dimensional CNS in vivo, a compact structure can be a mechanical obstacle, whereas this is not the case in monolayer, bi-dimensional cultures, where the axons grow on the surface of the cells. Recently, data showing a beneficial effect of reactive glia have also been reported (Mansour et al., 1990; Ridet et al., 1997; Nieto-Sampedro, 1998). Some authors distinguish ‘permissive’ and ‘nonpermissive’ reactive glia (Malhotra et al., 1993; Ho¨ke and Silver, 1994). The majority of data suggesting a ‘permissive’ effect of the reactive glia on axon growth were obtained in in vitro models (Hatten, 1985; Hatten et al., 1991; Ho¨ke and Silver, 1994; Ridet et al., 1997; Wang et al., 1994). Hatten et al. (1991) and Hunter and Hatten (1995) supposed that the glial reaction could represent a return to the stage of development, when the astroglia still supported axon growth, and that this return could be induced by the lesioned and/or growing axons themselves. In vitro, neurons affect glial cells and modify their activity to promote nerve growth (Hatten et al., 1984, 1991; Hatten, 1985; Wang et al., 1994; Hunter and Hatten, 1995; Colombo et al., 1995). Reactive glia has been found to release growth-promoting substances, such as thrombospondin, fibronectin, variable amounts of laminin (Liesi, 1985; Ho¨ke and Silver, 1994), growth promoting proteoglycan (heparan sulfate), PSA-NCAM, growth factors (Alonso and Privat, 1993; McKeon et al., 1991, 1995; Brodkey et al., 1993; Eddleston and Mucke, 1993; Ho¨ke and Silver, 1994; McMillian et al., 1994; Ridet et al., 1997; Goss et al., 1998; Moon et al., 2002) and perhaps also pleiotrophin, a heparin-binding

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growth-associated molecule, HB-GAM (see, e.g., Hampton et al., 1992). Reactive glia can become permissive in the presence of growth factors (Kawaya and Gage, 1991). According to McKeon et al. (1991) and Ridet et al. (1997), the inhibition exerted by reactive glia can be attributed to the presence of inhibitors, not the absence of the supportive factors. Therefore, the balance between these factors may either support axonal growth or, as in most cases, inhibit it. Fitch and Silver (1997a) attempted to unify the permissive and the nonpermissive effects of reactive glia on regeneration by the concept of the ‘trophic oasis’ in which neurotrophic factors are concentrated by the glia, and the axons are unwilling to leave. Nieto-Sampedro (1998) pursued a similar idea by suggesting the ‘misguidance’ phenomenon as an inhibitory mechanism. According to this suggestion, the axons prefer to grow in the gliotic tissue, thereby being ‘misguided’ from their natural targets. 11.2. Other inhibitors of regeneration Besides the reactive glia, other mechanisms have also been suggested to be responsible for the failure of regeneration in the mature CNS. Oligodendroglia and the myelin sheath produce several factors inhibiting axon growth (reviewed by GrandPre and Strittmatter, 2001). Such factors include janusin (tenascin-R), an ECM molecule associated with oligodendrocytes (e.g., Brodkey et al., 1993), the membrane proteins NI-35 and NI 250 (Caroni and Schwab, 1988; Schwab et al., 1993), the myelin associated glycoprotein MAG (Li et al., 1996), and Nogo, a transmembrane protein (Bandtlow and Schwab, 2000; Huber and Schwab, 2000). It is consistent with the adverse effect of oligodendrocytes that their removal (Moon et al., 2000), or replacement with Schwann-cells (Aguayo et al., 1987; Berry et al., 1992) promote the regeneration of CNS axons. The insufficient elimination of tissue debris in general (Section 10.2), and especially that of myelin-fragments and their inhibitory factors underlies the regeneration failure according to Sivron and Schwartz (1994), Schwartz et al. (1999) and Kipnis et al. (2001). Failure of astrocytes to repopulate the site of the lesion (Sivron and Schwartz, 1994), and insufficient laminin re-expression (Liesi, 1985; Sivron and Schwartz, 1994) have also been taken into consideration, which is consistent with the beneficial effect of reactive glia discussed above. Accordingly, it may not be the glial reaction itself, but the presence or absence of growth factors that determine the regeneration or the lack of it (Kawaya and Gage, 1991). In general, Sivron and Schwartz (1994), as well as GrandPre and Strittmatter (2001) regard the regeneration failure as a result of several factors, among them reactive glia and myelin, whereas according to Stichel and Mu¨ller (1995, 1998a), and Davies et al. (1999) the reactive glia, and especially the ECM, are responsible. 11.3. In the immature mammalian CNS A long series of studies show that no glial reactions, or only ‘moderate’ or ‘minimal’ forms follow CNS lesions during a so-called ‘critical period’ of the development of the CNS. The appearance of glial reactivity during development is supposed to be responsible for the disappearance of regenerative capability (Sumi and Hager, 1968a,b;

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Gearhart et al., 1979; Bernstein et al., 1981; Berry et al., 1983; Barrett et al., 1984; Sijbesma and Leonard, 1986; Smith et al., 1986; Moore et al., 1987; Rudge et al., 1989; Maxwell et al., 1990b; Trimmer and Wunderlich, 1990; Firkins et al., 1993; Janeczko and Kowalska, 1993). Transplants of immature tissues do not generate any glial reaction against the host CNS (Olson et al., 1982; Zhou et al., 1989). Immature astrocytes can even mitigate glial reactivity and promote regeneration (Smith and Miller, 1990; Smith et al., 1986), although transplantation of immature cells can also be demarcated by glial reaction of the host tissue (Azmitia and Whitaker, 1983). The pro-inflammatory cytokines, supposed to play a pivotal role in inflammation (see above) can provoke gliosis in rats only when injected after a few day post natum, but not when injected before or at birth (Eclancher et al., 1990; Sievers et al., 1993; Balasingam et al., 1994; Kahn et al., 1995; Scripter et al., 1997). The data referred to above are not consistent regarding the earliest age at which lesions provoke reactive gliosis (usually between P2 and P12—see the papers cited above). According to our investigations (Ka´lma´n et al., 2000) there are three phases, with different scenarios: (i) an immature response: healing without glial reaction, regardless of the remaining deformities (after severe lesions), or even implants; the glia lining the tissue defect is similar to that of the intact cortex; (ii) a transitory phase: depending on the size of the lesion there is either healing without residuum, or with remaining tissue defect plus reactive gliosis following severe lesions; i.e., reactive gliosis develops, unless the lesion disappears before the appearance of the glial reactivity; and (iii) the mature response: healing always occurs with reactive gliosis. The age boundaries between the three phases in the case of the rat cortex were: transition between phases (i) and (ii) at P0, and between phases (ii) and (iii) at P5; severe lesions, afflicted either at P0 or P5, result in reactive gliosis by P7/8. Persisting implants can provoke glial scar even when they are implanted before the maturation of CNS, i.e., in the rat cortex from P0 (Balasingam et al., 1996), but not earlier than that (Ka´lma´n et al., 2000). During the early postnatal period, it is the manifestation of the reactive gliosis that can be linked to a certain age (in the rat cortex P7/8), not the age at which the response can be elicited, which varies considerably according to the severity of the lesion. This may explain the controversy of literature data. Another question is, whether the appearance of the reactive gliosis depends rather on general factors (e.g., immunological) or local tissue factors. Our investigations (Ajtai et al., 1997; Ka´lma´n et al., 2000) suggested the latter one, since the glial reactivity appeared at different ages in the different brain regions, e.g., later in the cortex, than in the diencephalon. Surveying the developmental data (Ka´lma´n et al., 2000), we concluded that in the cerebral cortex the glial reactivity follows the cessation of neuronal migration, and it just precedes the massive transformation of the radial glia into astrocytes. In contrast to the classical data of Bignami and Dahl (1974), in our quite extensive study the lesions did neither provoke GFAP expression in the radial glia in the rat, nor did they induce an earlier transformation into astrocytes. No glial reaction was revealed by immunostaining for nestin and vimentin before the appearance of GFAP during development (Ajtai and Ka´lma´n, 2000). These data are in accordance with the supposition of Hatten et al. (1991). By estimating the relation between the onset of glial reactivity and the sequence of events occurring during CNS maturation we can adapt the experimental data to the human brain.

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In primates, including man, several events of cortical maturation occur prenatally, including the transformation of vimentin-containing radial glia into GFAP-containing astrocytes (e.g., Levitt and Rakic, 1980), not postnatally as in the rat (e.g., Pixley and de Vellis, 1984). In humans, reactive gliosis has therefore been found in fetuses (Roessmann and Gambetti, 1986a,b). On the other hand, the inflammatory phase of the immature rat brain is like that in nonCNS tissues during the entire first postnatal week, and only after P7 does the rat brain acquire the characteristic CNS inflammatory response (Lawson and Perry, 1995). A striking difference is observed between the immature and mature CNS in the invasion of meningeal connective tissue and formation of glio– meningeal scar (Berry et al., 1983; Moore et al., 1987). PSA-NCAM, constitutively present in immature astrocytes (see above) inhibits the invasion of fibroblasts (Harel, 1991). Another important factor is that the new surfaces formed by lesions in the CNS in young animals rapidly come into close contact with each other (Reier, 1986; Kruger et al., 1986). It is in accordance with our suggestion on the role of neuronal migration (with glial reactivity following the cessation of this migration), which may cover tissue defects in the developing CNS. In the immature cortex, at E20, however, even implanted meningeal tissue did not provoke gliosis (Ka´lma´n et al., 2000). This seems to be a result of the lack of reactivity of the immature glia to the stimuli causing glial reaction in the adult CNS, because prenatally IL-1b induces restitution of glia limitans, rather than a glial scar (Scripter et al., 1997). Another question is whether the cessation of regenerative capability really coincides in time with the appearance of glial reactivity, or whether there are other limiting factors for regeneration. According to experiments in the spinal cord, the end of the ‘critical period’, permitting regeneration, coincided with the myelination (e.g., Caroni and Schwab, 1989; Shimizu et al., 1990; Ghooray and Martin, 1993), which is consistent with a major role of the myelin-associated inhibitors (see above). Other model systems suggested a rapid change of the EMC following lesion at the time when the CNS environment becomes restrictive for regeneration (Ethel and Steeves, 1993; Hasan et al., 1993; Pindzola et al., 1993; Dow et al., 1994). Several observations suggest that the ‘permissive’ and ‘nonpermissive’ features depend on the age (maturity) of astrocytes (Smith et al., 1986; Rudge et al., 1989; Norenberg, 1994). Liesi et al. (1983) found considerable laminin production only in the immature astrocytes. Axon bundles seeking alternative pathways, the so-called Probst-bundles, have been described in developmental defects of the corpus callosum (Probst, 1901; cited by Lent, 1984), and in lesions of the immature spinal cord (Kalil and Reh, 1982; Bregman et al., 1989; Firkins et al., 1993; Ghooray and Martin, 1993; Nicholls and Saunders, 1996). The axons, however, grow through the intact tissue, bypassing the lesion, rather than going through it. This phenomenon demonstrated that (i) the axons preserved their intrinsic capability of growing; and (ii) the failure of axon growth is not a consequence of CNS tissue maturation in general, but rather due to a local alteration resulting from the lesion. Results by ourselves suggested that: (i) axon growth failure following lesions could precede the glial reaction in the immature brain, e.g., in the corpus callosum, between E18 and P0 (Ajtai and Ka´lma´n, 2000), the possible growth inhibiting factors being the destruction of the pioneer axons and/or that of the guiding astroglia, together with the disturbance of neuronal migration and maturation, which may form a

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nonpermissive cell mass; (ii) axons might induce the formation of growth-supporting astrocytes, as suggested by the observation that GFAP-immunopositive astrocytes could be seen along the axon bundles which sought alternative pathways around the lesion (Ajtai and Ka´lma´n, 2000), a finding which is consistent with a previous supposition by Hatten and co-workers, based on in vitro results (Hatten et al., 1984; Hatten, 1985; Wang et al., 1994); and (iii) reactive glia could promote and re-orient axon growth resulting in the formation of an unusual axon bundle, an event which in the diencephalon occurs between P0 and P5 (Ajtai and Ka´lma´n, 2001), and is in agreement with the ‘misguidance’ mechanism hypothesized by Nieto-Sampedro (1998). These results demonstrate (i) that the expression of GFAP indicates solely an activation of the glia, but it does not determine its permissive or nonpermissive nature; and (ii) that the correlation between the appearance of the glial reaction and the disappearance of capacity for axon regeneration is complex, depending upon age, brain area and type of lesion.

11.4. In the nonmammalian CNS In the so-called ‘lower vertebrates’ (i.e., different groups of fish as well as amphibia and reptiles) postlesional regeneration in the mature CNS has been widely observed (e.g., Holder and Clarke, 1988). There are divergent opinions on the causes: (i) that the ependymoglia, which is characteristic of these animals (see Fig. 2 in chapter by Laming), preserve the pathway (‘blueprint’) of the degenerated axons (Singer et al., 1979); (ii) that the absence of the orthogonal intermembranous particles (characteristic of mammals) from their glia facilitates regeneration (Kastner, 1987); (iii) that the glial reactivity is weaker or missing (e.g., Bignami and Dahl, 1976); (iv) that vimentin and/or keratin forms the intermediate filaments in these vertebrates (Giordano et al., 1989; Rungger-Brandle et al., 1989; Wasowicz et al., 1994; Druger et al., 1994; Maggs and Scholes, 1986); (v) that HBGP is missing (Bignami et al., 1992b); (vi) that myelin-associated growth inhibitors are only present at low levels (Sivron and Schwartz, 1994). Bignami et al. (1992b) tried to develop a comprehensive picture of the effects of glial evolution on the regeneration. In their opinion GFAP and HBGP had appeared in the Chondrichthyes together with oligodendrocytes and myelin and their associated inhibitory effect, described in Section 11.2. However, fish in general have a good regenerative capability, despite the presence of these factors (e.g., Holder and Clarke, 1988; Sivron and Schwartz, 1994). Whether the reactive gliosis of nonmammals is comparable to that of mammals is unknown, but several authors answer in the negative (Bignami and Dahl, 1976; Margotta et al., 1991). In the goldfish tectum, Bignami and Dahl (1976) found no glial reaction, but they also failed to detect GFAP-immunopositivity in the intact tectum, which is in contrast to the results of Ka´lma´n (1998). In the injured spinal cord of goldfish, Bignami et al. (1974), and Nona and Stafford (1995) found GFAPimmunopositive gliosis, as did Anderson et al. (1984) in a gymnotiform fish. Gliosis had previously been observed by Bernstein (1967) and Bernstein and Bernstein (1967) in the goldfish telencephalon and spinal cord, respectively, by the aid of classical impregnation methods.

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In our study (Ka´lma´n and Ajtai, 2000) in the brain of a teleost fish (goldfish, Carassius auratus) those areas were lesioned, which had been shown to be devoid of GFAP-immunopositivity in the intact animals. The lesions did not provoke GFAPexpression in contrast to what is seen in mammals and birds. This phenomenon suggests two possibilities. The first is that GFAP-expression does not follow lesion in the teleost brain. The second is that, in teleosts, the lack of GFAP-immunostaining is due to the absence of those astroglial elements which are capable of GFAPproduction, and not only to the suppression of the GFAP-production, as in GFAP-free regions of mammalian and avian brains. It has been suggested that the less effective immune system in fish does away with the requirement for separation between the CNS and the immune system by a complete BBB; accordingly, the blood-borne macrophages can take care of the elimination of tissue debris (Dowding and Scholes, 1993; Abbott, 1995), whereas the participation of the astrocytes in this task induces an intense glial reaction, as described in Sections 9.1 – 9.3, and by Eddleston and Mucke (1993), Lawson and Perry (1995), and Merrill and Benveniste (1996). In birds (chickens), as in goldfish, Bignami and Dahl (1976) reported only an ‘extremely limited’ glial reaction, but in our study (Ajtai and Ka´lma´n, 1998), the glial reaction in chicken brain was similar to that of mammals. Our results suggest that the typical astroglial reaction is a feature of astrocytes eo ipso, which are predominant in birds and mammals, but not of ependymoglia, which is characteristic for most lower vertebrates (except for rays and skates, see Ka´lma´n and Gould, 2001, in which the glial reaction, however, has not been studied yet).

11.5. Attempts to inhibit the inhibitors A review of the possible methods to inhibit the inhibitors has been published by Stichel and Mu¨ller (1998b). The approaches to eliminate the growth-inhibitory postlesional effects in the mature CNS numerous: (i) The ablation of scar-forming astrocytes was formerly attempted by administration of gliotoxins, e.g., a-aminoadipinate (Khurger and Ivy, 1996), whereas the up-to-date trials use genetic manipulations, e.g., tagging a herpes simplex virus gene to the GFAP promoter, followed by local treatment with the anti-herpes drug ganciclovir (Bush et al., 1999). The ablation of astrocytes, however, eliminates their control of leukocyte infiltration, repair of the BBB, and protection of neurons, whereas the potential improvement of nerve fiber growth is less obvious. (ii) The use of GFAP– vimentin double-null mutants (Menet et al., 2001) was promising in vitro, but it had adverse effects in vivo (Pekny et al., 2002) as it deprives the regenerating tissue of the glial demarcation as well as of the three-dimensional glial scaffold, which supports the axon growth and cell migration in vivo (in cell cultures the axons growth bi-dimensionally, see also Section 11.1, paragraph 10). (iii) A new trial has also been made to suppress the connective tissue scar but leave the glial intact (Klapka et al., 2002), which lead to massive axonal regeneration, but it still needs further experimental supports.

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(iv) Administration of such agents as glucocorticoids to decrease the inflammation and BBB-damage (Bracken et al., 1990; Guth et al., 1994); the inflammatory phase, however, eliminates the tissue debris that may also impede regeneration (see Sections 10.2 and 11.2). (v) Neutralization of the myelin-derived inhibitory factors described in Section 11.2 (Caroni and Schwab, 1989; Schnell and Schwab, 1993; Schwab et al., 1993; Huber and Schwab, 2000), and tissue debris (Schwartz et al., 1999); or depleting myelin in general (Moon et al., 2000). (vi) Neutralization of other inhibiting factors, e.g., chondroitin sulfate proteoglycans with chondroitinase (Yick et al., 2002). (vii) The establishment of a permissive glial environment, by applying Schwann-cells or immature glia or factors obtained from these (Aguayo et al., 1987; Smith et al., 1986; David et al., 1990; Berry et al., 1992; see also earlier). Although promising results have been reported by several authors (see, e.g., chapter by Dezawa), none of these approaches has been applied successfully in the clinic or replicated by other, independent research teams. 11.6. Is there a ‘biological rationale’ for regeneration failure? It is a question of utmost importance whether there is a biological rationale for the regeneration failure concomitant with the glial reaction. Since both phenomena appear approximately in parallel both during the evolution and during CNS maturation, they should carry advantages, justifying the loss of regenerative capacity. In contrast to the other functions of the glial reaction, however, the potential advantages of restricting nerve fiber growth after injury are not obvious. Identification of such potential advantages is a prerequisite for solving the problem of CNS regeneration satisfactorily. There are several possibilities. (i) There is no rationale. The reactive glia is an abortive attempt of regeneration (Hatten et al., 1984; Hatten, 1985; Wang et al., 1994). The inhibition therefore is a malfunction of the ‘guiding’ and ‘nursing’ capabilities of astroglia, which ‘misguides’ the growing axons (Nieto-Sampedro, 1998), or ‘enchants’ them in a ‘trophic oasis’ (Fitch and Silver, 1997a), and/or it is an ‘unfortunate side effect’ of the boundary formation between the necrotic and viable tissues (Fitch and Silver, 1997a, b). One must be, however, skeptical about Mother Nature being so negligent. (ii) The regeneration failure is a consequence of phylogenetic progressive abilities of the CNS, and especially of glia, which have been acquired at the expense of losing the regeneration capability (Lindsay, 1986). The charges against the GFAP, GHBP and myelin sheath are not convincing, and the reason may rather be the loss of the ‘blueprint’ during replacement of the guiding ependymoglia by astrocytes, and the inaccessibility of CNS due to the evolution of the BBB (see Section 11.4). However, why have compensatory mechanisms not been acquired during evolution?

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(iii) The mature mammalian CNS is too sophisticated a system to be regenerated, and the reactive glia prevents the formation of aberrant growth of axons and the formation of inappropriate connections (Lindsay, 1986; Fitch and Silver, 1997a). In this case the efforts to evoke regeneration menace with unexpected effects! (iv) The reactive glia is not, or not alone responsible for the failure of regeneration (Sivron and Schwartz, 1994– see also chapter by Schwartz); other factors are to be taken into consideration (e.g., myelin-derived inhibitors, see Section 11.2), which interfere with the relatively insufficient capability of axon growth and neuron formation in the mature CNS. CNS glia fail to provide suitable factors in proper amounts for axon growth, whereas PNS glia do (Liesi, 1985; Lindsay, 1986). The regeneration failure is a result (or a coincidence) of these, separately ‘minor’ effects, which not separately but collectively lead to regeneration failure. Conquering this inability requires a multilateral, complex approach, rather than searching for one key factor, a ‘philosopher’s stone’. 12. Concluding remarks The glial reaction is the form of the wound healing in the CNS. Similar molecular events follow the injuries in the CNS, as in the other organ systems, including the PNS. The expression of GFAP indicates astroglial activation in general, not only the ‘reactive glia’ responding to a lesion. Although the reactive gliosis seems to be the main impediment for axonal regrowth, it has beneficial effects, and its relation to axonal regeneration is more complex than a simple inhibition. Reactive astrocytes re-express some features of immature astroglia, but they are not ‘immature’ cells. The inhibitory effect of the glial reaction is acquired, both phylogenetically and ontogenetically. Its rationale must be understood, before it can be attempted to solve the problem of regeneration.

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Contributions of astrocytes to ischemia-induced neuronal dysfunction in vivo Asta Ha˚berga and Ursula Sonnewaldb,* a

Department of Circulation and Medical Imaging, Norwegian University of Science and Technology (NTNU), 7489 Trondheim, Norway b Department of Clinical Neurosciences, Norwegian University of Science and Technology (NTNU), 7489 Trondheim, Norway p Correspondence address: Department of Neurosciences, Medical Faculty, NTNU, Olav Kyrres gt. 3, N-7489 Trondheim, Norway. Tel.: þ47-73590492; fax: þ47-73598655. E-mail: [email protected](U.S.)

Contents 1. 2. 3.

4.

5.

6. 7.

Introduction The pathophysiology of cerebral ischemia Metabolic astrocytic – neuronal interaction 3.1. Synthesis of glutamate from glucose 3.2. The glutamate – glutamine and GABA – glutamate – glutamine cycles 3.3. Acetate metabolism 3.4. 13C MRS analysis Astrocytes in ischemia 4.1. Non-MRS studies 4.2. 13C MRS studies Glutamatergic neuronal –astrocytic interactions in ischemia 5.1. The ischemic core 5.2. The penumbra GABAergic neuronal – astrocytic interactions in ischemia Concluding remarks

Abbreviations CBF: cerebral blood flow; CSF: cerebrospinal fluid; g-GABA aminobutyric acid; GABA-T: g-aminobutyric acid transaminase; GAD: glutamate decarboxylase; GS: glutamine synthetase; MCAO: middle cerebral artery occlusion; MRS: magnetic Advances in Molecular and Cell Biology, Vol. 31, pages 837–855 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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resonance spectroscopy; PAG: phosphate activated glutaminase; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; TCA: tricarboxylic acid. Recent evidence has shown that astrocytes are more sensitive to ischemia than has been generally envisaged. However, the effects of altered astrocytic function on neighboring neurons were unknown. Our recent studies have demonstrated a relative increase in the use of astrocytic glutamine in glutamate formation in glutamatergic neurons, which may enhance glutamate excitotoxicity. In GABAergic neurons, reduction or discontinuation in the use of glutamine as substrate for the GABAergic TCA cycle and in GABA formation contributed to the complete halt in TCA-cycle activity and thus death of GABAergic neurons.

1. Introduction The concept of astrocytes as mere scaffolding has been replaced during the last decades with a more dynamic view. As described in other chapters, astrocytes actively maintain and support neuronal activity. One important function of astrocytes is to supply neurons with metabolic intermediates, since the neuronal metabolite pool/tricarboxylic acid (TCA) cycle is continuously drained by neurotransmitter release, and neurons on their own are incapable of synthesizing net amounts of the two major transmitters, glutamate and GABA from glucose, because they lack the enzyme pyruvate carboxylase (PC) needed for this synthesis (see below). Therefore, metabolites from astrocytes are used by the neurons for synthesis of both glutamate and its derivative, GABA (Hertz et al., 1999 and references therein—see also chapter by Schousboe and Waagepetersen). The major metabolite traveling from astrocytes to neurons and supplying the neurons with a precursor for transmitter glutamate is glutamine, which is synthesized in astrocytes but not in neurons, again because they lack the catalyzing enzyme, glutamine synthetase (GS). Although the importance of astrocytic glutamine in GABA synthesis has been questioned (Preece and Cerda´n, 1996), many in vitro and in vivo data imply that glutamine is also an essential precursor for GABA synthesis, via glutamate (Fonnum and Paulsen, 1990; Waagepetersen et al., 1999; Rothman et al., 1999). Moreover, astrocytes are also of major importance for subsequent oxidative degradation of glutamate (Waagepetersen et al., 2002—see also chapter by Schousboe and Waagepetersen). This close functional relationship between astrocytes and neurons indicates that any interruption in the performance of one of these cellular elements will influence the activity and well being of the other. Still, little is known about metabolic interactions between astrocytes and neurons in disease. Stroke is the most common life-threatening neurological disorder and the third leading cause of death in the Western World. Stroke encompasses thrombo-embolic occlusion of an intracerebral artery (referred to as focal cerebral ischemia), which represents more than 75% of the cases, with the remaining 25% being caused by intracerebral and subarachnoidal hemorrhage. In focal cerebral ischemia, excessive stimulation of glutamate receptors (glutamate excitotoxicity) is recognized as an important mediator of neuronal death (Rothman and Olney, 1986; Choi, 1992, 1997), whereas increased GABA receptor stimulation in the mature brain is considered neuroprotective

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(Schwartz-Boolm and Sah, 2001). Since, astrocytes are inextricably involved in the synthesis of both neurotransmitter glutamate and GABA, it follows that any disturbances in the metabolic interaction between astrocytes and neurons may have important implications for the final outcome after focal cerebral ischemia. In order to elucidate changes in astrocytic and neuronal metabolism as well as trafficking of metabolites between these two cell types during focal cerebral ischemia, a new method was recently applied (Ha˚berg et al., 1998, 2001). This method allows detailed analysis of astrocytic and neuronal metabolism during middle cerebral artery occlusion (MCAO) in the rat by combining in vivo injection of 13C labeled glucose and 13C labeled acetate, a substrate taken up and metabolized by astrocytes, but not by neurons, with ex vivo 13C magnetic resonance spectroscopy (MRS) of brain extracts. The main conclusions from these and other studies will be summarized below. In order to understand both the background for the MRS analyses and the importance of the conclusions drawn, crucial aspects of neuronal –astrocytic interactions in the formation of transmitter glutamate and GABA will also be reviewed.

2. The pathophysiology of cerebral ischemia The brain depends on a continuous supply of glucose and oxygen, since neither is stored in appreciable amounts. Thrombo-embolic occlusion of an intracranial artery immediately reduces cerebral blood flow (CBF) to the area normally supplied by the artery, and initiates a complex series of events transforming at least part of the ischemic tissue into infarction. The tissue damage spreads circumferentially from a central ischemic core where CBF is severely impaired and the brain tissue rapidly becomes irreversibly damaged (Symon et al., 1974; Touzani et al., 1995). Surrounding and probably also intermixed in this core are zones of less severely reduced CBF, which can potentially be rescued with reperfusion or neuroprotective drug treatment (Siesjo¨, 1992). This region has been termed the penumbra by Astrup et al. (1981), and it is of great interest, as reversing the metabolic failure here will reduce the final infarct volume and thus limit the physical and/or cognitive impairment suffered by the stroke victim. However, the potential for recovery of function is determined not only by the level of residual CBF, but also by the duration of the flow disturbance (Kohno et al., 1995; Zhao et al., 1997). This makes treatment, including possible revascularization, within the first few hours essential. A secondary consequence of reduced CBF is a substantial elevation of glutamate in both the ischemic core and penumbra (Benveniste et al., 1984; Shimada et al., 1989; Takagi et al., 1993). This increase, in turn, leads to excessive stimulation of glutamate receptors, initiating a range of detrimental biochemical pathways, which is considered a major contributor to neuronal death in ischemia (Rothman and Olney, 1986; Choi, 1992, 1997). The strongest evidence today implicating glutamate excitotoxicity in the pathogenesis of ischemic neuronal death was obtained in cell culture and animal studies. Decreased neuronal death has been demonstrated after administration of antagonists of several of the mechanisms involved in glutamate excitotoxicity (Scatton, 1994; Kobayashi and Mori, 1998; Lee et al., 1999). In humans, however, only blocking of the glutamate

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NMDA receptor using magnesium salts has been effective in improving outcome in stroke victims (Lampl et al., 2001; Muir, 2001). All other approaches towards blocking glutamate excitotoxicity have so far failed in clinical trials (Lees et al., 2000; Davis et al., 2000; Horn and Limburg, 2001). In order to study focal cerebral ischemia in detail, several animal models of focal cerebral ischemia have been developed. One of the most reliable and reproducible models of the human disease is the intraluminal occlusion of the MCAO in rats first described by Kozumi et al. in 1986, and later modified by Longa et al. (1989). In short, a monofilament is introduced into the internal carotid artery, via the external carotid artery, and advanced 19– 20 mm until it blocks the origin of the middle cerebral artery. Permanent MCAO induced with the intraluminal filament technique results in severe reduction of CBF in the lateral caudoputamen and lower part of the frontoparietal cortex, rapidly leading to irreversible ischemia (Longa et al., 1989; Mu¨ller et al., 1995). This region is often referred to as the ischemic core. In the upper frontoparietal cortex, CBF is moderately reduced (Memezawa et al., 1992; Mu¨ller et al., 1995), and this area is considered to represent the penumbra. However, as discussed above, cerebral ischemia is a dynamic process, and there is a gradual progression from reversible to irreversible ischemia. Also, the timeframe within which penumbral deterioration occurs is controversial.

3. Metabolic astrocytic – neuronal interaction 3.1. Synthesis of glutamate from glucose Glucose is the only metabolic substrate that rapidly crosses the adult blood – brain barrier. It is taken up by both neurons and astrocytes by powerful glucose transporters (see chapters by Drewes and by Roberts and Chih) and degraded oxidatively in both cell types after pyruvate dehydrogenase-(PDH)-mediated oxidative decarboxylation of the glucose metabolite pyruvate to acetyl coenzyme A (acetyl CoA). Acetyl CoA is transferred into the mitochondria for oxidation in the TCA cycle after condensation with oxaloacetate to citrate (Fig. 1A). During one turn of the TCA cycle, two carbon atoms are converted to CO2 and oxaloacetate is regenerated. Accordingly, this process does not give rise to any net formation of any TCA-cycle intermediate or any derivative of such intermediates, including glutamate. However, because there is extensive bi-directional exchange between the TCA-cycle intermediate a-ketoglutarate and glutamate in both neurons and astrocytes, administration of [1-13C]glucose gives rise to labeled glutamate, a process occurring so rapidly that it can be used for determination of the rate of TCA-cycle turnover in the intact brain (see chapter by Gruetter). Net synthesis of a TCA-cycle intermediate depends upon the additional entry of another molecule of pyruvate into the TCA cycle, in the brain mainly or exclusively by carboxylation, catalyzed by PC (Patel, 1974). This process generates ‘a new’ molecule of oxaloacetate, which then condenses in the normal manner with acetyl CoA to provide net synthesis of a molecule of the TCA-cycle intermediate a-ketoglutarate, from which glutamate can be formed by transamination (Fig. 1A). In contrast to the participation of labeling of glutamate (and other metabolites) via acetyl CoA (see above), participation of

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pyruvate carboxylation in glutamate labeling is an unequivocal indication of net synthesis, and 13C MRS analysis can distinguish between labeling from either pathway (see below). Subsequently glutamate formed from glucose can be transferred from astrocytes to neurons after amidation to glutamine by the glia- and perhaps astrocyte-specific enzyme GS (Norenberg and Martinez-Hernandez, 1979; Tansey et al., 1991—see also chapter by Derouiche), release of glutamine from astrocytes, and uptake of glutamine into neurons in the ‘glutamate – glutamine cycle’ as indicated in Fig. 1A. Glutamine is then hydrolyzed to glutamate, which can be released as transmitter glutamate. In addition, some aketoglutarate might be transferred as such from astrocytes to neurons and transaminated in the neurons to glutamate (see chapter by Schousboe and Waagepetersen).

Fig. 1. Schematic representation of changes in neuronal and astrocytic intermediary metabolism and metabolic interaction between astrocytes and neurons induced by MCAO. For simplicity, only metabolic pathways considered in the text are included in the figure. (A) Control condition. Glucose is metabolized by all three cell types, but acetate is exclusively taken up and metabolized in astrocytes. Acetate is converted to acetyl CoA by acetyl-CoA synthase (ACoAS). In neurons, glucose is exclusively metabolized via PDH-catalyzed formation of acetyl coenzyme A (acetyl CoA), but in astrocytes it is also metabolized by PC, yielding net synthesis of a TCAcycle intermediate (oxaloacetate) and thus of glutamate and GABA. Net synthesis of glutamate (GLU) occurs from the TCA-cycle intermediate a-ketoglutarate, which is in a bi-directional isotope exchange with glutamate. In astrocytes, glutamate is converted by GS to glutamine (GLN), but it can also be converted back to aketoglutarate and oxidized in the TCA cycle. Glutamine is transferred to glutamatergic neurons in the glutamate– glutamine cycle and converted in these neurons to glutamate by PAG. After release of transmitter glutamate, a small fraction is re-accumulated in neurons but a larger fraction is taken up in astrocytes, where it is converted to glutamate and either metabolized in the TCA cycle or carried back to the glutamatergic neurons in the glutamate– glutamine cycle. Although no net synthesis of glutamate occurs in the neurons from a-ketoglutarate, glutamate is labeled from [1-13C]glucose in a bi-directional isotope exchange with a-ketoglutarate, and this process is an indication of the rate of oxidative metabolism. Aspartate (ASP) is in equilibrium with oxaloacetate, but it is also not formed in net amounts. Glutamine formed in astrocytes is in addition transferred to GABAergic neurons in the GABA-glutamate– glutamine cycle, and a fraction is directly decarboxylated to GABA by GAD, whereas another fraction is metabolized to a-ketoglutarate and cycled in the TCA cycle, until glutamate is eventually re-generated and decarboxylated to GABA. Released transmitter GABA is mainly re-accumulated in neurons, but a minor fraction is taken up in astrocytes and converted by GABA transaminase (GABA-T) to a TCA-cycle intermediate (succinate), which probably mainly is oxidized, although a fraction may be turned over in the TCA cycle to form a-ketoglutarate and glutamate, which can be returned to the GABAergic neurons in the GABA–glutamate– glutamine cycle. GABA re-accumulated in GABAergic neurons is mainly re-utilized as a transmitter, but it can also be degraded via GABA-T. The exchange with aspartate is more pronounced in GABAergic than in glutamatergic neurons. (B) Changes induced by MCAO in the lateral caudoputamen and lower parietal cortex, which are referred to as the ischemic core. The figure depicts the changes at 240 min. Oxidative metabolism of both glucose and acetate is greatly reduced, as indicated by the thinner arrows. Formation of glutamate and glutamine is also reduced, although there is still some glutamate and glutamine synthesis after 240 min of MCAO. Trafficking in the glutamate–glutamine cycle is reduced and that in the GABA–glutamate–glutamine cycle abolished. TCA-cycle activity in GABAergic neurons has been halted, but GABA content is increased. (C) Changes induced by MCAO in the upper frontoparietal cortex, which is referred to as the penumbra. The figure depicts the changes after 240 min of MCAO. At this time point oxidative metabolism in astrocytes and neurons is reduced, but labeling of glutamine from [1,2-13C]acetate remained at control levels. The level of glutamine is increased, which may enhance formation and release of transmitter glutamate in glutamatergic neurons and thus excitotoxicity. Trafficking in the GABA–glutamate–glutamine cycle is reduced, but not abolished. Aspartate synthesis is reduced, and consumption increased. pEnzymes located in mitochondria, or in mitochondrial membrane (PAG). #Glycolysis is not included in the figure.

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3.2. The glutamate– glutamine and GABA – glutamate –glutamine cycles The glutamate –glutamine cycle (Berl and Frigyesi, 1969; Van den Berg, 1973) is operating not only to transfer newly synthesized glutamate from astrocytes to neurons, but, also to return previously released transmitter glutamate from astrocytes to neurons, after it has been taken up in astrocytes as means of transmitter inactivation. Synaptically released glutamate is primarily taken up by astrocytes (Erecinska, 1987) due to high-affinity glutamate transporters located predominantly on these cells (Gegelashvili, 1998; Tanaka, 2000). Once glutamate has been taken up by astrocytes it is rapidly converted to glutamine by the cytosolic enzyme, GS. Alternatively, glutamate can enter the astrocytic TCA cycle via transamination or NAD-requiring oxidative deamination by glutamate dehydrogenase (Westergaard et al., 1996). Whichever pathway is followed, the metabolism of glutamate is rapid, as indicated by the observation that the major part of glutamate in the brain is found in glutamatergic neurons, whereas the level of glutamate is very low in astrocytes (Ottersen et al., 1992). Glutamine is released from astrocytes as described above and subsequently taken up by high affinity glutamine transporters present on neurons (Varoqui et al., 2000), although it should be noted that astrocytes also possess glutamine transporters (Chaudhry et al., 1999). In the neuron, glutamine is converted to glutamate by phosphate activated glutaminase (PAG) (Kvamme et al., 2000) and it can subsequently be re-utilized as transmitter glutamate in glutamatergic neurons. In GABAergic neurons, glutamate is converted to GABA by glutamate decarboxylase (GAD), and glutamate is present in a very low concentration (Ottersen et al., 1992; Martin and Rimvall, 1993). However, only part of the glutamate from glutamine is immediately decarboxylated to GABA. A substantial amount of this glutamate enters into the TCA cycle in the neuronal compartment (Fig. 1A), where it cycles until glutamate eventually is re-generated and decarboxylated (Waagepetersen et al., 1999, 2001). Thus, although GAD is capable of operating under hypoxic conditions, formation of transmitter GABA in GABAergic neurons may be more sensitive than formation of transmitter glutamate in glutamatergic neurons to oxygen deprivation, because function of the neuronal TCA cycle is required for GABA synthesis. Synaptically released GABA is taken up primarily by GABAergic neurons, and only to a lesser extent by astrocytes (for reviews, see Borden, 1996; Schousboe, 2000). GABA is metabolized by GABA aminotransferase (GABA-T) in the so-called GABA-shunt (Bala´zs et al., 1970), which allows four of the five C-atoms from a-ketoglutarate to re-enter the TCA cycle as succinate. GABA-T activity is predominantly found in neurons, but is also present in astrocytes, making astrocytes and neurons equally capable of metabolizing GABA (Baxter, 1976). Once GABA-derived succinate has entered the TCA cycle, it can either be oxidized after conversion to malate and exit from the cycle via malic enzyme (see chapter by Schousboe and Waagepetersen), or it can be converted to glutamate and subsequently to GABA. 3.3. Acetate metabolism Acetate is an excellent marker of glial metabolism since it is metabolized by astrocytes, but not in neurons (Berl and Frigyesi, 1969; Van den Berg, 1973), due to the presence of a specific acetate carrier located specifically on astrocytes (Waniewski and Martin, 1998).

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Acetate is converted to acetyl CoA by the action of acetyl-CoA synthase (AcoAS) as shown in Fig. 1A, but since the PDH-catalyzed conversion of pyruvate to acetyl CoA is irreversible, it is not a precursor for pyruvate and can therefore not be metabolized by PC.

3.4.

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C MRS is a powerful tool to determine the position of 13C atoms within a molecule. Since the labeling of various carbon positions within a compound (usually an amino acid) depends on which enzymatic pathways were active, the metabolic history of the 13C labeled compounds can be analyzed in great detail. Labeling of a-ketoglutarate and all its derivatives, including glutamate, GABA and glutamine (present in brain in much larger amounts than the TCA-cycle intermediates) depends upon whether they were formed by pyruvate carboxylation, catalyzed by the astrocyte-specific PC, or by PDH-mediated entry into the TCA cycle via acetyl CoA. Since the former of these pathways is strictly astrocytic, the relative contribution from the neuronal and the astrocytic compartment in glutamate, glutamine and GABA formation can be derived from analysis of the total amount and the distribution of label in the different isotopomers (Cerdan et al., 1990). It has long been established that the brain predominantly metabolizes glucose (Sokoloff, 1976). Recent data suggest that approximately 65% of the acetyl CoA derived from glucose is metabolized in the neuronal TCA cycle versus 35% in the astrocytic TCA cycle (Qu et al., 2000). Such analyses can be further refined by the simultaneous administration of [1,2-13C]acetate (due to its selective metabolism in astrocytes) and [1-13C] glucose (which is metabolized by both neurons and astrocytes) (Taylor et al., 1996).

4. Astrocytes in ischemia 4.1. Non-MRS studies The role of astrocytes in ischemia is controversial, as is the susceptibility of astrocytes to ischemia. Astrocytes have been considered to die as a secondary consequence of neuronal death (Lee et al., 1999), because astrocytes are thought to obtain sufficient energy through glycolysis and thus be able to survive on glycolytically derived energy, including that derived from glycogenolysis, whereas neurons might depend more on mitochondrial function, due to a high degree of mitochondrial coupling (Almeida and Medina, 1997). However, some in vitro data have demonstrated that astrocytic metabolism and protein synthesis are even more susceptible than neuronal metabolism to hypoxic/ischemic/ excitotoxic conditions (Simantov, 1989; Tholey and Ledig, 1990; Sonnewald et al., 1994a, b; Bondarenko and Chesler, 2001). These findings are consistent with results from animal studies obtained using a variety of immunological/electronmicroscopy techniques, which have demonstrated rapid astrocyte death, in some cases preceding neuronal death, in focal cerebral ischemia (Garcia et al., 1995; Liu et al., 1999; Fern, 2001).

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Moreover, certain astrocytic subpopulations have been shown to be more vulnerable to hypoxic – hypoglycemic/ischemic conditions than others (Zhao and Flavin, 2000; Lukaszevicz et al., 2002). Neuronal regeneration after CNS injury has been considered to be impeded by the presence of astrocytes (Aguayo et al., 1981; Reier et al., 1983), but newer studies demonstrate that astrocytes may also promote neuronal survival after brain damage in vitro and in vivo through synthesis and release of glial cell-line derived neurotrophic factor (Ridet et al., 1997; Kitagawa et al., 1998; Louw et al., 1998—see also chapter by Kalman). Moreover, mice deficient in glial fibrillary acidic protein suffer significantly bigger cortical infarcts after focal cerebral ischemia than normal mice (Nawashiro et al., 2000). 4.2.

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In the ischemic core, astrocytic metabolism was reduced from 30 min of MCAO, which was the earliest time point evaluated, and remained at about the same level throughout the initial 120 min of ischemia. However, at 240 min severely deranged astrocytic metabolism was detected. The first sign of declining astrocytic metabolism was reduced acetate metabolism, as unmetabolized acetate was detected already after 30 min of MCAO (Ha˚berg et al., 2001). The glial specific enzymes PC and GS, as well as acetyl CoA synthetase, which is necessary for acetate metabolism, are all ATP dependent enzymes. In the ischemic core, acetyl CoA synthetase maintained a somewhat reduced level of activity at all times after MCAO (Fig. 1B). At 240 min GS activity was reduced, and signs that PC activity had been involved could no longer be detected in glutamine and GABA, but were still detectable at a low level in glutamate. Furthermore, the acetate/glucose utilization ratio (an indication of the distribution between glial and total metabolism [Taylor et al., 1996]) was markedly increased in glutamate, but not in glutamine after 240 min of MCAO. These findings suggest continued, although reduced, astrocytespecific glutamate synthesis from labeled acetate and glucose even after 240 min of MCAO, compared to an even larger reduction of average oxidative metabolism in all cell types, leading to a greatly reduced labeling of glutamate derived from all cellular pools via PDH-mediated glucose metabolism. However, the astrocytic compartment synthesizing glutamine appeared to be more susceptible to ischemia than astrocytically derived glutamate (Fig. 1B). This might arise from reduced transport of glutamate, produced in the astrocytic mitochondrion, over the mitochondrial membrane into the cytosol, where GS is located. This interpretation is supported by an immunocytochemical observation of mitochondrial glutamate accumulation in astrocytes in global ischemia (Torp et al., 1993). Synthesis of alanine can be considered a marker for astrocytic metabolism in cerebral cortex, since it is preferentially synthesized in cortical astrocytes in vitro (Westergaard et al., 1993). Alanine synthesis was significantly increased in the ischemic core between 60 and 120 min of MCAO, confirming an enhanced alanine synthesis specifically in astrocytes during cerebral ischemia reported by Bachelard (1998), and indicating an initially relatively well-maintained astrocytic metabolism. However, after 240 min of MCAO, alanine levels were markedly reduced indicating severely impaired astrocyte metabolism (Ha˚berg et al., 2001).

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In the penumbra, the astrocytic metabolism appeared relatively unaffected by MCAO until 90– 120 min of MCAO, when unmetabolized [1,2-13C]acetate appeared in the fully awake rats (Ha˚berg et al., 1998, 2001). This is considerably later than in the ischemic core, and utilization of [1,2-13C]acetate for glutamine synthesis was at the normal level during the entire 240 min of MCAO (Fig. 1C), indicating that amino acid synthesis via the TCA cycle was more resistant than oxidative metabolism. The activity of GS appeared unaffected by ischemia, and the glutamine content even increased steadily in the penumbra. As mentioned above, GS is an ATP dependent enzyme located in the cytosol, implying that ATP was present in sufficient amounts to convert glutamate to glutamine. ATP was probably partly provided by glycolysis, perhaps fueled by breakdown of glycogen, which takes place in the cytosol, and partly by the limited amounts of oxygen and glucose supplied to this area by a reduced, but not abolished blood flow. Part of the glutamine accumulation in the penumbra may have been caused by decreased glutamine consumption, as neuronal utilization of astrocytic glutamine was reduced at all times after MCAO. However, as total glutamate content was unchanged during the 240 min of ischemia, impaired glutamine oxidation probably contributed only marginally to the increase in glutamine content (Ha˚berg et al., 2001). This increase may rather have represented a response to elevated extracellular glutamate, since enhanced glutamine synthesis in astrocytes has been suggested to be a means of detoxifying glutamate taken up from the extracellular space (Cooper, 1988; Nissim, 1999). It is in further support of relatively well-preserved metabolism in astrocytes in the penumbra during several hours, that there was an increased alanine synthesis throughout the experimental period, and that PC activity did not fall dramatically until after 240 min of MCAO (Ha˚berg et al., 1998, 2001). There is an interesting temporal correlation between the onset of metabolic impairment in astrocytes and the transition from reversible to irreversible ischemia. The appearance of impaired acetate metabolism was the first indication of reduced astrocytic viability both in the ischemic core (after 30 min) and in the penumbra (between 90 and 120 min) (Ha˚berg et al., 1998, 2001). From reperfusion studies it is well documented that reperfusion within 30 min saves substantial volumes in the ischemic core from progressing to infarction, and that the upper frontoparietal cortex (belonging to the penumbra) can be salvaged by reperfusion before 120 min (Memezawa et al., 1992; Mu¨ller et al., 1995). Thus, it appears that unperturbed astrocyte metabolism may be a prerequisite for restoration of neuronal metabolism following an ischemic event. These results also suggest that infusion of labeled acetate in combination with MR techniques (MRS or molecular MR imaging) or PET may become a tool for predicting outcome and designing therapy for stroke patients. 5. Glutamatergic neuronal – astrocytic interactions in ischemia 5.1. The ischemic core As previously mentioned, incorporation of label into glutamate from glucose via acetyl CoA to a large extent represents exchange between a-ketoglutarate and glutamate, i.e., oxidative metabolism. Moreover, since most of the glutamate pool is present in neurons, especially glutamatergic neurons, information about glutamate labeling from

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[1-13C]glucose reflects predominantly, although not exclusively, events occurring in these cells. This labeling declined rapidly to a low level (Fig. 1B), although there was continued TCA-cycle activity and some glutamate labeling for the entire 240 min of MCAO. As glutamate labeling from glucose-derived acetyl CoA declined, the relative importance of astrocytic glutamine production increased throughout the 240 min of MCAO (Ha˚berg et al., 1998, 2001). Nevertheless, in the ischemic core, ischemia did also reduce the utilization of glutamine for glutamate formation, compared to normal conditions. The glutamate– glutamine cycle was severely disturbed from 30 min of MCAO and onwards, as clearly demonstrated by the changes in the metabolic pathways leading to glutamate synthesis and the disruption of the normal equilibrium between glutamate and glutamine content. The reduction in glutamate synthesis from glutamine probably resulted from ischemia-induced impairment of astrocytic or neuronal glutamine transporters, and/or reduced PAG activity due to ammonia accumulation and/or acidosis, which are known to occur in ischemia (Benjamin, 1981; Hogstad et al., 1988; Kvamme et al., 2000). Still, at all times after MCAO, astrocytic glutamine was a more important precursor for labeling of glutamate than precursors derived from neuronal TCA-cycle activity. The astrocytes continued to take up glutamate and converted accumulated glutamate to glutamine throughout the 240 min (Ha˚berg et al., 1998, 2001), which is in line with other studies (Aas et al., 1993; Takagi et al., 1993; Swanson, 1992; Torp et al., 1993). In agreement with observations by Pascual et al. (1998), there was also increased re-entry of glutamate into the TCA cycle during ischemia (Ha˚berg et al., 1998, 2001). Glutamate can be used in the TCA cycle as an alternate fuel, replacing glucose in ischemia. However, this small increase in glutamate consumption cannot account for the steady decline in glutamate content in the ischemic core, which was apparent from 30 to 240 min of MCAO. The decline in glutamate levels was probably multifactorial, resulting from a combination of reduced glutamate synthesis and increased glutamate consumption via the TCA cycle and for GABA and alanine synthesis. This may have been combined with a loss of glutamate into the systemic circulation and cerebrospinal fluid (CSF), since elevated glutamate levels have been demonstrated in both plasma and CSF in patients with acute ischemic stroke (Castillo et al., 1996). Exit of glutamate into CSF and the systemic circulation implies that the extracellular glutamate concentration exceeded the capacity of the astrocytic glutamate transporters, probably caused by the rapid and sustained massive glutamate efflux found in this region during MCAO (Butcher et al., 1990), at least partly reflecting release of nontransmitter glutamate by reversal of the uptake carriers (see chapter by Schousboe and Waagepetersen). These observations suggest that although astrocytes continued to accumulate glutamate, this uptake did not match the actual glutamate efflux in the ischemic core.

5.2. The penumbra The ischemia-induced effects in the penumbra showed some similarities with those in the ischemic core, but here were also distinct differences. These are not necessarily all a result of the differences in blood flow, since results from sham operated rats showed that astrocytic glutamine was a more important precursor for glutamate in cortical than in

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subcortical samples (Ha˚berg et al., 2001). This is in agreement with a higher PAG density and activity in neurons in cortex than in caudate-putamen/striatum (Aoki et al., 1991; Wallace and Dawson, 1993). Compared to control conditions, glutamate labeling from both neuronal and astrocytic precursors was reduced from 30 min of MCAO and onwards, but the astrocytes continued to take up glutamate and convert it to glutamine and some glutamate was also oxidized in the astrocytic TCA cycle (Ha˚berg et al., 1998, 2001). The conversion of glutamine to glutamate was 50% reduced from the onset of MCAO and it remained at this level at all times after MCAO, indicating a nonprogressing functional inhibition of glutamine transporters and/or PAG activity (Fig. 1C). In contrast, the glutamate labeling from the neuronally derived TCA cycle showed a continuous fall, probably reflecting a gradual decline in rate of oxidative metabolism, so that the relative importance of glutamine for glutamate labeling increased two-fold between 60 and 240 min of MCAO (Ha˚berg et al., 1998, 2001). The increased role of glutamine as a glutamate precursor was accompanied by significantly elevated glutamine levels between 60 and 240 min of MCAO (Ha˚berg et al., 1998, 2001). Several studies have demonstrated that glutamine specifically replenishes the neurotransmitter pool of glutamate (Bradford et al., 1978; Laake et al., 1995), which accordingly may be relatively well preserved in the penumbra. Since ischemia induced glutamate release in this area is considered to originate mainly from the neurotransmitter pool (Takagi et al., 1993; Obrenovitch, 1996), it follows that the preservation of the neurotransmitter pool of glutamate originating from glutamine, combined with reduced neuronal TCA-cycle metabolism may render the neurons very vulnerable to glutamate excitotoxicity. There is increasing evidence for a delayed component in glutamatemediated neuronal damage in cerebral ischemia which, based on the data reviewed above, may be connected to the continued astrocytic glutamine production and resulting formation of neuronal glutamate. Indeed, neuronal glutamate release may be increased in the penumbra due to the presence of an elevated extracellular glutamine concentration, which has been reported to stimulate glutamate release (Szerb and O’Regan, 1985). In fact, inhibiting GS activity has been shown to reduce infarct size in cortex in rats subjected to MCAO (Swanson et al., 1990). The glutamate content in the penumbra remained at control levels throughout the 240 min of MCAO. This reflects again the importance of astrocytic metabolites for glutamate formation in glutamatergic neurons, and it also shows that there was no loss of glutamate from brain parenchyma to blood or CSF in this region (Ha˚berg et al., 2001). Perhaps this is due to release of smaller amounts of glutamate and more efficient glutamate uptake in the penumbra than in the ischemic core (Takagi et al., 1993; Obrenovitch, 1996), which enables the astrocytic uptake better to keep pace with the release.

6. GABAergic neuronal – astrocytic interactions in ischemia Focal cerebral ischemia completely changed GABA metabolism. GABA synthesis from [1,2-13C]acetate was not detectable in either the ischemic core or the penumbra at any time after MCAO, demonstrating a dramatic reduction in conversion of astrocytic glutamine to GABA after induction of ischemia. In the ischemic core, PC activity was also

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undetectable in GABA from the onset of MCAO (i.e., absent already at the first measured time point of 30 min), suggesting complete cessation of the use of astrocytic precursors for GABA formation. However, in the penumbra PC activity was present in GABA from 30 to 120 min of MCAO, demonstrating that astrocytically generated precursors participated to some extent in GABA synthesis during this time period, but after MCAO for 240 min involvement of PC activity in GABA synthesis was undetectable also in this region. The cessation and decline in glutamine utilization for GABA formation observed in the ischemic core and penumbra, respectively, were at great variance with the increased use of glutamine for glutamate synthesis in the glutamatergic neurons in the same ischemic regions. This difference might at least partly reflect the need for glutamate cycling in the TCA cycle before its decarboxylation to GABA (see above). Considering the importance of glutamine for GABA formation under normal conditions (Waagepetersen et al., 1999; Rothman et al., 1999; Ha˚berg et al., 2001), interrupted utilization of glutamine for GABA synthesis will have severe consequences for GABAergic neurons. Concomitant with the dramatic reduction in glutamine utilization, but similar to the findings in glutamatergic neurons, the labeling of GABA from neuronally derived precursors was also markedly reduced at all times after MCAO in both the ischemic core and penumbra. The GABAergic neurons in the lateral caudoputamen and lower parietal cortex, representing the ischemic core, are very sensitive to ischemic conditions and showed no labeling above natural abundace after 240 min, whereas in the penumbra there was some residual GABA labeling from the neuronal TCA cycle at this time (Ha˚berg et al., 1998, 2001). In spite of the decreased GABA formation, GABA content increased in both the ischemic core and the penumbra with the duration of ischemia. In the penumbra, GABA build-up was present from 60 min of MCAO, and in the ischemic core it was observed after 240 min (Ha˚berg et al., 1998, 2001). As indicated in Fig. 1C, the GABA build-up arises from a combination of increased conversion of glutamate to GABA by GAD (Sze, 1979; Erecinska et al., 1996) and inhibited GABA breakdown via GABA-T (Schousboe et al., 1973; Baxter, 1976) under ischemic conditions. The increased GABA synthesis from glutamate can proceed without functioning mitochondria, and GABA accumulation has been reported both in the neuronal cytosol (Torp et al., 1993) and in the extracellular space (Matsumoto et al., 1993). In contrast, GABA does not accumulate inside astrocytes during ischemia (Torp et al., 1993), suggesting that astrocytic GABA uptake is limited under ischemic conditions. As GABA is regarded as an endogenous neuroprotectant, the limited astrocytic GABA uptake may be beneficial during ischemia. The mixture of beneficial and detrimental effects on GABAergic transmission seen under ischemia by Ha˚berg et al. (1998, 2001) may explain why GABAergic neurons in other studies both have been shown to be very sensitive (Francis and Pulsinelli, 1982; Shuaib et al., 1994), and particularly resistant to ischemic damage (Tecoma and Choi, 1989; Nitsch et al., 1989; Gonzales et al., 1992). A significant reduction in aspartate synthesis occurred in both ischemic regions from 30 min of MCAO and onwards, and the decline was steeper in the ischemic core, than in the penumbra (Ha˚berg et al., 2001). The reduced aspartate synthesis was probably related to the reduced glutamine utilization in GABAergic neurons, as in vitro data suggest that aspartate is predominantly synthesized from glutamine in cortical synaptic terminals

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(Sonnewald and McKenna, 2002), aspartate synthesis takes place mainly in GABAergic neurons (Johannessen et al., 2001), and aspartate is found mainly in GABAergic neurons (Ottersen and Storm-Mathisen, 1985). Aspartate consumption was also increased by MCAO, which might be linked to the reduced GABA-T activity since g-vinyl-GABA, an inhibitor of GABA-T, also has been shown to increase aspartate consumption (Hassel et al., 1998). 7. Concluding remarks Based on the studies by Ha˚berg et al. (1998, 2001), a complex picture of the role of astrocytes in neuronal dysfunction and possible death during ischemia emerged. Alterations in the use of astrocytic precursors appeared to contribute significantly to neuronal death, although through different mechanisms in glutamatergic and GABAergic neurons. In the glutamatergic neurons a relative increase in the use of astrocytic glutamine in glutamate formation probably contributed to glutamate excitotoxicity, thereby continuing the deleterious excitotoxic cascade and subsequent neuronal death. In GABAergic neurons, the reduction or discontinuation in the use of glutamine as substrate for the GABAergic TCA cycle and in GABA formation was probably an important factor leading up to the complete halt in TCA-cycle activity and consequently death of GABAergic neurons. Thus, it is evident that changes in the normal equilibrium between the dependence on astrocytic and neuronal metabolic activities in neurotransmitter synthesis significantly contributed to neuronal death. However, the results also imply that neuronal survival may be intrinsically connected to the well being of astrocytic metabolism. Acknowledgements This work was supported by the Norwegian Council on Cardiovascular Disease, the Norwegian Research Council and Inger Haldorsen’s Grant. We thank Hong Qu, PhD, and Oddbjørn Sæther, MSc for their excellent 13C MRS analyses.

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Differential vulnerability of oligodendrocytes and astrocytes to hypoxic – ischemic stresses Husnia Marrif and Bernhard H.J. Juurlink* Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 p Correspondence address: E-mail: [email protected]

Contents 1. 2. 3.

4.

Introduction Differences principally due to oxidative metabolism and management of strong oxidants Differences in gene expression responses to insult 3.1. Transcriptional response differences 3.2. Translational response differences Concluding remarks

Oligodendrocytes are much more vulnerable to hypoxia –ischemia than astrocytes. This appears to be due to several factors. Oligodendrocytes are much more dependent upon oxidative metabolism than astrocytes. They generate more strong oxidants than astrocytes and yet have less capability of scavenging such oxidants. Oligodendrocytes, cells with large surface areas of membranes containing polyunsaturated fatty acids, also have large iron stores that under conditions of stress can be released, thereby facilitating lipid peroxidation. Unlike astrocytes, oligodendrocytes neither have robust hypoxia-inducible responses, nor anti-oxidant responses. All of these features make the oligodendrocytes more vulnerable to hypoxic– ischemic insults than astrocytes.

1. Introduction Hypoxia – ischemia has both immediate and longer-term effects on cells. In general, the immediate consequence is on oxidative metabolism with resultant reliance on anaerobic metabolism with often resulting ATP deficits and all the problems to which this may give rise. Another immediate consequence is how readily cells can cope with the oxidative stress during the hypoxia –ischemia as well as during the immediate reperfusion period. The long-term effects are related in part to how the cells change gene expression in Advances in Molecular and Cell Biology, Vol. 31, pages 857–867 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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response to the hypoxic– ischemic insult and subsequent oxidative insult during the reperfusion period. Below is a brief and not comprehensive examination of differences in response between astroglia and oligodendroglia to such stresses.

2. Differences principally due to oxidative metabolism and management of strong oxidants Oligodendrocytes are more vulnerable to hypoxia and oxidative stress associated with reperfusion than astrocytes. This appears to be due to several factors, one of which is that oligodendrocytes are more dependent upon oxidative metabolism than astrocytes (Fig. 1). Fig. 2 clearly illustrates this difference in mature rat oligodendrocytes and astrocytes grown in culture in response to chemical hypoxia. Cultures were prepared as outlined in Juurlink and Walz (1998). There have been relatively few studies in vivo examining the effect of hypoxia– ischemia on oligodendrocytes. Waxman et al. (1990) demonstrated more than a decade ago that the rat optic nerve electrophysiological function was very resistant to anoxia but became susceptible to anoxia at the time that oligodendroglia were maturing between 10 and 20 days postnatum, i.e., as the axons became myelinated. That this vulnerability was associated with oligodendroglia was supported by the observation that the optic nerve remained resistant to anoxia in myelin deficient rats (Waxman et al., 1990). The vulnerability of axons appears to be mediated by glutamate release from oligodendrocytes as well as axons (Li et al., 1999). Glutamate release from oligodendrocytes is likely due to the depolarization that occurs as cellular ATP supplies no longer are adequate to maintain

Fig. 1. CO2 production from 1-14C-pyruvic acid in rat astrocytes and oligodendrocytes grown in culture. Results are means ^ SEM.

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Fig. 2. Cell death of rat astrocytes and oligodendrocytes exposed to 1 mM 2-dinitrophenol for differing periods of time. Results are means ^ SEM.

the function of the Na, K-pump; under these conditions glutamate is likely released due to the reversal of the sodium-dependent glutamate transporter as is the case with astrocytes (Gemba et al., 1994). Oligodendrocytes can be readily killed in vitro by glutamate due to activation of AMPA/kainate glutamate receptors (McDonald et al., 1998) or by depletion of cellular cystine because of the reversal of the cystine – glutamate antiporter (Oka et al., 1993). Hypoxia –ischemia simulated in vitro results in oligodendrocyte death via oligodendrocyte-released glutamate acting on AMPA/kainate glutamate receptors present on oligodendrocytes (Yoshioka et al., 2000). Oligodendrocytes are also much more susceptible to nitric oxide than astrocytes (Mitrovic et al., 1995). We have shown that immature oligodendroglia generate more strong oxidants than do astrocytes (Thorburne and Juurlink, 1996), due, at least in part, to their higher rate of oxidative metabolism. Hence, it is likely that the greater susceptibility of oligodendrocytes to nitric oxide is due to the greater formation of superoxide anion and thereby enhanced formation of peroxynitrite. McCulloch and colleagues have shown that hypoxic– ischemic insults in vivo cause the microtubule-associated protein tau to accumulate in oligodendrocytes and that this accumulation is in response to free radicals produced in oligodendrocytes (Irving et al., 1997). They have further shown that oligodendrocytes in vivo are very susceptible to strong oxidants such as 4-hydroxynonenal (McCracken et al., 2000). Additional in vivo evidence that demonstrate preferential vulnerability of oligodendrocytes is that chronic hypoperfusion of rat forebrain results in preferential death of oligodendrocytes (Masumura et al., 2001). We have demonstrated that oligodendrocyte precursor cells in vitro are very vulnerable to hypothermic and hypoxic insults (Juurlink and Husain, 1994; Husain and Juurlink, 1995) and subsequently demonstrated that this was due, at least in part, to a poor ability to

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scavenge strong oxidants produced during the reperfusion period (Thorburne and Juurlink, 1996); this has been reviewed in detail elsewhere (Juurlink, 1997). This vulnerability to oxidative stress is also seen with oligodendrocytes in vivo (Jelinski et al., 1998) where we demonstrated that a brief exposure to severe hypoxia –ischemia results in selective death of immature oligodendrocytes. This selective vulnerability of oligodendrocyte precursors has been confirmed by several other laboratories (Levison et al., 2001; Ness et al., 2001; Back et al., 2002). It is therefore no surprise that chronic exposure of the developing rat to hypobaric hypoxia results in profound effects on myelination in the corpus callosum (Langmeier et al., 1987; Kohlhauser et al., 2000).

3. Differences in gene expression responses to insult The longer-term effects of hypoxia– ischemia are on gene expression. Gene expression changes are generally examined in the context of changes in transcription; however, there are situations where changes in translation can occur without a necessary effect on transcription. 3.1. Transcriptional response differences Only changes in transcription due to lowered oxygen as well as due to the oxidative stress that is present during reperfusion will be examined. 3.1.1. Hypoxia-inducible responses In response to hypoxia, a major change in gene expression involves genes with a hypoxia-response element (HRE) in their promoter region; the consensus sequence for this element is: 50 -RCGTG-30 (Semenza et al., 1996). Genes that have an HRE in their promoter regions include endothelin-1 (Hu et al., 1998), vascular endothelial growth factor (Forsythe et al., 1996), erythropoietin (Semenza, 1994) glucose transporter-1 (Ebert et al., 1995) and the anaerobic isoforms of the enzymes of glycolysis (Semenza et al., 1996). HRE is bound by hypoxia-inducible factor 1 (HIF1), which is a heterodimer comprising a constitutively expressed aryl hydrocarbon receptor nuclear translocator protein (ARNT or HIF1b) and a hypoxia-inducible HIF1a protein (Wang et al., 1995). The levels of HIF-1a rise as oxygen tension lowers (Jiang et al., 1996); this is not due to increased transcription/translation, but rather due to stabilization of HIF-1a at low oxygen tensions. In the presence of oxygen, prolyl-4-hydroxylase and asparaginyl hydroxylase cause hydroxylation of proline and asparagine residues in HIF-1a (Bruick and McKnight, 2001; Dann et al., 2002) that results in ubiquination and subsequent degradation of the HIF-1a via the proteosome pathway (Kallio et al., 1999). Lower oxygen tensions interferes with hydroxylation, thereby increasing the half-life of HIF-1a. There are several isoforms of HIF-1a (Luo et al., 1997). For more details of this oxygen-dependent regulation, see Bruick and McKnight (2002). We have shown in vitro that hypoxia results in an increase of the protein signal for HIF-1a in astrocytes but not in oligodendroglia (Marrif, 2002). Similarly, a unilateral

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hypoxic– ischemic insult results in an increase in HIF-1a protein expression in ipsilateral cerebral astrocytes compared to contralateral astrocytes (Fig. 3). This difference in HIF1a protein levels in response to hypoxia in the two glial cell lines also correlates with differences in the ability to upregulate the anaerobic isoforms of the enzymes of glycolysis; we have shown, using in vitro experiments, that astrocytes but not oligodendrocytes upregulate the anaerobic isoforms in response to hypoxia (Marrif and Juurlink, 1999). We have also shown, using in vivo experiments, that glucose transporter-1 is upregulated in response to a hypoxic– ischemic insult in astrocytes but no other cell type (Marrif, 2002). One possible consequence of this is that under conditions of chronic hypoxia, astrocytes because they have the ability to upregulate anaerobic glycolysis have a better ability to survive than oligodendrocytes; indeed, as noted above, chronic cerebral hypoxia preferentially results in oligodendrocyte damage. 3.1.2. Anti-oxidant responses One consequence of oxidative stress is that the anti-oxidant response can be activated. The anti-oxidant response is controlled by activation of the anti-oxidant response element (ARE) found in the promoter region of certain genes; this element is also known as the electrophile response element (EpRE) (Zhu and Fahl, 2001). The ARE element comprises of an AP1-like element adjacent to a GC box and is flanked by one or more AP1-like elements; the consensus sequence is 50 -TGA C/T NNN GC A/G-30 (Wasserman and Fahl, 1997). Genes under the control of the ARE include NAD(P)H: quinone reductase, epoxide hydrolase, g-glutamyl-cysteine ligase (the rate-limiting enzyme for glutathione synthesis), glutathione reductase, Mn superoxide dismutase, Cu – Zn superoxide dismutase, heme oxygenase-1, aflatoxin B1 dehydrogenase, ferritin heavy and light chains, carbonyl

Fig. 3. Immunocytochemistry demonstrating HIF1a signal in corpus callosum of rats exposed to 30 min of 8% oxygen atmosphere following ligation of right common carotid artery. “A” is a representative area of the ispilateral corpus callosum while “B” is that of the contralateral corpus callosum. Animals were perfusion-fixed immediately after the exposure to hypoxia. Double staining with glial fibrillary acidic protein revealed that the positive cells were astrocytes.

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reductase, aldehyde dehydrogenase, various glutathione S-transferases (Ishii et al., 2000; Kwak et al., 2001; Park and Rho, 2002; Thimmulappa et al., 2002; Pietsch et al., 2003); these genes are now commonly referred to as phase 2 protein genes. The consequence of upregulation of phase 2 protein gene expression is that cells can more efficiently scavenge oxidants. Heterodimers of the transcription factors Nrf2 and jun family proteins bind to the ARE and promote transcription (Venugopal and Jaiswal, 1998; Jeyapaul and Jaiswal, 2000). There is some evidence that heterodimers of Nrf2 and small maf proteins bind to the ARE but inhibit transcription (Dhakshinamoorthy and Jaiswal, 2000; Nguyen et al., 2000) whereas other researchers report that heterodimers of Nrf2 and small maf proteins promote transcription (Itoh et al., 1997). There is evidence that Nrf2 requires phosphorylation for its ability to promote transcription (Huang et al., 2002). The contradictory evidence for the role of small maf proteins in controlling phase 2 protein gene induction may be dependent upon the phosphorylation status of Nrf2 or possibly whether the coactivator protein CREB-binding protein is present (Katoh et al., 2001). Nrf2 is normally bound to the cytoskeleton by a protein known as Inhibitory Nrf2 protein (INrf2 protein) or Keap1 (Itoh et al., 1999). Strong oxidants or phase 2 protein inducers allow dissociation of Nrf2 from Keap1, thereby allowing translocation of Nrf2 to the nucleus where it heterodimerizes with either jun family or small maf family proteins. Phase 2 protein inducers are mainly the Michael reaction acceptors, quinones and certain isothiocyanates that at low micromolar and submicromolar concentrations selectively activate the anti-oxidant response; the maximal induction of phase 2 proteins by the isothiocyanate sulforaphane is at a concentration as low as 50 nM (Wu and Juurlink, 2001). How strong oxidants and phase 2 protein inducers appear to cause release of Nrf2 from Keap1 is through either oxidizing or alkylating critical sulfhydryls in Keap1, thereby allowing release of Nrf2 (Dinkova-Kostova et al., 2002). We have previously shown that astrocytes can better cope with perturbations such as oxidative stress than can oligodendrocytes (Juurlink, 1993; Juurlink and Husain, 1994; Husain and Juurlink, 1995; Thorburne and Juurlink, 1996; Juurlink et al., 1998); this is due, in part, to the ability of astrocytes, but not oligodendrocytes, to upregulate a number phase 2 protein genes (Jordan and Juurlink, 1999). Thus we have shown that phase 2 protein inducers such as tertiary butylhydroquinone readily upregulate glutathione, glutathione reductase and thioredoxin reductase levels in astrocytes (Eftekharpour et al., 2000); however, this is not seen with oligodendrocytes when oligodendrocytes are administered this inducer (unpublished observations). Similarly, the laboratory of J.A. Johnson has shown that ARE-mediated gene expression preferentially occurs in astrocytes (Murphy et al., 2001).

3.2. Translational response differences Only one example will be given. Hypoxia has been shown to induce ferritin synthesis in oligodendrocytes (Qi and Dawson, 1994). It is known that oligodendrocytes have high iron stores both in vivo (Connor et al., 1995; Cheepsunthorn et al., 1998) and in vitro (Thorburne and Juurlink, 1996). This iron is mostly Fe3þ sequestered in ferritin

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(Cheepsunthorn et al., 1998). All ferritins have 24 subunits comprising ferritin heavy (H) and ferritin light (L) chains that give rise to a hollow shell that can store up to 4500 Fe3þ atoms (Harrison and Arosio, 1996). Somewhat counterintuitively, hypoxia increases superoxide anion production by mitochondria (Dawson et al., 1993); this is because the mitochondrial electron carriers tend to become saturated during hypoxia increasing the likelihood of an encounterance with an oxygen molecule and thereby forming superoxide anion. Protonated superoxide anion can enter ferritin and reduce Fe3þ to Fe2þ (Funk et al., 1985), thereby releasing iron into the cytosol. The released Fe2þ binds to an iron-binding protein that would otherwise bind to 50 untranslated regions of the mRNAs for L and H ferritin chains interfering with translation; in the presence of free Fe3þ, the proteins dissociate from the mRNAs allowing translation of ferritin peptides (Haile, 1999). Like the other susceptible cell-type in the central nervous system, the neuron, the oligodendrocyte is characterized by a high rate of oxidative metabolism and thereby a high rate of strong oxidant production, but the oligodendrocyte also has very large surface area of membranes (i.e., the myelin) that contains an abundance of polyunsaturated fatty acids. The large surface area of membrane together with the high iron stores is a dangerous combination when there is a perturbation that results in an increase of protonated superoxide production since this will facilitate lipid peroxidation cascades of the polyunsaturated fatty acids of these membranes (Juurlink, 2001).

4. Concluding remarks One can ask the question why oligodendrocytes are so much more susceptible to hypoxic– ischemic insults, but perhaps a better question is why astrocytes are so resistant to such insults. One possibility is innate to the functional relationship between astrocytes and neurons, with neurons having the greater oxidative metabolism. The greater oxidative metabolism is related to the greater demand for ATP by neurons. One consequence of this is that in neurons the electron store in the form of NADH is used mainly for developing membrane potentials across the inner mitochondrial membrane rather than be used for the formation of redox buffers such as glutathione. Because of the high demand for pyruvate by neurons, neuronal glycolysis often does not suffice during times of intense neuronal activity and a metabolic coupling has been developed with astrocytes, where transmitters such as glutamate and norepinephrine acting on metabotropic receptors activate glycogenolysis and glycolysis in astrocytes, e.g., O’Dowd et al. (1995). The specialization of astrocytes for such a function is emphasized by the presence of substantial glycogen stores in these cells (Peters et al., 1991). A high rate of glycolysis results in the production of substantial electron stores in the form of NADH and because of the presence of transhydrogenases, also of NADPH. Because of the presence of anaerobic isoforms of the enzymes of glycolysis, astrocytic pyruvate is converted to lactate with astrocytes, having high lactate levels, exporting lactate to neurons with low lactate levels. Almost by default, astrocytes act as a redox buffer for neurons (and probably oligodendrocytes) since the lactate in neurons is converted to pyruvate with the concomitant reduction of NAD to NADH - this is discussed in some detail in a previous review (Walz and Juurlink, 2001). This energy coupling function is dictated to a great extent by astrocytes having the

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capacity for anaerobic glycolysis, i.e., converting glucose to lactic acid. The lactic acid would flow down its concentration gradient, i.e., flow from astrocytes to neurons. In neurons the lactic acid is converted to pyruvic acid that is acted upon by pyruvate dehydrogenase, thereby entering the Kreb cycle. To carry out anaerobic glycolysis necessitates that astrocytes have high NADH/NAD ratios, with the consequence that astrocytes have a high redox buffering capacity, thereby a high capacity to scavenge strong oxidants (see Juurlink, 1997).

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Glial heme oxygenase-1 in CNS injury and disease Hyman M. Schipper McGill University and Center for Neurotranslational Research, Lady Davis Institute for Medical Research, S.M.B.D. Jewish General Hospital, 3755 Cote St. Catherine Rd., Montreal, QC, Canada H3T 1E2 Correspondence address: Tel.: þ 514-340-8260; fax: þ 514-340-7502. E-mail: [email protected](H.M.S.)

Contents 1. 2. 3. 4. 5. 6.

7. 8. 9. 10.

Introduction Heme oxygenases Heme oxygenase-1 Neuroprotective role of HO-1 Dystrophic effects of HO-1 in neural tissues HO-1 and glial iron deposition 6.1. HO-1 induction and mitochondrial iron sequestration 6.2. Oxidative stress: a common pathway for glial HO-1 induction 6.3. HO-1 upregulation is necessary and sufficient for mitochondrial iron trapping 6.4. HO-1 promotes intraglial oxidative stress 6.5. Role of the mitochondrial permeability transition pore A model for pathological neuronal –glial interaction HO-1 expression in AD and PD brain Is glial HO-1 induction relevant to the pathogenesis of human neurodegenerative disorders? Concluding remarks

Heme oxygenase-1 (HO-1) is a 32 kDa stress protein that degrades heme to biliverdin, free iron and carbon monoxide. The upregulation of HO-1 in brain may either be cytoprotective (biliverdin and its metabolite, bilirubin have antioxidant properties) or neuroendangering (free iron and carbon monoxide may promote intracellular free radical generation) contingent upon the extent and duration of HO-1 over-expression, the prevailing redox microenvironment and the experimental model employed. Our laboratory has shown that cysteamine, dopamine, b-amyloid, IL-1b and TNF-a upregulate HO-1 followed by mitochondrial sequestration of nontransferrin-derived 55iron in cultured rat astroglia. In these cells and in rat astroglia transfected with the human HO-1 gene, Advances in Molecular and Cell Biology, Vol. 31, pages 869–882 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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mitochondrial iron trapping was abrogated by the HO-1 inhibitors, tin-mesoporphyrin and dexamethasone. We determined that HO-1 immunoreactivity is greatly enhanced in neurons and astrocytes of the hippocampus and cerebral cortex of Alzheimer subjects and co-localizes to senile plaques and neurofibrillary tangles. HO-1 staining is also augmented in astrocytes and decorates neuronal Lewy bodies in the Parkinson substantia nigra. Collectively, our findings suggest that HO-1 over-expression contributes to the pathological iron deposition and mitochondrial damage documented in these agingrelated neurodegenerative disorders. HO-1 induction may also participate in the biogenesis of corpora amylacea, glial and extracellular glycoproteinaceous inclusions that progressively accumulate in the aging and degenerating mammalian nervous system. 1. Introduction Oxidative stress (free radical damage) and mitochondrial insufficiency (ATP depletion) have been implicated in mammalian brain senescence and in the pathogenesis of Alzheimer disease (AD), Parkinson disease (PD) and other aging-related neurodegenerative disorders (Reichmann and Riederer, 1994; Beal, 1995; Mattson, 1997). The excessive deposition of redox-active iron (an important generator of reactive oxygen species (ROS)) in the basal forebrain and association cortices of AD victims (Schipper, 1998a; Sayre et al., 2001) and the substantia nigra/basal ganglia of subjects with idiopathic PD (Jenner, 1992; Youdim, 1994; Riederer et al., 1989; Jellinger et al., 1990) may contribute to oxidative mitochondrial injury in these conditions. In the diseased tissues, astrocytes, microglia, macrophages and microvessels appear to be more important repositories for the excess iron than the affected neuronal populations (Gelman, 1995; Youdim, 1994; Morris and Edwardson, 1994). Expression of tissue ferritin, the major intracellular iron storage protein, parallels the distribution of the metal deposits and further implicates nonneuronal (glial) cellular compartments (Youdim, 1994). In contrast, there appears to be no (or an inverse) relationship between concentrations of cellular transferrin receptors and sites of abnormal tissue iron accumulation. The latter observations suggest that, in contradiction to most peripheral tissues, the transferrin/transferrin receptor pathway may play little or no role in pathological brain iron sequestration. Indeed, evidence is accumulating implicating alternative iron-regulating proteins in this process, including lactoferrin and its receptor, melanotransferrin and the heme oxygenases (Mash et al., 1991; Connor and Menzies, 1992; Kalaria et al., 1992; Faucheux et al., 1993; Gelman, 1995; Schipper, 1998a). In the following sections, the putative role(s) of glial heme oxygenase-1 (HO-1) expression in brain aging and neurodegeneration is discussed, with particular consideration of the enzyme’s potential contribution to aberrant brain iron deposition and attendant oxidative mitochondrial injury. 2. Heme oxygenases Heme oxygenases (E.C. 1:14:99:3; heme-hydrogen donor:oxygen oxidoreductase) constitute a family of rate-limiting enzymes of the heme degradative pathway. Virtually in all mammalian tissues, heme oxygenases are localized to the endoplasmic reticulum,

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where they operate in conjunction with NADPH cytochrome P450 reductase to oxidize heme (ferriprotoporphyrin IX) to biliverdin (biliverdin IXa), free iron and carbon monoxide (CO). Biliverdin is metabolized further by biliverdin reductase to the yellow bile pigment, bilirubin (Ryter and Tyrrell, 2000; Fig. 1). Three isoforms of heme oxygenase, HO-1 (also referred to as heat shock protein32), HO-2 and HO-3, have been identified. HO-1 and HO-2 share a 24-amino acid motif that imparts robust heme catabolic activity to these enzymes. The HO-3 protein appears to be far less efficient in metabolizing heme and may serve primarily as a heme-sensing molecule involved in the maintenance of intracellular heme homeostasis (McCoubrey et al., 1997). Although each HO enzyme displays similar substrate and cofactor specificities, they are encoded by separate genes and exhibit important differences with respect to molecular weight, antigenicity, electrophoretic mobility, susceptibility to proteolysis, tissue distribution and regulation (Dennery, 2000). HO-2 mRNA and protein are widely distributed in neurons of the mammalian brain, with highest concentrations in olfactory epithelium and olfactory bulb, hippocampal pyramidal cells and dentate gyrus, and cerebellar granule and Purkinje cell layers

Fig. 1. The heme catabolic pathway. M ¼ methyl; V ¼ vinyl; P ¼ propionate. (Modified after Ryter and Tyrrell, 2000, with permission).

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(Verma et al., 1993). HO-2 expression appears not to be changed under oxidative challenge or in disease states. The topography of HO-2 expression in rat brain exhibits considerable overlap with the distribution of soluble guanylate cyclase. Heme-derived CO, like nitric oxide, may modulate neuronal activity by stimulating guanylate cyclase and raising intracellular levels of the second messenger, cGMP (Verma et al., 1993). In contrast to HO-2, HO-1 expression in the normal, unstressed brain is limited to neuroglia and small populations of scattered neurons (ZZBaran˜ano et al., 2001). HO-3 has thus far only been reported in rat brain and its pattern of expression remains to be delineated (McCoubrey et al., 1997).

3. Heme oxygenase-1 HO-1 is a highly inducible 32 kDa member of the stress protein superfamily that was first described in 1968. In humans, the ho-1 gene is localized to chromosome 22q12 and consists of four introns and five exons. The regulatory region of the mammalian ho-1 gene comprises a 500 bp promoter, a proximal enhancer and two or more distal enhancers (Fig. 2; Dennery, 2000). These regulatory regions contain heat shock consensus (HSE) sequences, metal (MTRE, CdRE) and antioxidant (ARE) are response elements and AP-1, AP-2, nuclear factor kappa B (NF(B) and HIF-1 binding sites. These response elements render the ho-1 gene exquisitely sensitive to upregulation by heme, dopamine, b-amyloid, H2O2 and other oxidants, UV light, transition metals, proinflammatory cytokines, lipopolysaccharide and prostaglandins (Dennery, 2000; Schipper, 2000). The ho-1 promoter region also contains a 56 bp sequence (STAT-3 acute-phase response factor binding site) that confers susceptibility to transcriptional suppression by glucocorticoids (Lavrovsky et al., 1996). Additional binding sites, indicated in Fig. 2 and its legend, are less germane to the present discussion. In certain tissues, such as rat liver and lung, HO-1 expression appears to be developmentally regulated at both transcriptional and posttranscriptional levels (Dennery, 2000). The HO1 (but not the constitutively expressed HO-2) protein contains a destabilizing PEST (proline (P) – glutamic acid (E) –serine (S) – threonine (T)) sequence at the carboxy terminus that renders the peptide sensitive to rapid proteolysis (Dwyer et al., 1992). The half-lives of HO-1 mRNA and protein are approximately 3 h and 15– 21 h, respectively (Dennery, 2000). Induction of HO-1 in the face of oxidative challenge may protect cells by fostering the conversion of pro-oxidant heme and hemoproteins to the radical-scavenging bile pigments, biliverdin and bilirubin (Stocker et al., 1987; Nakagami et al., 1993; Llesuy and Tomaro, 1994; Dore´ et al., 1999; Baran˜ano et al., 2001). On the other hand, free iron and CO released during heme degradation may, under certain circumstances, perpetuate intracellular oxidative stress and predispose the mitochondrial compartment to free radical damage (Zhang and Piantadosi, 1992; Frankel et al., 2000). This disparate behavior has stimulated lively controversy as to whether HO-1 upregulation in the CNS is neuroprotective or contributory to cellular injury and degeneration. These opposing perspectives should not be viewed as mutually exclusive; the timecourse and intensity of HO-1 induction and the chemistry of the local redox

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Fig. 2. Regulatory elements of the mammalian HO-1 gene. P ¼ promoter region; PE ¼ proximal enhancer region. DE ¼ distal enhancer. Patterned bars represent various regulatory protein/transcription factor binding sites: C/EBP ¼ sequence for CCAAT-enhancer binding protein; AP-1 ¼ activator protein-1 (c-fos/c-jun dimer) binding site; SP-1 ¼ GC boxes recognized by sequence-specific transcription factor; NFKB ¼ nuclear factor kappa B consensus sequence; HypoRE ¼ hypoxia response element (or HIF-1); CdRE ¼ cadmium response element; PRE ¼ prostaglandin response element; IL-GRE ¼ IL-6 response element (or STAT-1); HSE ¼ heat shock element; MTRE ¼ metal response element; AP-2 ¼ activator protein-2 binding site; STAT-3 ¼ acutephase response factor binding site; USF ¼ upstream regulatory factor binding site. (Modified after Dennery, 2000, with permission).

microenvironment may determine whether free radical damage accruing from intracellular liberation of iron/CO or the antioxidant benefits of a diminished heme – bilirubin ratio predominate (Galbraith, 1999; Suttner and Dennery, 1999). Specific neuroprotective and neuroendangering aspects of central HO-1 expression are discussed in the following sections.

4. Neuroprotective role of HO-1 There exists ample evidence implicating a neuroprotective role for HO-1 both in intact animals and in tissue culture. Following exposure to a variety of noxious stimuli, HO-1 induction occurs in many types of nonneuronal cells (e.g., astrocytes, oligodendrocytes, microglia, brain endothelial cells, ependyma cells and choroid plexus epithelial cells) and

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some neuronal cells, although it has been argued that the greater capacity of astrocytes than neurons to mount a robust HO-1 (and other heat shock protein) response may partly account for the relative preservation of the former in the face of oxidative challenge (Dwyer et al., 1995; Manganaro et al., 1995; Snyder et al., 1998). Cerebellar granule cells harvested from transgenic (tg) mice designed to over-express HO-1 in neurons (Maines et al., 1998) appear to be relatively resistant to glutamate- and H2O2-mediated oxidative damage in vitro (Chen et al., 2000). Analogously, neuroblastoma cell lines transfected with HO-1 cDNA were less vulnerable than control cells to oxidative damage resulting from exposure to (b-amyloid1 – 40 (Le et al., 1999) or H2O2 (Le et al., 1999; Takeda et al., 2000). In an in vivo study, HO-1 tg mice subjected to cerebral ischemia manifested decreased tissue staining for lipid peroxidation end-products, enhanced expression of the anti-apoptotic factor, bcl-2, and smaller infarct volumes relative to normal littermates (Panahian et al., 1999). HO-1 may also confer neuroprotection in animal models of traumatic brain injury (Fukuda et al., 1996; Beschorner et al., 2000). As described above, rapid heme/hemoprotein degradation and the intracellular accumulation of antioxidant bile pigments may be responsible, at least in part, for the observed neuroprotection associated with the induction of HO-1. Alternatively or in addition, CO liberated in the course of heme catabolism may mediate some of the cytoprotective effects of HO-1. For example, by engendering smooth muscle relaxation (Verma et al., 1993), hemederived CO has been postulated to ameliorate cerebral vasospasm, a cause of significant morbidity in patients with subarachnoid hemorrhage (Matz et al., 1996; Suzuki et al., 1999; Tanaka et al., 2000).

5. Dystrophic effects of HO-1 in neural tissues As described above, the antioxidant effects of heme-derived bile pigments may provide some degree of cytoprotection to stressed CNS tissues. On the other hand, the excessive and prolonged hyperbilirubinemia of untreated neonatal jaundice may incur irreversible neurological injury (kernicterus). In these children, kernicterus can be obviated by photodegradation of circulating bilirubin or administration of synthetic metalloporphyrins, competitive inhibitors of heme oxygenase activity (Qato and Maines, 1985). Even physiological augmentation of heme breakdown resulting from transient upregulation of endogenous HO-1 may, under certain conditions, promote rather than prevent neural injury. Thus, metalloporphyrin suppression of heme oxygenase activity has been shown to diminish tissue necrosis and edema formation following focal cerebral ischemia in intact rats (Kadoya et al., 1995), confer neuroprotection in an experimental model of intracerebral hemorrhage (Koeppen and Dickson, 1999) and alleviate traumatic CA1 insults in rat hippocampal slices (Panizzon et al., 1996). Differences in species, experimental models and pharmacological protocols employed in these studies and those described in Section 4 may partly account for the conflicting data regarding the role(s) of HO-1 induction in ischemic, hemorrhagic and traumatic neural damage.

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6. HO-1 and glial iron deposition The molecular mechanisms by which augmentation of HO-1 may endanger neural tissues remain ill-defined. In this and the following section, we review evidence that induction of HO-1 in astrocytes perturbs patterns of cellular iron sequestration that may, secondarily, render nearby neuronal constituents prone to oxidative injury. 6.1. HO-1 induction and mitochondrial iron sequestration In cultures of neonatal rat astroglia, cysteamine (CSH; 880 mM), dopamine (1 mM; but not equimolar norepinephrine), b-amyloid40/42 (3 – 15 mM), tumor necrosis factor-a (TNFa; 20 ng/ml) and interleukin-1b (IL-1b; 20 ng/ml) upregulated HO-1 mRNA, protein and/or activity levels within 3– 12 h of treatment. After 3 –6 days of exposure to these stimuli, we observed augmented sequestration of nontransferrin-derived 59Fe (or 55Fe) by the mitochondrial (but not the lysosomal) compartment relative to untreated control cultures; enhanced iron uptake by whole-cell compartments in these studies was variable (Chopra et al., 1995; Manganaro et al., 1995; Mydlarski and Schipper, 1998; Schipper, 1999; Schipper et al., 1999; Ham and Schipper, 2000; Mehindate et al., 2001). These stimuli had no appreciable effect on mitochondrial trapping of diferric transferrin-derived iron (Chopra et al., 1995; Manganaro et al., 1995; Mydlarski and Schipper, 1998; Schipper, 1999; Schipper et al., 1999; Ham and Schipper, 2000; Mehindate et al., 2001), commensurate with the fact that pathological brain iron deposition in situ may be mediated independently of transferrin and its receptor (see Section 1). 6.2. Oxidative stress: a common pathway for glial HO-1 induction Hydrogen peroxide (300 – 500 mM) and menadione (100 mM) administration recapitulated the effects of CSH, dopamine, b-amyloid and pro-inflammatory cytokines on glial HO-1 expression and mitochondrial iron trapping. Furthermore, the effects of the latter stimuli on astroglial HO-1 expression were attenuated by coadministration of antioxidant compounds such as ascorbate, melatonin or trans-resveratrol (Schipper et al., 1999; Mehindate et al., 2001). These observations strongly suggest that oxidative stress is the final common pathway mediating induction of the ho-1 gene in these cells. 6.3. HO-1 upregulation is necessary and sufficient for mitochondrial iron trapping In cultured astrocytes exposed to dopamine, b-amyloid, TNFa or IL-1b, mitochondrial iron sequestration was suppressed by administration of tin mesoporphyrin (SnMP; 1 mM), a competitive inhibitor of heme oxygenase activity, or dexamethasone (DEX; 50 mg/ml), a glucocorticoid transcriptional suppressor of the ho-1 gene. Moreover, overexpression of the human ho-1 gene in cultured rat astroglia by transient transfection markedly increased mitochondrial 55Fe deposition relative to sham-transfected controls, an effect that was completely abrogated by treatment with SnMP or DEX (Schipper et al., 1999; Ham and Schipper, 2000; Mehindate et al., 2001). These findings suggest that upregulation of HO-1

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is a pivotal event leading to excessive mitochondrial iron deposition in oxidatively challenged astroglia. 6.4. HO-1 promotes intraglial oxidative stress Transient transfection of human HO-1 cDNA in cultured rat astroglia stimulates late, presumably compensatory induction of the manganese superoxide dismutase gene. The latter can be attenuated by antioxidant treatment (ascorbate, melatonin or resveratrol) indicating that HO-1 over-expression, at least in astroglia, may exacerbate intracellular oxidative stress (Frankel et al., 2000). 6.5. Role of the mitochondrial permeability transition pore In the HO-1-transfected cells and in astrocytes challenged with dopamine or proinflammatory cytokines, mitochondrial iron trapping was significantly curtailed by treatment with cyclosporin A or trifluoperazine, potent inhibitors of the mitochondrial permeability transition pore (Schipper, 2000; Mehindate et al., 2001). Conceivably, intracellular free ferrous iron and CO, products of HO-1-mediated heme degradation, subject the glial mitochondria to heightened levels of oxidative stress (Zhang and Piantadosi, 1992). Oxidative stress is known to favor maintenance of the mitochondrial permeability transition pores in the open configuration (Petronilli et al., 1993; Bernardi, 1996). The open pores, in turn, may facilitate influx and sequestration of low-molecularweight iron within the mitochondrial matrix. Using transmission electron microscopy and X-ray microanalysis, we noted that metal-laden mitochondria in CSH-exposed astroglial cultures and in aging subcortical astroglia in situ are often markedly distended and acristic, morphological features consistent with prior engagement of the permeability transition pore (Brawer et al., 1994a,b). In Section 7, implications of this astroglial iron deposition for neuronal survival are considered. 7. A model for pathological neuronal – glial interaction The literature (including this volume) is replete with data attesting to the beneficial role of astrocytes in neuritic outgrowth and neuronal survival. However, virtually all the effects have been demonstrated in studies using young, healthy astroglia. Considerably less is known concerning the behavior (or misbehavior) of ‘stressed’ astroglia in the senescent and diseased nervous system. For example, although ATP-dependent uptake of extracellular glutamate by healthy astroglia is an ‘asset’, failure of this glial action in ischemic brain may constitute an important ‘liability’ predisposing to excitotoxic neuronal injury (Schipper, 1998c—see also chapter by Schousboe and Waagepetersen). In Section 6, we reviewed evidence that upregulation of HO-1 in cultured astrocytes promotes the sequestration of nontransferrin-derived iron by the mitochondrial compartment. Using electron spin resonance spectroscopy, we demonstrated that this mitochondrial iron behaves as a nonenzymatic (or pseudo-) peroxidase activity capable of oxidizing catechol-containing compounds, such as dopamine and 2-hydroxyestradiol,

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to potentially neurotoxic ortho-semiquinone radicals (Schipper et al., 1991). Redoxactive glial iron may also facilitate the bioactivation of the pro-toxin, MPTP to the dopaminergic neurotoxin, MPP þ in the face of monoamine oxidase blockade (DiMonte et al., 1995). The accumulation of various stress (heat shock) proteins within the iron-laden astrocytes (Chopra et al., 1995; Mydlarski and Schipper, 1998), the compensatory upregulation of MnSOD observed in these cells, and the natural abundance of reduced glutathione in astrocytes relative to other CNS cell types (Manganaro et al., 1995; Frankel et al., 2000) may protect against oxidative damage within the glia themselves. However, the toxic effects of ROS need not be confined to the cellular compartment in which they originate. Superoxide, for example, can egress from neuroglial cells via anion channels (Kontos et al., 1985), and the stability and lipid solubility of hydrogen peroxide permit this molecule to readily traverse plasma membranes and gain access to the intercellular space. Experiments conducted in our laboratory revealed that catecholamine-producing PC12 cells grown atop monolayers of astrocytes replete with mitochondrial iron (induced by CSH pre-treatment) exhibit far greater vulnerability to dopamine/H2O2-related killing than PC12 cells cocultured with control (iron-poor) astroglia (Frankel and Schipper, 1999). PC12 cell death in this model was significantly attenuated by coincubation with antioxidants or deferoxamine, further attesting to the role of oxidative stress and catalytic iron in the demise of these cells. Presumably for reasons outlined above, astroglial death was not appreciably increased by dopamine– H2O2 exposure in either the experimental or control coculture paradigm. To the degree that similar neuronal – glial interactions operate in situ, it is conceivable that, far from providing neuroprotection, HO-1 induction and attendant mitochondrial iron deposition in astrocytes stressed by aging or disease create a redox microenvironment inimical to the survival of indigent neuronal constituents.

8. HO-1 expression in AD and PD brain Using immunolabeling techniques and laser scanning neurons, confocal microscopy, we observed consistent colocalization of HO-1 protein to neurons, GFAP-positive astrocytes, neurofibrillary tangles, senile plaques and corpora amylacea (CA) in postmortem AD brain specimens (Schipper et al., 1995). Approximately 86% of GFAP-positive astrocytes in the AD hippocampus expressed HO-1, whereas only 6.8% of hippocampal astrocytes in normal senescent control tissues exhibited HO-1 immunoreactivity. Furthermore, intense 32 kDa bands corresponding to HO-1 were detected by Western blotting of protein extracts derived from the AD hippocampus and temporal cortex after SDS-PAGE, whereas HO-1 bands were faint or absent in protein extracts prepared from control specimens (Schipper et al., 1995). Our findings indicate that HO-1 is significantly upregulated in astrocytes of Alzheimer-diseased hippocampus and cerebral cortex relative to corresponding tissues derived from elderly, nondemented controls. In contrast, there is no discernible augmentation of HO-1 staining in brain endothelial cells, ependyma cells or choroid plexus cells (Anthony et al., in press).

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In PD, the fraction of GFAP-positive astroglia expressing HO-1 in the substantia nigra (the brain region most heavily affected in this condition) was found to be significantly greater (77.1%) than that observed in the substantia nigra of age-matched control subjects (18.7%). In the caudate, putamen and globus pallidus (subcortical nuclei extensively interconnected with the substantia nigra), percentages of GFAP-positive astroglia coexpressing HO-1 were relatively low and did not differ substantially between PD and control specimens (Schipper et al., 1998).

9. Is glial HO-1 induction relevant to the pathogenesis of human neurodegenerative disorders? The results of in vitro studies suggest that (i) dopamine released from dying nigrostriatal neurons may be a prime stimulus for glial HO-1 induction in PD-affected substantia nigra (Schipper et al., 1999); and (ii) upregulation of glial HO-1 in AD cerebral cortex and hippocampus may be the consequence of excessive b-amyloid and/or cytokine provocation (Ham and Schipper, 2000). In light of the data reviewed in Sections 6– 8, we submit that, in AD and PD, over-production of HO-1 protein in affected astrocytes may contribute to the (transferrin-independent) iron overload and mitochondrial insufficiency, characteristic of these neurodegenerative disorders (Reichmann and Riederer, 1994; Beal, 1995). In addition to the mitochondrial compartment, augmentation of HO-1 activity in the degenerating CNS may be deleterious to other subcellular organelles. Smith and coworkers reported downregulation of tau expression in a neuroblastoma cell line transfected with human HO-1 cDNA (Takeda et al., 2000). Suppression of this microtubulestabilizing protein could theoretically compromise the integrity of the cytoskeleton and thereby predispose to cytopathological changes. We also conjecture, based on in vitro and whole-animal evidence, that sustained or repeated induction of HO-1 in astroglia may play a role in the biogenesis of CA. CA are glycoproteinaceous inclusions that accumulate extracellularly and within subpial and periventricular astrocytes of the AD brain and, to a lesser extent, in normal aging neural tissues (Schipper et al., 1995; Cavanaugh, 1999). CA can be induced experimentally in rat astroglial cultures (Cisse´ and Schipper, 1995) and in rat subcortical astrocytes in situ (Schipper, 1998b) by long-term exposure to CSH, a potent inducer of glial HO-1. Human and experimentally induced CA contain immunoreactive HO-1 (Schipper et al., 1995; Sahlas et al., 2002) and administration of the HO-1 transcriptional suppressor, DEX attenuates the accumulation of CA in CSH-treated glial cultures (Sahlas et al., 2002). Taken together, these data strongly suggest that the accelerated formation of CA in AD astroglia may be dependent upon antecedent over-expression of HO-1 in these cells. Finally, heme-derived CO may facilitate the development of cognitive (Liebson and Albert, 1994), olfactory (Doty, 1991) and neuroendocrine (Sapolsky, 1992) disturbances characteristic of AD by increasing cGMP concentrations in olfactory neurons (Verma et al., 1993), modulating the secretion of corticotropin-releasing hormone (Parks et al., 1994), altering central vasoregulatory mechanisms (Marks et al., 1991) and impacting hippocampal long-term potentiation (Stevens and Wang, 1993; Zhuo et al., 1993).

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10. Concluding remarks HO-1 is a 32 kDa member of the stress protein superfamily that mediates the degradation of heme to biliverdin, free ferrous iron and CO. Robust upregulation of HO-1 occurs in astrocytes within AD- and PD-affected brain tissues, in various animal models of CNS injury and disease, and in response to a host of oxidative and other noxious stimuli. The downstream cytophysiological implications of this HO-1 response, however, remains a hotly debated topic. One school marshals experimental evidence that HO-1 induction in the injured CNS is beneficial (reviewed in Section 4) and posits that the intracellular catabolism of pro-oxidant heme molecules to antioxidant bile pigments helps restore a local redox milieu that favors cell survival. Conversely, evidence presented in Sections 5– 9 argues that sustained over-expression of HO-1 in astroglia with attendant liberation of intracellular free iron and CO may contribute to the pathological iron deposition, oxidative damage and mitochondrial insufficiency documented in AD, PD and other agingassociated neurodegenerative disorders. The disparate behavior of HO-1 under various neuropathological conditions may be reconciled by the fact that intracellular heme degradation may exert net antioxidant or pro-oxidant effects, contingent upon the intensity and temporal profile of HO-1 induction and its interplay with the prevailing redox microenvironment. Further delineation of the role(s) of glial HO-1 expression in CNS injury and disease is clearly warranted and may suggest novel molecular targets for future therapeutic intervention.

Acknowledgements The skillful secretarial assistance of Mrs Lucia Badolato is greatly appreciated. The work described herein was supported by grants from the Canadian Institutes for Health Research, the Fonds de la Recherche en Sante´ du Que´bec and the Alzheimer’s Association (US).

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Astrocytes and microglia in Alzheimer’s disease Steven W. Barger Donald W. Reynolds Department of Geriatrics, Department of Anatomy & Neurobiology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Geriatric Research Education and Clinical Center, Central Arkansas Veterans Healthcare System, Little Rock, AR 72205, USA Correspondence address: E-mail: [email protected]

Contents 1. 2. 3.

4.

Introduction The senile plaque: a city under quarantine Glia in Alzheimer’s disease: just whose side are they on? 3.1. The ‘inflammatory hypothesis’ 3.2. Indictment of the bad guys 3.3. The defense does not rest Concluding remarks

The role of inflammatory processes in disease in general—and neurodegenerative diseases, in particular—has garnered increasing attention in the last decade. In the central nervous system, many of these inflammatory events are initiated or propagated by glial cells. As the resident monocytic phagocytes of the brain, microglia are especially important to these processes, but astrocytes have well-documented roles, as well. The evidence that these cells contribute to the pathogenesis of Alzheimer’s disease has been mounting, but data coming to light most recently cast doubt on this interpretation and support the proposition that certain inflammatory events may slow or even combat the incipient disease triggers. These debates and their implications are discussed in this chapter.

1. Introduction Functional deficits in Alzheimer’s disease have naturally led to hypotheses and investigations concerning the neuronal elements of the affected brain regions. Memory loss and eventual elaboration of dementia almost certainly involves the functional Advances in Molecular and Cell Biology, Vol. 31, pages 883–899 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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compromise of neurotransmission, and this assumption is borne out by the reduction in numbers of neurons and their synapses in the earliest and most dramatically affected regions. However, the first neuropathological descriptions of Alzheimer’s disease noted the concomitant alterations of glial elements. A subset of amyloid plaques are surrounded by astrocytes and are invaded by microglia. These glia appear altered in morphology and function, generally showing the signs of inflammatory activation. This chapter seeks to describe classical findings about glial contributions to neuropathology and to synthesize this information with recent findings regarding the biology of normal astrocytes and microglia and their potential roles in Alzheimer’s disease.

2. The senile plaque: a city under quarantine A striking neuropathological presentation, the amyloid plaque has been the focus of Alzheimer’s disease research from the outset. Even as therapeutic hopes are pinned on rational drug design and cutting-edge molecular biology, these strategies are aimed at the amyloid b-peptide (Ab) that comprises the primary proteinaceous mass of these plaques. However, clinical signs of dementia and the official diagnosis of Alzheimer’s disease depend, more or less, on a structure more complex than a simple peptide deposit. Alzheimer and Cajal both reported glial elements present in amyloid plaques. Silver staining and the later application of amyloidophilic dyes like Congo red revealed amyloid deposits in which the Ab had achieved a specific conformation of b-pleated sheets. But most of these also contained astrocytes and microglia. Since those early descriptions, the application of immunohistochemistry has revealed a broader array of Ab deposits, in both the brains of Alzheimer’s victims and neurologically normal individuals. A popular opinion among modern neuropathologists is that these different forms of Ab deposits reflect different stages of progression that each individual plaque undergoes (Rozemuller et al., 1989). In this staging scheme, the earliest deposits take the form of diffuse, disorganized Ab that is nevertheless concentrated by self-affinity (aggregation) or deposition upon some other extracellular structure. The second stage is marked by the presence of dystrophic neuronal processes. Whether derived from synaptic terminals (e.g., axon boutons) or from en passant axons severed by the pathogenic events occurring in and around the plaque, these dystrophic neurites are swollen and contain inclusions of membrane remnants and elevated levels of the very protein from which Ab is derived: b-amyloid precursor protein (bAPP). Plaques of this second, or ‘neuritic,’ stage are those that best correlate with the presence and degree of clinical dementia (Nagy et al., 1995). Analyses conducted in the last decade (Griffin et al., 1995; Sasaki et al., 1997) reveal that most neuritic plaques contain microglia—often intimately dispersed throughout the Ab deposit – and astrocytes—which have sealed off the deposit much as they would the boundaries of an acute lesion. Some investigators have advised a further subdivision into neuritic plaques that contain a dense core of Ab that has assumed the b-pleated sheet conformation, and is thus ‘congophyllic.’ The final stage of the plaque-progression scheme was envisioned to account for those dense, congophyllic deposits of Ab that have no nearby neurons or neurites. It has been supposed that this reflects a point in the lifetime of an individual plaque by which all neurons within its sphere of influence have been

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killed, inspiring the colorful moniker ‘the burned-out plaque’. Presumably, the debris of victimized neuronal bystanders has been cleared by glial cells, which have since moved on or disintegrated themselves; few astrocytes or microglia are found associated with the burned-out plaque (Griffin et al., 1995; Sasaki et al., 1997; Sheng et al., 1995). The apparent expurgation of neuronal elements around the burnt-out plaque provides compelling support to the argument that plaques are responsible for neuronal loss and functional deficits in Alzheimer’s disease. If factual, the plaque progression scheme must either occur at varying rates or be initiated anew at various points in the disease process, as plaques of various categories can be found close to one another in an affected brain region (Joachim et al., 1989). Support for the latter idea of staggered initiation has been provided by mathematical models that describe a turnover of plaques, suggesting that their formation and removal occurs with kinetics approaching equilibrium (Cruz et al., 1997). One can detect diffuse Ab deposits in the cerebella of Alzheimer brains and in the cerebral cortices of cognitively intact older persons (Joachim et al., 1989). Are these simply situations in which the deposits do not progress? More detailed examination suggests otherwise. When immunohistochemistry is performed with antibodies that detect specific Ab termini, it becomes apparent that diffuse Ab deposits and neuritic plaques are comprised of different forms of Ab. Most consistently, it has been documented that diffuse Ab deposits only contain forms of Ab that end at Ala42; Ab with Val40 as the carboxyterminus is exclusive to neuritic plaques (Iwatsubo et al., 1994; 1996). Furthermore, some analyses suggest that diffuse plaques contain substantial amounts of Ab beginning at Leu17, a peptide also known as p3 (Gowing et al., 1994; Kida et al., 1995). A product of the so-called a-secretase processing of bAPP, p3 is less efficient in activating glial responses in vitro (below). Thus, the different characteristics of diffuse Ab deposits, including reduced glial reactivity and neurotoxicity, may be attributed to a structural difference in the Ab peptide. Regardless of whether neuritic plaques represent an intermediate stage in plaque evolution or a distinct type of plaque, the glial involvement is worth dissecting. Detrimental influences on synapses, neurites, and neurons may be related to any of the unique attributes of neuritic plaques, including their glial elements. Indeed, degeneration may arise from a glial activation that persists too long. Astrocytes and microglia appear to have different strategies for responding to injury or foreign bodies. Most often, these strategies are cooperative: microglia are recruited to the site and begin phagocytosis; their response includes the release of IL-1 and other cytokines that activate nearby astrocytes to wall off the injured tissue or foreign body (Sievers et al., 1993). The latter step usually includes the secretion of a mortar of proteoglycans. When the instigator is Ab, these strategies may be working at cross purposes. Ab deposited on the substratum of a microglial cell culture can be cleared quite efficiently. However, if astrocytes are plated before the microglia, this clearance does not occur, even if the astrocytes have been killed (Hoke et al., 1994; Shaffer et al., 1995). Astrocytes produce chondroitinsulfate proteoglycans (CSPGs) in the presence of Ab, and enzymatic degradation of these CSPGs can return the Ab to a microglia-sensitive state (Shaffer et al., 1995). If these relationships exist in vivo, astrocytic production of PG could render microglia impotent in their attempts to clear Ab deposits, leaving them in what some have termed a state of ‘frustrated’ activation. Notably, proteoglycan is quite abundant in neuritic

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plaques, and its coinjection with synthetic Ab can generate plaque-like deposits in rat brain (Snow et al., 1994). During the 1972 smallpox outbreak in Yugoslavia, hope was abandoned for some communities. To contain the threat posed by the contagion, entire neighborhoods were fenced off and placed under quarantine. No one was allowed out, but physicians and other healthcare professionals were sent in for two-week periods to do the best they could. Had they not been working under a communist dictatorship, some workers might have complained that their efforts were hindered by the quarantine barricade itself, which doubtlessly complicated the delivery of life-sustaining supplies. The neuritic plaque may be a microscopic analogy of this situation; the metaphor may be even more complete for the 1893 smallpox outbreak of Muncie, Indiana, where a quarantine dragged on long enough to engender a homicidal desperation. 3. Glia in Alzheimer’s disease: just whose side are they on? 3.1. The ‘inflammatory hypothesis’ It was assumed for some time that the glia associated with Alzheimer’s disease lesions were reactive, responding to the plaque itself or to the damage the amyloid caused directly to neurons. But in the late 1980s, several investigators began to suggest that the inflammatory sequelae apparent in neuritic plaques played a more intimate role in the disease process. Eikelenboom reported elevation of complement factors in Alzheimer brains (Eikelenboom and Stam, 1982), and the McGeer’s focused on histocompatibility proteins (McGeer et al., 1988). Griffin et al. (1989) reported an elevation of interleukin-1 (IL-1) in AD and in Down syndrome; notably, elevation of this inflammatory cytokine in trisomy 21 occurred even in fetuses, decades before the inevitable onset of Alzheimer-type pathology in these individuals. These phenomena were moved to a more primary status by the discovery that IL-1 could elevate expression and processing of bAPP to form more Ab (Buxbaum et al., 1992; Goldgaber et al., 1989), evoking hypotheses of positive feedback loops such as the ‘cytokine cycle’ (Griffin et al., 1998). Furthermore, epidemiological data indicated that regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) was associated with a reduced risk for Alzheimer’s disease (Breitner et al., 1994; in’t Veld et al., 1998; Rogers et al., 1993; Zandi et al., 2002). Thus, credible weight has been afforded the ‘inflammatory hypothesis,’ the supposition that unknown circumstances—perhaps including Ab accumulation—lead to the activation of inflammatory events that are harmful to the function and viability of neurons. However, it is somewhat counterintuitive that evolution of a sophisticated organ like the brain would have retained such a broadly acting selfdestructive capacity, and evidence of beneficial roles for glia has been assembled. 3.2. Indictment of the bad guys Among the advocates of the inflammatory hypothesis, some were careful to note that ‘inflammation’ with all its peripheral sine qua non is a poor descriptor of what happens in the Alzheimer brain. There is no evidence of swelling, vasodilation, or extravasation of

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neutrophils. But the molecules of inflammation, including cytokines and other products of activated macrophages, are the agents elevated in Alzheimer microglia, and their potential for tissue damage suggests that compromise of neuronal function in Alzheimer’s disease might be dependent on such factors. Studies on this topic progressed in parallel with a broader development of research on inflammatory mechanisms in the brain, and most studies have concentrated on mechanisms of microglial neurotoxicity that appear to be shared between Alzheimer’s disease and conditions as diverse as stroke, trauma, multiple sclerosis and Parkinson’s disease. Most speculation about the neurotoxic properties of microglia has arisen from studies of cells in culture. As described more extensively elsewhere in this volume, a variety of stimuli produce a predictable repertoire of responses in cultured microglia (see chapter by Benveniste). Indicative of their role in fighting infectious agents, activated microglia are capable of an oxidative burst, a specific production of superoxide mediated by NADPH oxidase and orchestrated by a defined signal transduction system (Kang et al., 2001). Other signal transduction pathways activated by proinflammatory stimuli lead to the induction of transcription factors like NF-kB (Akama et al., 1998), JAK/STAT (Liva et al., 1999) and cAMP-responsive element binding protein (CREB) (McDonald et al., 1998). Genes controlled by these factors include those of cytokines such as IL-1, IL-6, and tumor necrosis factor (TNF). Transcription of inducible nitric oxide synthase (iNOS) is also strongly elevated by a broad array of proinflammatory agents (see chapter by Garcia and Baltrons), resulting in a robust production of nitric oxide (NO). Human microglia obtained from autopsied Alzheimer brains show a predilection for potentiated responses in some regards, as compared to microglia taken from normal, aged brain (Lue et al., 2001). 3.2.1. Activation of glia by Ab The conspicuous activation of microglia in and around neuritic plaques led to the logical supposition that Ab is itself the relevant proinflammatory stimulus in Alzheimer’s disease. The reductionism possible in cell culture experiments has documented that Ab is sufficient to evoke at least some components of microglial activation, including neurotoxicity. Some of the initial demonstrations of this effect utilized the 25 –35 portion of Ab (Bonaiuto et al., 1997; Meda et al., 1995), which had been shown in previous studies to be sufficient for direct neurotoxicity (Yankner et al., 1989). Most of these studies applied the peptide in a soluble form and reported a requirement for coapplication of interferon-g (Goodwin et al., 1995; Meda et al., 1995), which can prime monocytic phagocytes for potentiated responses; other cytokines were also shown to be cooperative with Ab (Rossi and Bianchini, 1996). However, subsequent studies documented differences between the bioactivity of Ab25 – 35 and full-length (40 – 42 a.a.) Ab (Goodwin et al., 1995; Klegeris et al., 1994). The most complete proinflammatory activation has been found to require the aminoterminal 16 amino acids of Ab (Giulian et al., 1996; Hu et al., 1998), consistent with the low levels of glial activation by p3 in and around diffuse plaques (above). More refinement of the system followed, as investigators reported responses to substrate-bound Ab (Ard et al., 1996; Giulian et al., 1996; Hoke et al., 1994) and autonomous activation by fibrillar or aggregated Ab (Casal et al., 2002; Muehlhauser et al., 2001). Several receptor molecules have been implicated as important for microglial

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activation by Ab, including the receptor for advanced glycation endproducts (RAGE) (Yan et al., 1996), class A and B scavenger receptors (Coraci et al., 2002; El Khoury et al., 1996), and formyl peptide receptor-like 1 (FPRL1) (Le et al., 2001). Cultured astrocytes react to the application of Ab with responses that partially overlap those of microglia. The morphology of astrocytes is altered transiently but quite dramatically in culture when Ab is applied (Hu et al., 1998). The iNOS enzyme is also reported to be induced in astrocytes by Ab (Akama et al., 1998). Furthermore, IL-1, IL-6, S100b, and several chemokines are elevated in Ab-treated astrocytes (Araujo and Cotman, 1992; Bales et al., 1998; Pena and Marshak, 1993). Any neurotoxicity exhibited by astrocytes does not appear to be as fulminant as that seen with microglia. Therefore, contributions of astrocytes to Alzheimer pathogenesis are probably more subtle, such as production of molecules that facilitate Ab fibrillogenesis (Styren et al., 1998). It is tempting to look for specificity in the inflammatory events occurring in Alzheimer’s disease to explain the unique constellation of neuropathological findings. It is possible that these distinctions arise simply from regional specificity due to the expression patterns of bAPP and its secretases or from temporal issues related to the prolonged duration over which inflammatory processes presumably simmer before the onset of clinical dementia. There is a correlation between the neurofibrillary pathology of Alzheimer’s (neurofibrillary tangles and hyperphosphorylation tau) and IL1-positive microglia (Sheng et al., 1995; 2001). Furthermore, conditions such as neuro-AIDS and epilepsy lead to similar indications (Stanley et al., 1994). But, by and large, the activation of cultured microglia by Ab does not produce any outcomes that are remarkably distinct from those evoked by lipopolysaccharide (LPS), opsonized zymogen, or other proinflammatory agents: phagocytic activity, NO, IL-1, TNF, and glutamate release. One notable exception may be the production of D -serine. This amino acid is produced by an enzyme that racemizes L -serine, and it is a potent agonist of the NMDA class of glutamate receptors (Snyder and Kim, 2000). While LPS can stimulate the release of modest amounts of D -serine from microglia, Ab promotes much higher levels (Fig. 1). This effect suggests that Ab may specifically activate a microglial response that could elevate NMDA receptor activation, putting neurons at risk for excitotoxic damage. 3.2.2. Other potential proinflammatory stimuli In the quest for specificity, it is useful to consider other Alzheimer-relevant agonists besides Ab. Activated microglia can be found adjacent to neurofibrillary tangles in vivo (Afagh et al., 1996), suggesting that this component of Alzheimer pathology may contribute to glial activation. Although less specific for Alzheimer’s disease, microglia may also react to the major histocompatibility complex (MHC) proteins expressed by neurons suffering reduced electrophysiological activity (Neumann et al., 1995). Advanced glycation endproducts (AGE) have been advocated as age-related moieties that could be relevant (Dickson et al., 1996; Yan et al., 1996). One of the striking features of neuritic plaques is the dramatic elevation of bAPP present in the dystrophic neurites. Because Ab is not the only fragment of bAPP, we explored the proinflammatory effects of large, secreted portions of bAPP. These are

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Fig. 1. D -serine levels in microglial culture medium. Primary microglia were incubated 20 h with no addition (Con), 15 mM Ab, or 300 ng/ml LPS. Aliquots of media were assayed for D -serine by HPLC. Values represent the mean ^ SEM of triplicates ðpp , 0:01Þ; and results are representative of three separate experiments. Inset: Development of chromatographic analysis for D -serine. A mixture of amino acid standards were injected onto the column (2: L -Ser, Rt ¼ 24:7 min; 4: D -Ser, Rt ¼ 27:8 min) to demonstrate resolution of D - and L -serine.

termed sAPPa and sAPPb due to their production by the a- or b-secretase, respectively. When applied to primary cultures of microglia or microglial cell lines, both sAPPa and sAPPb stimulate the expression of IL-1 and iNOS and the manifestation of a neurotoxic profile (Barger and Harmon, 1997). The latter effect may involve the release of glutamate, a response evoked by sAPP with much greater potency and efficacy than seen with Ab itself (Barger and Basile, 2001). Another interesting distinction of sAPP is its ability to stimulate the expression of apolipoprotein E (ApoE), a lipoprotein which presence in the CNS is due to endogenous synthesis, mainly in astrocytes (see Chapter by Ito and Yokoyama). This protein has been implicated in Alzheimer’s disease genetically: compared to homozygotes for 13, persons carrying an 14 allele of the ApoE gene have a 3- to 7-fold elevated risk for developing Alzheimer’s (depending on race and homozygosity). It is still not clear how this distinction influences disease etiology, but one possibility is suggested by the direct physical interaction of ApoE with sAPP. The protein product of the e3 allele of ApoE (ApoE3) appears to bind sAPP more avidly than does ApoE4 (Barger and Harmon, 1997; Barger and Mattson, 1997). The binding results in a dampening of sAPP’s proinflammatory effect, and both microglial activation and ApoE binding are dependent upon the aminoterminal amino acids of sAPP (Barger and Harmon, 1997). Interestingly, sAPP can induce the expression of ApoE protein (Fig. 2). Elevation of ApoE expression is not a common element of inflammatory activation of microglia (LaDu et al., 2001), and LPS can actually suppress ApoE levels (Werb and Chin, 1983). This relationship suggests a feedback

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Fig. 2. Induction of ApoE protein (A and B) and mRNA (C) by sAPP. N9 microglial cells were exposed to indicated concentrations of sAPPa for 18 h (A) or to 5 nM sAPPa for various times (B). Cells were lysed and subjected to Western blot analysis with an anti-ApoE monoclonal antibody. The relative mobilities of protein standards are indicated (kD £ 1023); that of monomeric ApoE is ,35 kD (a fraction of the ApoE appears to have migrated in a dimeric form). (C): N9 microglial cells were exposed to 5 nM sAPPa for the indicated times. Total RNA was extracted for analysis by northern blotting with an ApoE cDNA probe. The upper panel shows hybridization signal, and the lower panel shows ethidium bromide staining of 18S and 28S ribosomal RNA to demonstrated equivalency of loading.

pathway whereby ApoE levels are tied rather closely to sAPP levels, perhaps to limit specifically the proinflammatory activity of its binding partner. Therefore, conditions that permit elevations of bAPP and, consequently, sAPP that outpace ApoE’s suppressive activity could predispose that tissue to inflammatory complications; this would be exacerbated by the 14 genotype because it produces an ApoE product with diminished anti-inflammatory activity.

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3.2.3. A vowel wind this way blows: aging, excitotoxicity, inflammation, oxidation and you The ability of sAPP to evoke the production of neurotoxins from microglia led us to consider the possible contribution of excitotoxity. Indeed, conditioned medium from microglia treated with sAPP rapidly trigger a robust activation of NMDA receptors, as indicated by intraneuronal calcium concentrations (and their responsiveness to an NMDA receptor antagonist) (Barger and Basile, 2001). Like Ab, sAPP can elevate the levels of D -serine in microglial medium, but initial assays indicate that it is less effective than Ab in this regard. However, glutamate release is stimulated by sAPP with a potency that is 300-fold greater than that of Ab and a maximal efficacy that is almost six-fold higher. This release appears to involve the activity of a glutamate– cystine XC antiporter (Barger and Basile, 2001). The role of a glutamate –cysteine antiporter in microglial glutamate release suggests a paradigm of specific events that connect inflammation, oxidation, and the excitotoxic activation of glutamate receptors. Activated microglia produce an oxidative burst that generates hydrogen peroxide and probably a marginal level of lipid peroxidation. Microglia must protect themselves from this oxidative stress, utilizing glutathione. This important cytosolic antioxidant can detoxify peroxide and hydroxyl radicals directly, and cellular defense mechanisms may be able to satisfy the demand for re-synthesis of reduced glutathione through the regenerative power of glutathione reductase. However, the use of glutathione by peroxidases that quench lipid peroxidation (e.g., GSTA4-4) (Berhane et al., 1994; Hubatsch et al., 1998) is a one-way conjugation of the molecules. To replenish this loss of glutathione requires new synthesis, and an important reactant in this synthesis is cysteine, imported by the glutamate– cystine antiporter from extracellular sources as cystine. In the balance, the microglia can only dump glutamate into the extracellular space. Glutamate is, of course, the most intensively studied excitotoxin. Robust activation of its receptors, particularly those of the NMDA class, result in several processes that compromise the health, function, and structure of the neuron. As these receptors are localized primarily at post-synaptic elements, the dose- and time-dependent continuum of glutamate toxicity begins at dendritic spines and progresses through the entire dendrite to the soma. Therefore, doses of glutamate (or times of exposure) that are subtoxic for the cell as a whole can cause degeneration of the dendrites alone (Mattson and Barger, 1993). This is consistent with the pruning of dendritic arbors (Ferrer et al., 1990; Paula-Barbosa et al., 1980) and the loss of synapses that correlates well with dementia in Alzheimer’s disease (DeKosky and Scheff, 1990; Terry et al., 1991). An intermediate level of dystrophy hypothetically limited to the dendritic elements may be expected in the face of glutamate, a toxin that is so efficiently removed by astrocytes (Anderson and Swanson, 2000). However, the combination of Ab and sAPP being elevated would mean that a substantial glutamate release would be accompanied by an equally significant release of D -serine. Although transport kinetics for the reuptake of D -serine are not well characterized, it is likely that the simultaneous presence of these cooperative NMDA-R agonists would cause considerable excitotoxic stress quickly and at concentrations that would be low for either alone. Furthermore, Ab appears capable of inhibiting glutamate uptake by astrocytes (Harkany et al., 2000; Harris et al., 1996). It is notable that indications of excitotoxic mechanisms have been observed in Alzheimer’s

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disease, including an apparent drop-out of NMDA-R-bearing cells (Jansen et al., 1990) and evidence of a large calcium elevation in the remaining neurons (Masliah et al., 1998; Saito et al., 1993). Memantine, a particularly well-tolerated NMDA antagonist, has shown modest benefit in Alzheimer treatment trials (Winblad and Poritis, 1999), so earlyintervention trials with persons suffering mild cognitive impairment may be justified. Finally, delivery of Ab into the brains of rats produces evidence of neurodegeneration, and these effects are blocked by NMDA-R antagonists (Harkany et al., 2000; Parks et al., 2000).

3.3. The defense does not rest Despite the fact that activated glia can produce neurotoxicity in culture, it is not clear that these events are important in Alzheimer’s disease. The presence of microglia in neuritic plaques may reflect an ongoing clearance attempt. The distribution of plaques across Alzheimer stages suggests a near equilibrium in the steady-state levels of deposited Ab. The phagocytic function of microglia towards Ab is apparent in cell culture (Ard et al., 1996; Giulian et al., 1996; Hoke et al., 1994), and microglia are known to produce two proteases capable of hydrolyzing Ab (Iwata et al., 2000; Qiu et al., 1998). There is even an under-appreciated body of work suggesting that astrocytes can contribute significantly to phagocytosis (Bechmann and Nitsch, 1997). Whatever the precise mechanism(s), Ab is generally cleared from the cerebral cortices of experimental animals efficiently. Synthetic Ab peptide injected directly into rat cortical tissue rapidly disappears (Price et al., 1992), especially if it is not fibrillar (Weldon et al., 1998). In some cases, this removal is accompanied by markers of glial activation (Weldon et al., 1998), suggesting that such indications may be a common element of a phagocytic phenotype. The failures of many early attempts at making a transgenic mouse model of Alzheimer’s disease (Lannfelt et al., 1993) may be related to such efficient clearance. The mouse lines that were eventually most successful for modeling in vivo plaque formation show astronomically high levels of Ab production (Games et al., 1995; Hsiao et al., 1996) and/or express a version of Ab that is chemically predisposed to a high rate of aggregation (Moechars et al., 1999). Much ado has been made about preventing or reversing amyloid deposition through a strategy of immunization. In several of the transgenic mouse models of Alzheimer neuropathology, a humoral immune response to Ab in young animals can largely prevent the accumulation of plaques at later ages, and immunization of older mice shows signs of reversing existing deposition (Schenk et al., 1999). Early reports of this paradigm mentioned that immunization was associated with the appearance of Ab antigenicity within microglia (Schenk et al., 1999), suggesting that that the vaccination had facilitated microglial phagocytosis of Ab. This idea was supported by ex vivo experiments in which clearance of Ab deposits in brain sections by mouse microglia appeared to be dependent on Fc receptors (Bard et al., 2000), which monocytic phagocytes use to bind and internalize antibody-bound material. Two lines of evidence now suggest that microglial phagocytosis of parenchymal Ab is not the primary means of plaque suppression (Bacskai et al., 2002; DeMattos et al., 2001), but a contribution by this mechanism has not been excluded.

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bAPP-overexpressing transgenic mice have also been used in another paradigm that hints at a beneficial activation of microglia: treatment with NSAIDs, the very class of drugs that appear to stave off Alzheimer’s disease in humans. Similar to a previous report using ibuprofen (Lim et al., 2000), Morgan and coworkers have demonstrated an inhibition of Ab deposition in transgenic mice fed nitroflurbiprofen (Jantzen et al., 2002). But unlike other reports, this study documented a surprising increase in markers of microglial activation in the drug-treated animals. In addition to phagocytosis of Ab, other reactions of activated microglia may promote positive outcomes. NO has been much maligned as a mediator of neurotoxicity, beginning with its early connection to excitoxicity (Dawson et al., 1991). However, the NO that contributes to neuron cell death may be generated within the cytosol of the very neuron that is being impacted, as neurons contain a NO synthase that is activated by calcium/ calmodulin. NO diffusing to a neuronal membrane in a paracrine fashion may actually have beneficial effects, such as attenuation of NMDA receptor activation (Lei et al., 1992). TNF has also been maligned, largely because of a misunderstanding of the data that led to its name. True, it can be cytotoxic, but that action is primarily expressed towards transformed cells, which have an inappropriate mitotic signal (Carswell et al., 1975). Bioassays of TNF that rely on its ability to kill cells are performed in the presence of a macromolecular synthesis inhibitor; healthy cells rarely die from TNF alone. On the contrary, considerable evidence indicates that TNF can protect neurons from other toxic insults (Barger et al., 1995; Mattson et al., 2000). It is possible that the deposition of Ab in aged brains and the presumably consequential development of Alzheimer’s disease arises not from the harm done by glia in these unfortunate individuals but rather from the failure of preventative functions assumed by glia in younger or otherwise resistant brains. Phagocytosis and degradation could be compromised in elderly microglia; proteasome activity is documented to have age-related deficiencies (Ponnappan, 1998), which could hamper the responsiveness of the proinflammatory transcription factor NF-kB, as well as general degradation of ingested material. With regard to astrocytes, declines in the glutamate uptake and conversion systems that squelch excitotoxic events have been noted with advanced age (Saransaari and Oja, 1995; Smith et al., 1991).

4. Concluding remarks While changes in glial elements in Alzheimer’s disease were noted in the earliest reports, their role in fostering or combating the key pathogenic mechanisms remains elusive. Within the last decade, an emerging interest in molecules connected to inflammation has driven a great deal of productive research. Regardless of whether inflammatory events contribute to Alzheimer’s disease or delay its severity, we now know a great deal more about what responses are elicited in astrocytes and microglia by derivatives of bAPP and the more general aspects of neurodegeneration. Furthermore, mechanisms by which glia contribute to more obvious cases of neuroinflammation (multiple sclerosis, brain abcesses, viral encephalitis, etc.) have become apparent through investigations of Alzheimer’s disease and related disorders. In the end, Alzheimer’s

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disease victims will likely be found to both benefit and suffer from the events propagated by glia; as with most conditions of physiology and pathology, glia probably play complex roles in Alzheimer’s. Indubitably, they produce neurotrophic factors and otherwise engage in responses that provide neuroprotection, yet the insidious disease process almost certainly recruits them to its ends. The challenge now and in the near future will be to find ways to shape glial responses in the direction of the former. This will require an even more detailed understanding of the integrated effects of glial responses and actions, an undertaking steadily yielding to the growing co-operation of neuroscientists and immunologists. Acknowledgements Dr Barger and his laboratory are supported by funds from the National Institutes of Health (1R01AG17498-01A1 and 2P01AG12411-06A10003). The technical contributions of Dr. Anthony Basile (Alkermes, Inc.) are greatly appreciated.

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Non-neuronal interactions in HIV-1-associated dementia Anuja Ghorpadep and Howard E. Gendelman Center for Neurovirology and Neurodegenerative Disorders, Department of Pathology/Microbiology, Omaha, NE 68198-5215, USA p Correspondence address: Center for Neurovirology and Neurodegenerative Disorders, University of Nebraska Medical Center, 985215 Nebraska Medical Center, Omaha, NE 68198-5215 E-mail: [email protected]

Contents 1. 2. 3.

4.

5.

6.

Introduction HIV-1 Encephalitis (HIVE) Mediators released from microglia 3.1. Tumor necrosis factor-alpha (TNF-a) 3.2. Other cytokines 3.3. Chemokines 3.4. Other microglial neurotoxins Microglial activation begets astrocyte activation 4.1. TNF-a as a mediator of microglia – astrocyte interactions 4.2. IL-1b as a mediator of microglia – astrocyte interactions Astroglial dysfunction 5.1. HIV infection of astrocytes 5.2. Role of viral proteins (HIV-1gp120 and tat) 5.3. Astrocytic function 5.4. Astrocyte-mediated neurotoxicity Concluding remarks

The pathogenesis of HIV-1-associated dementia (HAD) and the accompanying damage is known to involve inflammatory events within the central nervous system (CNS). HAD is a devastating complication of progressive viral infection leading to cognitive, behavioral, and motor impairments in a significant number of infected people (Navia et al., 1986; Dore et al., 1999; Sacktor et al., 2001). Microglia and astrocytes produce a battery of pro-inflammatory and neurotoxic mediators that all contribute to neurodegeneration in unique ways, when compared to other forms of viral encephalitis. Almost all of the mechanisms proposed to affect CNS pathogenesis also participate in Advances in Molecular and Cell Biology, Vol. 31, pages 901–920 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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tissue homeostasis. It is precisely the dysregulation or over-production of inflammatory factors that affect neuronal function. Understanding the complete network of secretory molecules produced by activated nonneuronal cells in HAD is pivotal for deciphering the mechanism of neuropathogenesis of the disease and ultimately reaching the goal of protecting the brain or reversing the disease process in the infected human host.

1. Introduction Glia (specifically microglia and astroglia) play a principal role in the neuropathogenesis of HIV-1 infection. These are the target cells and reservoirs for persistent infection by HIV-1 and the source of neurotoxic activities during HAD. Indeed, it is the MP [(microglia, blood-derived perivascular macrophages and multinucleated giant cells (MGC))] that serve as the principal productively infected cells within the nervous system (Takahashi et al., 1996). Neurons are infected rarely, if at all, yet, are ultimate targets of disease. This has led to the concept that the pathogenesis of HAD is caused not by direct infection of neurons but by secretory and cell-associated products (viral and cellular), manufactured by glia that disrupt neuronal function. Support for this idea comes from clinical, pathological, therapeutic, and laboratory studies. HAD is a subcortical dementia manifested by behavioral, motor, and cognitive impairments. HIV-1 commonly invades the brain early in disease, yet the neurological manifestations occurs as a part of latestage disease (Navia et al., 1986; Davis et al., 1992; Garden, 2002). Cognitive dysfunction predominates, and mental disorders are manifest over weeks to months, not days, as in other forms of viral encephalitis. Pathologically, the presence of microglial nodules, MGC, myelin pallor, dendritic vacuolization and reactive astrogliosis characterize the histological aspects of the disease complex (Sharer et al., 1985; Budka, 1991; Wiley et al., 1991; Masliah et al., 1997; Everall et al., 1999). Treatment of HAD patients with anti-retroviral and anti-inflammatory drugs can reverse many of the clinical symptoms. Moreover, following the advent of highly active anti-retroviral therapy (HAART), the incidence, not prevalence, of HAD has decreased to less than 7% of infected individuals (Maschke et al., 2000; Sacktor et al., 2001). Disease is commonly associated with high levels of inflammatory glial products in the cerebrospinal fluid (CSF) and the brain. Altogether, these findings reflect the involvement of microglia and astrocytes in affecting neuronal function. Notably, this involvement precludes productive infection. It is now widely accepted that HIV-1-infected and immune activated microglial cells play a pivotal role in the neuropathogenesis of disease (Dickson et al., 1991; Gendelman et al., 1994; 1997; Brew et al., 1996; Kolson and Gonzalez-Scarano, 2000; Kaul et al., 2001; Williams and Hickey, 2002). The elucidation of mechanisms of HAD by deciphering the role played by interaction of the non-neuronal cells in CNS disease remain critical towards developing preventative and/or ameliorative strategies for brain disease in the infected human host. The key mediator for neurodegeneration in HAD is the microglial cell, the principal target cell and source of neurotoxins in disease. The microglia was first described by del Rio Hortega in 1932, but it was shown as a primary source of neurotoxins

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in neurodegenerative diseases only within the past decade. The chapter will review the regulation of many of the toxins that microglial cells produce, and it will address a critical unresolved question in HAD, i.e., how a few perivascular macrophages infiltrating the brain lead to extensive neural dysfunction. The hypothesis supported in this review is that glial cell interactions lead to amplification of immune-effector responses and ultimately damage neuronal networks. Clearly, over the last two to three years, the view that astroglial cells are simply supportive cells has been put to rest. The data reviewed in this report strongly support the idea that activated astrocytes contribute to neurodegenerative disease processes. To address the issues of glial neurotoxic activities, this chapter will be divided into three sections. First, it will review the neuropathological findings of HIV-1 encephalitis, the best-known glial-mediated neurodegenerative disorder (Section 2). Second, it will review the classical pathway of production of deleterious metabolites by infected/activated microglial cells that lead to neurotoxicity. In doing so, the chapter will restrict itself to certain classes of molecules and emphasize those that are involved in interactions with astrocytes (Section 3). Third, and perhaps the most interesting, it will reflect on the possible mechanisms of microglial – astrocyte interactions (Section 4) and their effect on neurons (Section 5).

2. HIV-1 Encephalitis (HIVE) The late stages of HIV infection are frequently associated with three disorders of the nervous system: HIV-1-associated subcortical dementia (HAD), vacuolar myelopathy, and sensory neuropathy. HAD remains the most thoroughly studied and HIVE is the best known pathological correlate of HAD. It is characterized by multifocal microglial nodules with variable numbers of MGC (Dickson et al., 1994) that are often juxtavascular to vessels (Fig. 1A). The presence of activated CD68-positive microglial cells, which are positive for the Major Histocompatibility Antigen Class II (HLA-DR) in areas of infiltration of macrophages (Fig. 1B, C), coincide with reactive astrogliosis (Fig. 1D). Notably, there is an overall paucity of lymphocytes in contrast to other types of viral encephalitis. This reflects the fact that HIVE usually occurs in association with a profound immunosuppression and lymphopenia. Moreover, the cell types that are directly responsible for the pathology associated with HIVE are microglia and monocyte-derived macrophages (MDM). However, the reactions of astrocytes and presence of dystrophic neurites in the areas of this pathology (Masliah et al., 1997) lead to the conclusion that the full spectrum of HIV-related disease involves interactions between the virus, virus-infected cells and other activated cells of the nervous system. One may argue that reactive astrogliosis is a stereotypical response to all types of CNS injury from ischemia to a plethora of degenerative disorders (Gabuzda et al., 1986; Koenig et al., 1986; Eddleston and Mucke, 1993; Norenberg, 1994; Ridet et al., 1997; Martin et al., 2001). Certainly, the specific role played by reactive astrocytes in mediating neuronal damage is poorly understood. However, newer insights into its role in disease processes have been elucidated and will be reviewed in the third section of this chapter.

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Fig. 1. Neuropathological changes in HIVE. Fifteen brain autopsy cases were analyzed including three controls (without CNS disease), six severe cases of HIVE, three with mild HIVE and three HIV-1 seropositive cases without nervous system disease. In the severe HIVE cases, HIV-1-infected MGC (A) were prominent in zones of significant monocyte infiltration. Serial sections stained with CD68 (B) and HLA-DR (C) demonstrated activated/HIV-1-infected cells of microglial nodules in the areas of macrophage infiltration. In addition, prominent astrogliosis as identified by GFAP immunoreactivity was observed (D). Control mouse IgG served as the negative control (data not shown). Immunoreactivity was detected by Vectastain Elite Kit using DAB as a substrate. Tissue sections were counterstained with Mayer’s hematoxylin. Original magnification, panels B–D £ 200; panel A £ 400. (Adapted from Ghorpade et al., 2001, included with permission from the American Society for Microbiology).

Figure 2 provides a schematic representation of the ongoing events in HAD pathogenesis. It begins by infiltration of macrophages into the brain (Fig. 2, Step 1). Neuro-pathological observations of HIVE reveal infection of macrophages and microglial cells (Fig. 2, Step 2). Isolates so far obtained from brain tissue are commonly macrophage-tropic. In vitro productive infection of microglia cells by macrophage-tropic HIV-1 isolates has been demonstrated by several groups including our own (Lee et al., 1993; Strizki et al., 1996; He et al., 1997; Ghorpade et al., 1998a; 2001). These include human fetal and adult microglial cells. Indeed, this is intriguing, since microglial cells express low levels of CD4, the primary receptor for HIV-1. Nonetheless, they express a variety of chemokine viral co-receptors, and

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Fig. 2. Schematic model of the non-neuronal interactions in HAD. This model shows the pathways of non-neuronal participation in HAD. Step 1: The BBB is compromised and HIV-1-infected monocyte-derived macrophages (MDM) infiltrate the brain. Perivascular multinucleated giant cells, as shown juxtaposed to the vessel, are often observed. Step 2: Resident microglial cells are infected and activated. This leads to an ongoing inflammation in the CNS. Steps 3a and 3b: Activated MP (macrophages and microglia) produce a variety of immune mediators as discussed in the text (3a). These include, but are not limited to, proinflammatory cytokines, chemokines, eicosanoids, glutamate, proteases, arachidonic acid metabolites and other ROS. Platelet activating factor (PAF), in particular is involved in a feed –forward loop leading to increased levels of TNF-a from activated MP (3b). Step 4a: One or more of the MP immune mediators interact with astrocytes that accumulate at the site of injury and contribute to reactive gliosis. Step 4b: These immune-activated astrocytes in turn produce glutamate, proteases, and other potential toxic molecules such as soluble Fas ligand (FasL) in addition to showing an impaired glutamate uptake. Step 5: The virus by itself may have an additive or synergistic effect on the astrocytes and neurons as well. All reactions, together, lead to neuronal malfunction and ultimately death even though neurons are not productively infected with the virus.

different isolates of HIV-1 are known to use these receptors differentially (Rucker et al., 1997; Ghorpade et al., 1998b; Li et al., 1999). The question remains as to how HIV-1-infected, immunologically activated macrophages/microglia lead to disease? The answer to this question revolves around the soluble mediators of disease produced by infected/activated microglial cells and macrophages (Fig. 2, Step 3a) that directly and indirectly cause neuronal damage. A discussion on the principal compounds implicated in this process is presented below.

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3. Mediators released from microglia 3.1. Tumor necrosis factor-alpha (TNF-a) Pro-inflammatory cytokines such as TNF-a, Interleukin (IL)-1b and IL-6, all have been implicated in neuropathogenesis of HAD (Dickson et al., 1991; Sebire et al., 1993; Gendelman and Tardieu, 1994; Persidsky et al., 1999). TNF-a production by activated microglial cells in the context of HIV-1 has been the most extensively studied of all cytokines (Benveniste, 1994; Benveniste and Benos, 1995; Cheret et al., 1996; Petito et al., 1999) and TNF-a is an important mediator in immunologic and inflammatory responses in a variety of diseases (Aggarwal and Natarajan, 1996). In HAD, the roles that TNF-a can play as a potential neurotoxin are diverse. It can be neurotoxic and can lead to apoptosis of neuronal cells via specific receptors and induction of caspases (Gelbard et al., 1993; Chao et al., 1995a). TNF-a and its receptor levels are significantly elevated in HAD as compared to HIV-1-seropositive individuals without cognitive impairment (Wesselingh et al., 1993; 1994). Overexpression of TNF-a correlates well with the severity of neurological complications (Garden, 2002). TNF-a is an interesting candidate for neurotoxicity, since its effects can range from neurotrophic to neurotoxic, depending on its concentration and the target cell (Zhou et al., 2002). Indeed, this seems to be true for a variety of members of the TNF-family, and a fine balance seems to exist in the tissue microenvironment. It has been repeatedly shown that cells of the MP lineage, MDM and microglial cells in vitro can produce copious amounts of TNF-a, when activated with endotoxin (Persidsky et al., 1999; Cotter et al., 2001). Other immune activators, such as CD40L, and other pro-inflammatory cytokines also lead to TNF-a expression in MP (Cotter et al., 2001). It is noteworthy that HIV-1 infection itself leads to only minimal elevation in TNF-a levels, and that HIV-1 infection and immune stimulation by an activator, together, lead to synergistic effects on TNF-a production. This led to the notion that HIV-1 infection of MP causes priming of these cells, such that they are more receptive to immune activation, and that the primed and activated MP produce high levels of neurotoxins.

3.2. Other cytokines IL-6 is another pro-inflammatory cytokine, implicated both in disease and neuroprotection. One of the effects of IL-6 is that in combination with IL-1 and TNF-a, it can lead to proliferation of astrocytes and contribute to the astrogliosis and immune activation of astrocytes in the CNS. Several groups have demonstrated that activated microglia, both human and rodent, produce IL-6 in response to lipopolysaccharide (LPS) and viral infection. In the case of human fetal microglial cells, IL-1 is a potent inducer of IL-6 and IL-1 itself is one of the first immune molecules generated upon any cell activation. Therefore, the pro-inflammatory cytokines themselves, namely TNF-a, IL-1b and IL-6, have their own autocrine and paracrine loops that participate in the microglial activation in HAD.

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3.3. Chemokines Chemokines and their receptors play a pivotal role in the regulation of HIV-1 infection. A flurry of studies in recent years have shown that chemokines and their receptors are implicated in the neuropathogenesis of HIV-1 infection (Bjorndal et al., 1997; Connor et al., 1997; Ghorpade et al., 1998b; Gabuzda and Wang, 1999; 2000). It was known prior to studies of HIV-neural interactions that chemokines produced by endothelia, astrocytes and activated MP affected traffic of leukocytes across the blood – brain barrier (BBB, see chapter by Couraud et al.), and that they regulated neuronal signaling events. Certainly, and in addition to playing a role in recruitment of immune cells in the brain, chemokines are also known to activate resident cells of the CNS, even the neurons, and they may contribute to neurotoxicity. The sources of chemokines in the CNS are varied and depend on the type of chemokine. Several excellent reviews address the diversity of chemokines and their effects on target cells (Gabuzda and Wang, 1999; 2000; Miller and Meucci, 1999). It should suffice to say that both a and b chemokines are produced by microglial cells and astrocytes, and they further complicate the web of secretory molecules that leads to neurodegeneration through affecting trans-migration of MP through the BBB and neurotoxic pathways, critical for the development of disease.

3.4. Other microglial neurotoxins 3.4.1. Arachidonic acid metabolites and platelet activating factor Arachidonic acid metabolites, quinolinic acid, platelet activating factor (PAF), proteases, and glutamate are all neurotoxins that can be produced by microglia. Macrophages exposed to viral proteins, such as HIV-1gp120, also produce arachidonic acid metabolites. Viral infection is a stronger stimulus for production of leukotriene B4, D4, PAF and lipoxin A4, all of which have been detected in supernatants of infected cells. Interestingly, formation of arachidonic acid metabolites is subject to regulation through interactions with astrocytes, revealing yet another cross talk between the glial cells. PAF is produced simultaneously by processing of phosphoglycerocholine by phospholipase A2. A variety of cells are known to produce PAF, including endothelial cells, basophils, macrophages, eosinophils, neutrophils and even neurons. However, macrophages secrete PAF, whereas other cells store it intracellularly (Braquet and Rola-Pleszczynski, 1987). PAF and TNF-a are participants in a feed – forward loop (Fig. 2, Step 3b), in that, TNF-a stimulation of macrophages leads to secretion of PAF, and PAF can increase the levels of TNF-a production by macrophages. For that reason, PAF can also be regarded as a proinflammatory stimulus for macrophages. PAF is directly toxic to neurons, and its toxicity can be partially reversed by NMDA antagonists. Although HIV-1 infected patients have higher levels of PAF in the CSF, this does not necessarily correlate with the severity of disease. Increased levels of quinolinic acid are found in the CSF of AIDS patients, however, microglial cells have not been shown to produce copious amounts of it. Their counterparts in the periphery, MDM, are known to generate significant amounts of quinolinic acid, which is implicated in neurotoxicity.

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3.4.2. Proteases Proteases such as matrix metalloproteinases are produced by monocytes as they differentiate. Increased metalloproteinase production is implicated in transmigration through the BBB. In addition, metalloproteinase production by infected/activated macrophages and microglia is increased. Taken together, infected/activated macrophages can actively digest the extracellular matrix proteins that constitute the BBB. Increased levels of metalloproteinase are found both in CSF and brain parenchyma of patients suffering from HAD (Conant et al., 1999; Ghorpade et al., 2001). Since metalloproteinases play an important role in maintenance of CNS extracellular matrix, their dysregulation can have devastating effects on its integrity. Indeed, analysis of extracellular matrix proteins in late stage HAD disease has clearly demonstrated its significant loss (Belichenko et al., 1997). Since microglia and astrocytes both produce metalloproteinases and tissue inhibitors of metalloproteinases, a delicate balance exists between the interactions of these two non-neuronal cells in the tissue microenvironment; interactions that can compromise the BBB and brain extracellular matrix and facilitate disease. 3.4.3. Reactive oxygen species Nitric oxide (NO) is a nitrogen free radical, generated in many tissues, including the CNS (Culotta and Koshland, 1992). The immunologic or inducible NO synthase, which is expressed and upregulated in macrophages and astroglial cells, contributes to neurotoxicity, partly through stimulation of glutamate production. It has also been observed that patients suffering from HAD are under a chronic oxidative stress. This includes a depletion of anti-oxidants and an increase in reactive oxygen species (ROS), of which NO is a member. There are a number of other ROS that accompany this damage. In particular, the oxidative damage by NO is complemented by peroxynitrite. This leads to Snitrosylation of tyrosine residues in structural proteins, such as neurofilament, which is an important component of neuronal architecture. Oxidative stress in the pathogenesis of HAD involves other ROS, such as superoxide dismutase, catalase, free radical overproduction, which has been reviewed in detail (Mollace et al., 2001). 4. Microglial activation begets astrocyte activation 4.1. TNF-a as a mediator of microglia– astrocyte interactions In section 3.1, we reviewed the regulation of TNF-a by MP and emphasized that TNFa is released from activated microglia in HAD. TNF-a has diverse effects on multiple cell types in the CNS. It can affect function of MP, astrocytes, endothelial cells and also neurons (Gelbard et al., 1993; Chao et al., 1995b; Rajavashisth et al., 1999; Zhou et al., 2002). Since its levels are known to be upregulated in HAD, the varied effects of TNF-a have led this to be considered as a very important mediator in neuropathogenesis of HAD (Fig. 2, Step 4a). Transgenic animals that overexpress TNF-a develop a chronic demyelinating disease, which includes astrogliosis and microglial activation as histopathologic findings (Probert et al., 1997). Some of these effects can be partially reversed by administration of TNF-a neutralizing antibodies (Probert et al., 1996).

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Astrocytes make very limited amounts of TNF-a (Persidsky et al., 1999), yet they respond to TNF-a in different ways. TNF-a along with IL-b, can lead to astrocyte proliferation (Giulian and Lachman, 1985; Giulian et al., 1994), which is implicated in reactive gliosis observed in the CNS (see chapter by Kalman). It can also affect ion transport in astrocytes such as Naþ/Kþ exchange and glutamate uptake/efflux, and it leads to production of chemokines and different colony stimulating factors (Benveniste, 1994). 4.2. IL-1b as a mediator of microglia –astrocyte interactions 4.2.1. Production of IL-1b IL-1b is produced by a variety of cells including endothelial cells, B cells, astrocytes, activated microglia and macrophages. In addition, it acts as a neuromodulator by assisting neural function and regulation of sleep (Vitkovic et al., 2000b). Although IL-1b itself is not toxic to neurons, it has activation effects on microglia and astrocytes that contribute to its potential neurotoxicity. Indeed, in the CNS, the majority of the cell types, including neurons, astrocytes, microglia and oligodendrocytes, can produce IL-1b (Arai et al., 1990). Thus, IL-1b seems to spin a web of cascading interactions among the different cell types that generate it and the cells that respond to it. One such pathway is through the interactions of IL-1b with the astrocyte and is discussed below (Fig. 2, Step 4a). First, activated and/or HIV-1-infected MP are known to produce elevated IL-1b levels (Yamato et al., 1990). As mentioned, production of IL-1b is one of the first responses observed upon activation of immune cells (Pellegrini et al., 1996). This finding suggests that peripheral activation in HAD may provide soluble IL-1b that penetrates the BBB (Vitkovic et al., 2000a,b). IL-1 expression is upregulated in a number of disorders of the brain including epilepsy and HAD (Vitkovic et al., 2000b). Brain tissue from simian immunodeficiency virus infected rhesus monkeys (Laverda et al., 1994), CSF (Gallo et al., 1991; Vitkovic et al., 1995) and CNS tissue from patients with HAD (Tyor et al., 1993; Vitkovic et al., 1995; Boven et al., 1999) demonstrates elevated levels of IL-1b. HIV-1 is known to regulate the expression of brain IL-1b (Ilyin and Plata-Salaman, 1997). 4.2.2. Effects of IL-1b on astrocytic function IL-1b induces biosynthesis of a number of immunologically important proteins during infection, tissue damage and/or stress. IL-1b regulation of astrocyte function is multifaceted and ranges from activation of NO synthase (Lee and Brosnan, 1996; Hua et al., 1998; Zhao et al., 1998) to calcium wave regulation (John et al., 1999). Cellular proliferation (Kasahara et al., 1990; Vigne et al., 1993), induction of an autocrine loop leading to further production of IL-1b and other cytokines (Vitkovic et al., 1995), increased expression of intercellular adhesion molecule (ICAM)-1 (Vigne et al., 1993), and changes in the m-opioid receptor mRNA (Ruzicka et al., 1996) are all observed in astrocytes activated with IL-1b. It has also been suggested that astrocytes may have dichotomous effects on HIV-1 replication in MDM and may either enhance or reduce toxin production from infected MP (Nottet et al., 1995; Hori et al., 1999). Astrocyte –neuron co-cultures utilized for assay of neurotoxicity have shown that a combination

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of IL-1b/IFN-g activation leads to neurotoxicity (Dragic et al., 1996). In addition to these studies, our unpublished observations show that primary human fetal microglial cells respond to a variety of stimuli and produce IL-1b. Also, studies performed in our laboratories with microglial cells obtained from postmortem adult HIV tissue patients demonstrate that interactions with activated astrocytes may augment microglial activation. These interactive loops will provide additional sources of IL-1b, or other proinflammatory stimuli, relevant to astrocyte activation in the context of HAD. Furthermore, activation of astrocytes by IL-1b initiates an autocrine loop for IL-1b production. This reduces the requirement of IL-1b for initial activation of astrocytes and it is conceivable that the finite amount of IL-1b that penetrates the BBB represents a pathway independent of microglial activation.

4.2.3. IL-1b-mediated signal transduction pathways in astrocytes Different portions of a variety of signal transduction pathways activated by IL-1b in a variety of cell types have been worked out (Moynagh et al., 1994; Cao et al., 1999; Guo et al., 1999; Heyninck and Beyaert, 1999; Jeon et al., 2000; Sanz et al., 2000; Yang et al., 2000; Funakoshi et al., 2001; Li et al., 2001). Signal transduction can be initiated at the cell membrane by complex formation between extracellular IL-1b and the trans-membrane IL-1 receptor type 1 (IL-1R1) and IL-1R accessory protein (IL-1RacP). IL-1b also stimulates mitogen-activated protein kinase kinase (TAK1), which in turn mediates activation of c-Jun N-terminal kinase (JNK). It is generally accepted that IL-1R signal transduction pathways merge at the activation of transcription factor NF-kB and Activating Protein (AP)-1. The activation of NF-kB is achieved via phosphorylation of its inhibitor IkB. This in turn is performed by IkB kinases (IKKs) (Umehara et al., 1998; Nomura et al., 2000; 2001). Interestingly, NF-kB is a transcription factor that is involved in HIV-1 long terminal repeat (LTR) promoter activation (Taylor et al., 1994; 1995; Lapointe et al., 1996; Miller et al., 1997; Millhouse et al., 1998; McAllister et al., 2000), FasL promoter activation (Chan et al., 1999) and Fas –FasL pathway to apoptosis (Ponton et al., 1996; Ravi et al., 1998; Borset et al., 1999; Cheema et al., 1999; Dudley et al., 1999; Irie et al., 1999; Teixeiro et al., 1999; Harwood et al., 2000; Ivanov and Ronai, 2000; Li-Weber et al., 2000; Zheng et al., 2001). In summary, IL-1b in the context of HIV-1 should also be considered an important mediator of astrocyte-mediated disease processes in neurodegeneration.

5. Astroglial dysfunction Astrocytes might contribute to the symptomatology of HAD in different fashions (Fig. 2, Steps 4b&5): (i) by being infected by the HIV virus; (ii) by showing adverse responses to viral proteins; (iii) by immune activated astrocytes being unable to carry out their normal function; and (iv) by release of toxic compounds from immune activated astrocytes.

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5.1. HIV infection of astrocytes Restrictive infection of astrocytes has been demonstrated beyond any doubt (Saito et al., 1994; Tornatore et al., 1994; Ranki et al., 1995; Bagasra et al., 1996; Takahashi et al., 1996; Messam and Major, 2000; Thompson et al., 2001). In some of these studies, the frequency of HIV-1-positive astrocytes reached 1% in specific regions (Takahashi et al., 1996). In vivo, HIV-1 infected astrocytes express viral regulatory genes rather than structural genes associated with virus production (Ranki et al., 1995; Takahashi et al., 1996), which indicates viral entry into the cells occurs, but the productive replication process is lacking. The HIV-1-astrocyte infection pattern is atypical, to say the least. Many argue that this restrictive infection, given the numerical superiority of astrocytes, is significant, and that it is another factor that contributes to disease and may have a significant impact on the progression of neurodegeneration. In comparing the numbers of restrictively infected cells with those of immune activated cells, the reader should bear in mind that immune activation is a major impetus and should be a more important modulator of astrocyte function, especially given the number of activated cells, than the restrictive infection, per se. A more important question is whether restrictive infection and immune activation together cause a synergistic regulation of astrocyte function. The answer to this question will shed better light on what is most pertinent to disease. The following discussion on the role of the astrocytes in HAD will focus on the effects of ongoing inflammation that ensues viral infection of microglial cells and how it may participate in the disease.

5.2. Role of viral proteins (HIV-1gp120 and tat) Although HIV-1-infected MP release viral proteins such as gp120 and tat, their levels in the brain are not very high. Several investigators have studied how these free viral proteins may contribute to disease (Giulian et al., 1993; Bagetta et al., 1994; Aggoun-Zouaoui et al., 1996; Lannuzel et al., 1997; Hesselgesser et al., 1998; Zheng et al., 1999). It is now widely accepted that gp120 and tat in vitro are toxic to primary human neuronal cultures. Several transgenic models have been developed to study in vivo effects of de novo expression of these proteins (Toggas et al., 1994; Berrada et al., 1995). In vitro HIV-1gp120 affects several different functions of astroglial cells (see chapter by Kovacs et al.). Even though astrocytes do not express CD4, the receptor for HIV-1, a binding site for gp120 has been reported on astrocytes in addition to a number of chemokine co-receptors for viral entry. HIV-1 gp120 can modulate ion transport in astrocytes (Patton et al., 2000), glutamate influx, expression levels for adhesion molecules, cytokines and chemokines, and can stimulate signal transduction pathways including the protein kinase C, tyrosine kinase, JAK/STAT pathway, cyclic adenosine monophosphate and intracellular Ca2þ levels (Toggas et al., 1994; Bubien et al., 1995; Aggoun-Zouaoui et al., 1996; Lannuzel et al., 1997; Scorziello et al., 1998; Banks et al., 2001). Interestingly, some of these pathways can be modulated both by HIV-1 and immune activation, thus bringing us back to the question of whether a synergy exists in HIV-1, in which viral proteins and immune activation mutually enhance each other’s contribution to disease progression.

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5.3. Astrocytic function 5.3.1. Astrocytes and tissue homeostasis In the normal CNS, astrocytes serve in the maintenance of neuronal architecture and carry out a plethora of immune and regulatory functions critical for CNS homeostasis. Essential astroglial functions include, regulation of the levels of extracellular glutamate, maintenance of the BBB, and responsiveness to pathogens and brain injury (Thoenen, 1995; Danbolt, 2001; Dong and Benveniste, 2001). On one hand they provide neurons with essential precursors for transmitter glutamate and GABA (see chapter by Schousboe and Waagepetersen), and on the other hand, they “clean up” these transmitters as well elevated levels of Kþ (see chapter by Walz). They express receptors for a multitude of transmitters (see chapter by Hansson and Ro¨nnba¨ck), and recent research also suggests that they play a critical role in neuronal signal transmission by enhancing synaptic activity and strength, increasing the number of synapses, and modulating neuronal functions (Bezzi et al., 2001a; Bezzi and Volterra, 2001; Vesce et al., 2001). Indeed, the brain should not be considered a complex array of neuronal circuits alone, but represents an integrated circuitry of neurons and glia (Haydon, 2001). This impacts on views regarding the role astrocytes play in the pathogenesis of a variety of diseases. 5.3.2. Disruption of astrocytic functions by HIV Indeed, if the glia play an important role in the fundamental function of the nerve cells circuits, a corollary to this conclusion is that their malfunction could cause primary neuronal impairment. It is increasingly accepted that in HAD, some of the homeostatic functions of astrocytes are disrupted. It is known that the BBB is compromised, and astrocytes contribute to the maintenance and modulation of the BBB. The role of astrocytes in homeostasis of transmitters and ions is also impaired, and many of these effects may be caused by the toxicity of a glycoprotein in the viral envelope, gp120 (see chapter by Zsembery et al.). Increased astrocyte apoptosis is also believed to correlate with HAD and may participate in disease process (Thompson et al., 2001). One could argue that if activated astrocytes cause more neuronal damage than neuroprotection against disease, then their apoptosis may be beneficial. However, on the other hand, if high level of activation and the damage it causes precedes apoptosis, it will correlate with disease process (Thompson et al., 2001), and in either case essential astrocytic functions will not operate normally. The virus and the immune activation together affect the astrocytes and alter their normal neuroprotective behaviors to neurotoxic effects. 5.3.3. Impairment of glutamate uptake Impairment of glutamate uptake by astrocytes has been implicated in several diseases including schizophrenia, where there may be an excessive uptake of glutamate, and HAD, where there is reduction in glutamate clearance (Danbolt, 2001). TNF-a leads to a dosedependent inhibition of glutamate uptake in primary human fetal astrocytes (Fine et al., 1996). HTLV-1-infected human T-cells impair the glutamate transporters GLAST and GLT via production of TNF-a. HIV-1gp120 is known to increase levels of extracellular

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glutamate both by increasing its production (see below) and reducing its uptake. Radiolabeled-ligand studies with postmortem human brain tissue from patients with HAD have demonstrated reduced levels of glutamate transporters (Reynolda et al., 1995; Sardar et al., 1999).

5.4. Astrocyte-mediated neurotoxicity 5.4.1. Glutamate release Amplification of microglial inflammatory reactions by astrocytes lead to neurotoxic exocytosis of glutamate from astrocytes (Bezzi et al., 2001b). The details of this process include chemokine receptor activation on astrocytes, followed by a set of signal transduction events that lead to secretion of TNF-a, which in turn leads to the generation of prostaglandin, that ultimately triggers a third series of events leading to release of glutamate. This highly complicated and interesting signal transduction cascade, leading to exocytosis of glutamate from astrocytes, proposes astrocytes can be a source of active neurotoxin production and damage neurons directly. The activation and signal transduction events can form a network by working on neighboring cells and serving to amplify the neurotoxic cascade.

5.4.2. Activated astrocytes and death proteins Recently, soluble Fas ligand (FasL), a death protein was included in the list of ‘prognostic’ markers for disease in patients with HAD (Sabri et al., 2001). In addition, work from McArthur’s group and ours has shown that activated astrocytes are a source for this death protein. Needless to say, the overall concept of a killer astrocyte in the context of neuronal cells is highly provocative and challenges all pre-conceived notions about their protective functions. Expression of FasL on astrocytes may represent one pathway of maintaining the immune-privileged status of the brain as an organ. Human glioma cells have been known to produce FasL in vitro. Our data show that activation of astrocytes with IL-1b leads to significant upregulation in FasL levels (A. Ghorpade, unpublished data). In addition, significantly higher levels of sFasL are observed in CSF and brain tissue specimens from patients with HAD (A. Ghorpade, unpublished work).

6. Concluding remarks HAD is a devastating complication of progressive viral infection leading to cognitive, behavioral and motor impairments. Disease occurs despite the fact that neurons are not infected by virus. Activated and HIV-1-infected microglia and astrocytes produce a battery of pro-inflammatory and neurotoxic mediators that all contribute to neurodegeneration in unique ways resembling a metabolic encephalopathy.

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Glycoprotein gp120-mediated astrocytic dysfunction Eva Z. Kovacs,a Beverly A. Bushb and Dale J. Benosa,* a

Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA Correspondence address: Professor and Chair of Department of Physiology and Biophysics, MCLM 704 1918 University Boulevard Birmingham, Al 35294-0005, Tel.: þ205-934-6220; fax: þ205-934-2377 E-mail: [email protected] b Department of Psychiatry, University of Alabama at Birmingham, Birmingham, AL 35294, USA

p

Contents 1. 2. 3. 4. 5. 6.

7.

Introduction Morphology of the mature HIV virion The role and morphology of envelope proteins Cellular introduction and localization of HIV-1 and gp120 Possible mechanisms of gp120– astrocyte interaction Mechanisms of gp120-mediated astrocytic dysfunction 6.1. Alterations in astrocyte transport functions 6.2. Gp120-induced changes in astrocyte adhesion molecule expression 6.3. Modification of cytokine and soluble mediator secretion 6.4. Other cellular mechanisms influenced by gp120 in astrocytes Concluding remarks: future prospects

Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) or HIVassociated dementia (HAD), presents as a subcortical dementia with cognitive, motor and behavioral disturbances. The pathogenesis of ADC is still unknown. Because neurons are not directly infected with HIV-1, the causes of neuronal dysfunction are evidently indirect. It is believed to involve interactions among virally infected macrophages/microglia, astrocytes and neurons. We address the role of the astrocyte in the development of HAD. Astrocytes are the most numerous of the glial cells and have a complex function in maintaining homeostasis in the central nervous system (CNS). Astrocyte function is altered by HIV-1 infection, exposure to viral proteins and soluble factors secreted by HIV-1 infected macrophages. The HIV-1 surface envelope glycoprotein gp120 is Advances in Molecular and Cell Biology, Vol. 31, pages 921–949 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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considered to be one of the principal mediators of viral toxicity. The alterations in astrocyte function in connection to gp120 exposure are described in this chapter. The findings indicate that astrocytes are directly involved in the pathogenesis of HAD.

1. Introduction The human immunodeficiency viruses (HIV-1 and HIV-2) belong to the subgenus ‘primate lentiviruses’ and are the etiologic agents of acquired immunodeficiency syndrome (AIDS). Currently, more than 42 million individuals are infected worldwide (see ‘AIDS epidemic update: December 2002’; http://www.UNAIDS.org). The majority of these infections are due to HIV-1. The prevalence of HIV-2 infection is significantly less frequent and varies from region to region. It tends to occur in distinct geographical locations (e.g., West African countries, Portugal, Southwestern India) (for the epidemiology of HIV-2, see review by Schim van der Loeff and Aaby, 1999). Although the two strains are similar in many ways, there are important differences between them: HIV-2 has a reduced transmission rate, reduced pathogenicity and lower mortality rate (Reeves and Doms, 2002). With its higher transmission rate, HIV-1 presents a greater and more devastating public health challenge, in part because of the neurophysiological changes it causes (Atwood et al., 1993). In this chapter, we describe the effects of the HIV1 envelope glycoprotein (gp120) on astrocytes and the potential ramifications for HIV-1 neuropathogenesis. The central nervous system (CNS) is a prime target for HIV-1. The virus enters the brain early following systemic infection (Levy et al., 1986; Navia et al., 1986a,b; 1998; Navia and Price, 1987; Achim et al., 1991; Kolson et al., 1998; Nath, 1999). In the early 1990s, it was estimated that up to 30% of HIV-1 infected persons develop symptomatic neurological difficulties (McArthur et al., 1993; Sacktor et al., 1996). This percentage is even higher in the pediatric population (Vallat et al., 1998). HIV-1 is considered to be the most common cause of dementia among people under 60 years of age, and HIVAssociated Dementia (HAD) is a risk factor for death due to AIDS (Ellis et al., 1997). While the use of highly active antiretroviral therapy (HAART) has prolonged the lives of those infected with HIV-1 and appears to have positive effects on HAD (Price et al., 1999; Sacktor et al., 2001), the growing cohort of people with HIV-1 is resulting in an increase in the incidence of HIV-related neurologic disease and cognitive impairment (Sacktor et al., 2002). The spectrum of neurological complications associated with HIV-1 has been referred to as ‘AIDS dementia complex (ADC)’ (Price and Sidtis, 1990), ‘HIV-1-associated cognitive/motor complex’ (Anon., 1991) and ‘HAD’ (Gendelman et al., 1998). All these terms refer to a distinct neurological syndrome that may lead to dementia. The symptoms range from mild cognitive and motor slowing or subtle attentional difficulties to a subcortical dementia characterized by impaired memory, attention and concentration, and motor slowing that significantly impairs daily functioning. The symptoms progress in severity over time if the disease is untreated. Patients who begin with mild difficulties may experience quadriparesis, incontinence, and mutism in the late

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stages of the infection (Navia et al., 1986a,b; Price, 1996). The neurological complications of HIV-1 infection are accompanied by high morbidity and mortality, and result in impaired quality of life for the affected individuals. While HAD is caused by HIV infection, neither the extent of CNS histological abnormalities, nor the presence of HIV-encephalitis (HIVE) correlates with the severity of clinical symptoms (Glass et al., 1993). How HIV causes neuronal injury and apoptosis has not been clarified. The signs of HIV infection in the CNS are widespread: reactive astrocytosis, with an increase in both the number and size of astrocytes, diffuse myelin pallor, demyelinization, infiltration with blood-derived macrophages, resident microglia, and multinucleated giant cells (MGCs), simplification of synaptic contacts and neuronal loss (Navia et al., 1986b; Wiley et al., 1991; Petito et al., 1999). The neuropathology does not appear to be due to HIV-1 infection of neurons. Although HIV-1 gene sequences have been detected in neurons (Nuovo et al., 1994; Bagasra et al., 1996; Torres-Munoz et al., 2001), productive viral infection has not been demonstrated. This observation has led investigators to conclude that the neuronal damage is induced by indirect mechanisms, such as proinflammatory cytokines, chemokines, viral products, platelet activating factor, quinolinic acid, arachidonic acid metabolites, prostaglandins and free radicals, to name a few (Heyes et al., 1989; Genis et al., 1992; Guilian et al., 1993; Wesselingh et al., 1993; Gelbard et al., 1994; Nath et al., 1999). These factors are released into the CNS by productively infected cells—mainly macrophages and microglia (Koenig et al., 1986; Wiley et al., 1986; Pumarola-Sune et al., 1987; Fauci, 1988; Price et al., 1988; Stanley et al., 1994; Nottet and Gendelman, 1995). Some of the secreted cytokines and chemokines are directly toxic to neurons (see chapter by Ghorpade and Gendelman). However, there are several in vitro and in vivo observations supporting the hypothesis that astrocytes play a major role in the process of neurotoxicity. Astrocytes are the largest and most numerous of the glial cells, and outnumber neurons 10:1 (Benveniste, 1992; Norenberg, 1994—see also chapter by Wolff). Astrocytes retain the ability to divide and multiply. Not only do they provide structural support for neurons, they are essential for maintaining a proper microenvironment for all cells in the CNS by regulating extracellular pH and neuronal metabolite and neurotransmitter levels (Walz, 1989; see chapters by Bevensee; and by Schousboe and Waagepetersen). For example, astrocytes function to regulate extracellular [Kþ] by a process called Kþ spatial buffering and by active uptake (see chapter by Walz). Astrocytes have well-defined ion transport systems: one for the coupled exchange of Naþ for Hþ ions, one for the counter-transport of þ þ 2 Cl2 and HCO2 3 ; one for the co-transport of Na and HCO3 ; and a wide variety of Na , þ 2 2þ K , Cl , and Ca channels (Barres et al., 1990; Walz, 1989). Numerous recent findings draw attention to the active role of astrocytes, glia –glia interactions, and glia –neuron interactions mediated by calcium waves propagating through an astrocytic syncytium and giving rise to glutamate release from astrocytes, which in turn modulates synaptic transmission and might be an important contributor to information processing (Bezzi et al., 1998; Carmignoto et al., 1998; Vesce et al., 1999; Bezzi and Volterra, 2001; Haydon, 2001; Mazzanti et al., 2001; Pasti et al., 2001). Astrocytes also play a major role in neurotransmitter metabolism; for example, astrocytes possess a specific ATP and Naþdependent uptake system that functions to remove the excitatory amino acid

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neurotransmitter glutamate from the extracellular medium (Benveniste, 1992). Astrocytic end-feet on CNS capillaries are a major structural component to the blood – brain barrier (BBB). They also act as immune effector cells, playing a vital role regulating immune response (for review, see Dong and Benveniste, 2001 and chapter by Benveniste). Thus, astrocytes are indispensable for normal CNS function.

2. Morphology of the mature HIV virion HIV-1 is a retrovirus. Two single-stranded RNA molecules encode its genome. The HIV genome encodes a total of three structural proteins, two envelope proteins, three enzymes and six accessory proteins (Turner and Summers, 1999). Fig. 1 illustrates the morphology of the mature HIV virion. Like all retroviruses, an envelope, consisting of a host cell-derived lipid bilayer and virus-encoded envelope glycoproteins, surrounds the mature HIV-1 virion. The lipid bilayer contains several cellular membrane proteins derived from the host cell, including major histocompatibility antigens and adhesion molecules. One of the three major viral genes, env, encodes the envelope glycoproteins: the exposed surface glycoprotein (SU, gp120) and the transmembrane protein (TM, gp41).

Fig. 1. Morphology of the mature HIV virion. An envelope, consisting of a host cell-derived lipid bilayer and virus-encoded envelope glycoproteins, surrounds the mature HIV-1 virion. The lipid bilayer contains several cellular membrane proteins derived from the host cell. One of the three major viral genes encodes the envelope glycoproteins: the exposed surface glycoprotein (gp120) and the transmembrane protein (gp41). The matrix protein (MA) lines the inner surface of the viral membrane. The capsid protein (CA) builds up the cone-shaped capsid shell, which contains viral enzymes protease (PR), integrase (IN) and reverse transcriptase (RT), and the genome centrally.

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The gene gag encodes three types of structural proteins: the matrix protein (MA, p17), which lines the inner surface of the viral membrane, the capsid protein (CA, p24), the most abundant protein and the nucleocapsid protein (NC, p7). The latter two build up the coneshaped capsid shell, which contains viral enzymes and the genome centrally. The third major gene, pol, encodes some essential replication enzymes: protease (PR, p10), integrase (IN, p32), reverse transcriptase (RT, RNA dependent DNA polymerase, p66). These three enzymes and three of the accessory proteins [negative effector (Nef, p24), viral infectivity factor (Vif, p23) and viral protein R (Vpr, p15)] are located in the capsidshell. The other three accessory proteins that function in the host cell [regulator of viral gene expression (Rev, p19), transcriptional activator (Tat, p14) and viral protein U (Vpu)] appear to be located outside of the capsid. Small genes encode the accessory proteins. The reverse transcriptase transcribes the viral RNA into double stranded DNA. The IN carries out the integration of the viral DNA into the host-cell chromosome, while PR is required for proteolytic ‘maturation’ of the viral particle (Katz and Skalka, 1994). The replication cycle of the HIV virus has two phases. The early phase begins with the recognition of the target cell by the mature virion and involves all processes leading to and including integration of the genomic DNA into the chromosome of the host cell. The late phase begins with the regulated expression of the integrated proviral genome and involves all processes leading to virus budding and maturation. A detailed review about the HIV replication cycle can be found in Greene and Peterlin (2002).

3. The role and morphology of envelope proteins In the late phase of viral replication, the envelope precursor polyprotein is synthesized in the endoplasmic reticulum (ER). The precursor protein contains 845 – 870 amino acids; it undergoes posttranslational modifications in the ER and Golgi apparatus. It is heavily glycosilated to form the gp160 glycoprotein, is transported to the Golgi apparatus and cleaved into two parts: the exterior surface unit (gp120) and the transmembrane unit (gp41). Some carbohydrates on the gp120 glycoprotein are subsequently modified by the addition of complex sugars. Most of the surface-exposed elements are located on gp120, thus this glycoprotein mediates virus – host cell attachment, receptor binding, and determines viral tropism. The transmembrane protein, gp41, mediates membrane fusion between the virion and the attacked host cell. Gp41 has a flexible, coil-like ectodomain that interacts with the surface unit and is largely responsible for the trimerization with gp120 (see below). Following conformational changes during virus entry, gp41 has the potential to bridge the viral and target cell membranes by inserting its hydrophobic NH2-terminus (the ‘fusion peptide’) into the membrane of the target cell (Chan et al., 1997; Weissenhorn et al., 1997). The two constituents, gp120 and gp41 form a (TM-SU)3 trimeric glycoprotein complex (Chan et al., 1997; Wyatt and Sodroski, 1998a). This mature envelope glycoprotein complex is then transported to the host cell surface. While a fraction of these envelope glycoprotein complexes are incorporated into budding virus particles, a large number disassemble, releasing free gp120 and exposing the previously

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buried gp41 ectodomain. The shedding viral particles bind to circulating antibodies and thus attenuate the host-immune defense through productively infected cells (Wyatt and Sodroski, 1998a). The exposed surface glycoprotein, gp120, contains five variable regions (V1 – V5) interposed among more conserved regions. The structural basis for the ability of HIV-1 to evade immune response and hinder also the development of an effective vaccine is largely due to its envelope—glycoprotein complex. X-ray crystallography studies suggest that in gp120 the receptor-binding, discontinuous, conserved regions are located internally, surrounded by the externally accessible, heavily glycosylated, variable loops (Kwong et al., 1998; Wyatt et al., 1998b). Entry of primate immunodeficiency viruses into the host cell involves the binding of the gp120 envelope glycoprotein to the CD4 glycoprotein on the target cell. The CD4 glycoproteins have otherwise important roles in immune-defense mechanisms, but in HIV-infection they serve as the primary receptor for viral entry. CD4 binding induces conformational changes in the gp120 glycoprotein resulting in formation and exposure of a secondary binding site for specific chemokine (chemotactic cytokine) receptors (Miller and Meucci, 1999). These chemokine receptors serve as obligate co-receptors for virus entry. Different immunodeficiency virus strains use distinct co-receptors. In case of HIV-1, the gp120 third variable (V3) loop is the principal determinant of chemokine receptor specificity and thus viral tropism. Nakagawa and Schwartz describe chemokine receptors in a chapter. The chemokine receptor CCR5 acts as the co-receptor for macrophage-selective HIV-1 strains (M-strains). For the infection of T-lymphocytes by T-lymphocyte selective (T-topic) strains, the presence of CXCR4 chemokine receptor is essential (Clapham and Weiss, 1997). CCR5 and CXCR4 are the major co-receptors for HIV-1. However, some strains exhibit dual tropism while others use different chemokine receptors. For example, CCR3 co-receptor is involved in the infection of neural microglia. At least 10 chemokine receptors have been identified so far that can act as a co-receptor for HIV-1 (Littman, 1998). Simian Immunodeficiency Virus (SIV) strains and some cultured HIV-1 and HIV-2 isolates do not depend on the presence of additional CD4 receptor for successful viral entry, suggesting that chemokine receptors are the primary, obligate receptors for immunodeficiency viruses (Reeves et al., 1999). The use of CD4 as a coreceptor is probably a product of subsequent evolution that resulted in higher viral pathogenicity.

4. Cellular introduction and localization of HIV-1 and gp120 In the natural course of HIV-1 infection, the CD4 positive macrophages and dendritic cells in the skin and mucosa are the first targets of the virus. Examination of the phenotypes of HIV strains sampled at different times in the course of the infection showed that isolates present during the early, acute phase were macrophage-tropic, CCR5 chemokine-receptor specific. The significance of this finding was accentuated by the discovery that 32bp depletion in the CCR5 gene results in an abnormal CCR5 receptor, which is not displayed on the plasma membrane. The 1% of Caucasians with this

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homozygous depletion are almost completely resistant to HIV-1 infection. Those who are heterozygous to this mutation show a slower disease progression (Stewart, 1998; Kostrikis et al., 1998). During disease advancement, there is a general broadening of viral tropism. CXCR4, dual-tropic or additional chemokine receptor tropic strains emerge and/or prevail (Horuk, 1999). The ability of various HIV-1 strains to use different chemokine co-receptors and their diversity in cellular tropism, together with host factors, probably affects viral neuropathogenicity. Research findings have been inconsistent and the nature of this relationship is still in question (McCarthy et al., 1998; 2002; Littman, 1998; Zheng et al., 1999; Ohagen et al., 1999; Shapshak et al., 1999; Price, 2000; Smit et al., 2001; Gorry et al., 2001). During peak viraemia, within weeks of systemic infection, the virus is present in the brain parenchyma (Davis et al., 1992; Sopper et al., 2002). However, how HIV-1 crosses the BBB is not entirely clear. One widely accepted hypothesis suggests that HIV enters the nervous system through infected macrophages that migrate into the CNS (‘Trojan horse mechanism’) (Edinger et al., 1997). Another way HIV may penetrate the BBB and replicate is through gp120-induced adsorptive endocytosis (Banks et al., 1998; Huang and Jong, 2001). Disruption or dysfunction of the BBB has been implicated as means of facilitating viral entry (Nottet, 1999; Didier et al., 2002). How HIV replicates within the CNS is also not completely clear. The percentage of cells that are productively infected in the brain is relatively small, composed of macrophages, microglia and MGCs (Gosztonyi et al., 1994; Glass and Wesselingh, 2001). It is generally accepted that HIV-1 does not replicate in neurons. The infection of astrocytes and oligodendrocytes also does not result in replication; only a few types of viral proteins (for example Nef, Rev) and nucleic acid fragments are synthesized in these cells (Kohleisen et al., 1999; Robichaud and Poulin, 2000). Abundant in vitro evidence for gp120-mediated toxicity exists (Brenneman et al., 1988; Dreyer et al., 1990; Lipton, 1991; Toggas et al., 1994; Dawson and Dawson, 1994; Viviani et al., 2001). Because gp120 long escaped detection in the CNS, the in vivo toxic potential of this external glycoprotein has been questioned. However, gp120 is released from the surface of infected macrophages and leukocytes in the serum of patients with AIDS and can be found as circulating antigen (Gilbert et al., 1991; Oh et al., 1992) in the plasma. Nath and co-workers have recently provided an important missing link for the role of gp120 in neuropathogenesis. They have reported the presence of gp120 in MGCs, infiltrating monocytes, and microglial cells (Jones et al., 2000; Nath et al., 2000) in the brains of HIV-infected patients. Gp120 positive cells were primarily present perivascularly and occasionally in the vessel lumen. This is consistent with the hypothesis that infection may spread through blood vessels, penetrating the vessel walls and infected cells subsequently accumulate at perivascular sites. Gp120 positive MGCs observed in the brain parenchyma were most abundant in the basal ganglia. This finding concurs with neuropathological examinations of HIV-1 infected individuals, which found that the HIV-1 is not uniformly distributed in the brain. The basal ganglia carry the heaviest viral load (Kure et al., 1990a; 1991; Fujimura et al., 1997; Wiley et al., 1998). The predilection for basal ganglia

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involvement in HIV-1 infection has also been suggested by in vivo studies on brain circulation and metabolism (Berger and Nath, 1997; Berger et al., 2000; Ernst et al., 2000; von Giesen et al., 2001). Altmeyer et al. (1999) also reported the detection of macrophage-tropic HIV-1 gp120 in vitro, using conformation-dependent antibodies, with the subsequent successful detection of gp120 at the plasma membrane in rat brain. The intrabrain concentration of gp120 in vivo is not known, and whether or not the amount of gp120 detected by Jones et al. (2000) would be sufficient to cause neuronal and/or glial dysfunction or death has not been determined. However, the intraparenchymal detection of gp120 is an important step forward in understanding HIV neuropathogenesis. Because relatively few cells in the brain harbor the replicating virus and allow a productive infection, some type of amplification is needed to explain the widespread neuropathology found in HAD. This amplification probably involves the astrocytes. Astrocytes are the most numerous glial cells and have a central role in maintaining homeostasis in the CNS. Numerous studies have demonstrated that astrocyte function is altered by HIV-1 infection, exposure to viral proteins, and soluble factors secreted by HIV-1 infected macrophages. These alterations are described below.

5. Possible mechanisms of gp120– astrocyte interaction There are at least three mechanisms for gp120 and astrocyte interaction. One is infection of astrocytes by HIV virus; the second is interaction between soluble gp120 and astrocyte membrane receptors; and the third is interaction between the astrocytes and membrane-bound gp120 on the surface of infected cells. We will review each of these mechanisms. Human astrocytes can be infected with HIV-1 both in vitro and in vivo, but the significance and extent of astrocyte infection is a matter of debate (Kure et al., 1990b; Blumberg et al., 1994; Ma et al., 1994; Saito et al., 1994; Tornatore et al., 1994a; Nath et al., 1995; Sharer et al., 1996; Takahashi et al., 1996; Sabri et al., 1999). Because astrocytes constitute nearly 40% of the total CNS population (Rutka et al., 1997), a relatively small percentage of astrocytes carrying the HIV-1 genome would reflect a substantial infected population. Studies have suggested that under certain circumstances HIV-1 infection of astrocytes can be productive (Taylor et al., 1994). Once the limitation of viral entry into astrocytes has been overcome, a sustained viral protein synthesis and the release of infectious virions result (Canki et al., 2001; Schweighardt and Atwood, 2001). However, other studies maintain that astrocytic infection is ‘restricted’, characterized by a very low level and transient virus production, lack of fusion and giant cell formation (Brack-Werner et al., 1992; Brengel-Pesce et al., 1997; McCarthy et al., 1998; reviewed by Brack-Werner, 1999). Tornatore and co-workers found that following stimulation with soluble factors (tumor necrosis factor alpha or interleukin-1 beta) the persistent, otherwise unproductive infection of astrocytes changes and the cells resume viral protein synthesis and allow viral multiplication (Tornatore et al., 1991; 1994b). Although the exact nature of HIV-1 infection of astrocytes awaits further analysis, it has been hypothesized that viral

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binding, viral protein up-take or partial viral transcription may modify astrocyte function and lead to neurological disease, even in the absence of productive infection (reviewed by Brack-Werner, 1999) HIV-1 primarily infects CD4 positive (CD4þ) immune cells. In the brain macrophages and microglia express CD4 receptors, together with CXCR4, CCR5, or CCR3 obligate chemokine co-receptors on their plasma membrane. In these cells a productive and cythopatic infection occurs. Astrocytes and neurons also express CXCR4, CCR5, and/or CCR3 surface chemokine receptors, but they do not possess CD4 receptors (He et al., 1997a; Lavi et al., 1997; Ghorpade et al., 1998; Vallat et al., 1998; Boutet et al., 2001). Astrocytes do, however, have various receptors capable of binding HIV-1 envelope proteins. Glycosphingolipids located on the plasma membrane have long been considered as alternative primary receptors for HIV-entry in CD4 negative (CD42) cells. Harouse et al. (1991) have suggested a role for galactosylceramide (GalCer) in entry into two CD42 neural cell lines. GalCer is a glycosphingolipid found in particularly high concentrations on the surface of oligodendrocytes. Other groups have further supported the hypothesis that GalCer might be involved in the infection of CD42 cells, by showing (i) that antibodies against this glycosphingolipid have blocked the infection of several HIV lines from the brain and colon: and (ii) in high performance thin layer chromatography (HPTLC) binding assays, recombinant gp120 bound GalCer with high affinity (Bhat et al., 1991; Yahi et al., 1992; Cook et al., 1994; Fantini et al., 1998). Recent studies have confirmed the presence of the alternative epithelial receptor GalCer on CD42 epithelial cell lines from the gastrointestinal tract expressing chemokine co-receptors. Involvement of GalCer or other related glycosphingolipids in the productive viral infection of epithelial cells, and the potential role of glycosphingolipids in oral-genital and vertical transmission of HIV-1 has been suggested by different groups (Han et al., 2000; Meng et al., 2002; Moore et al., 2002). Others have postulated that glycosphingolipid microdomains located in the outer leaflet of the plasma membrane stabilize the attachment of viruses to the host cell through multiple low affinity interactions. Subsequently, the virus is conveyed to an appropriate co-receptor by moving freely in the plasma membrane, and thus the glycosphingolipid microdomains serve as crucial elements in organizing gp120-gp41, CD4, and the chemokine receptor into a membrane fusion complex (Fantini et al., 2000; Hug et al., 2000; Mahfoud et al., 2002). By promoting the virus– host cell interaction during CD4 dependent HIV-1 fusion glycosphingolipids may be essential both in CD4-dependent and independent infection. However, the literature regarding the affinity of the specific interactions between gp120 and various glycosphingolipids is contradictory (Long et al., 1994; McAlarney et al., 1994; McReynolds et al., 1999). The inconsistency may also be due to technical difficulties in measurement (Conboy et al., 2002), and the contribution of glycosphingolipids as viral receptors to HIV-1 infection of different cell types under physiological circumstances is yet to be defined. Several other researchers have detected gp120 and astrocyte interactions. Hao and Lyman (1999) have described the presence of an approximately 65 kDa molecule on the membrane surface of astrocytes. This molecule is neither CD4 nor galactocerebrosid. However, gp120 and this molecule bound in a specific, concentration-dependent manner.

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(Hao and Lyman, 1999). Meulen and co-workers detected a 180 kDa protein receptor on glioma cells (Schneider-Schaulies et al., 1992). The specific binding of gp120 induced a tyrosine-specific protein-kinase activity and tyrosine phosphorylation. Other than HIV-1 infection of astrocytes, additional possible ways of direct interaction between gp120 and astrocytes exist. For example, HIV envelope proteins induce signals through CCR5 and CXCR4 chemokine receptors in different cells either elevating intracellular [Ca2þ], inducing tyrosine phosphorylation of proteins, or modifying cAMP synthesis. In these ways, gp120 may increase the activation state of target cells, and influence viral replication, viral cytopathicity, cellular protein synthesis or apoptosis even in the absence of HIV-1 infection of those cells (Weissman et al., 1997; Davis et al., 1997; Meucci et al., 1998; Bajetto et al., 1999). Finally, cell-to-cell contact probably contributes to viral transmission in astrocytes (Nath et al., 1995). The increased expression of cellular adhesion molecules (intercellular adhesion molecule, ICAM and vascular endothelial cell adhesion molecule-1, VCAM-1) on astrocytes in HIV-1 infection has been observed and facilitates the entry of infected immune cells into the brain. Boutet et al. have compared the ability of gp120/41 found on the surface of HIV infected cells versus soluble external HIV envelope glycoprotein to cause astrocyte damage by measuring lactate dehydrogenase (LDH) release and assessing morphologic alterations. They found that membrane-bound gp120 is more efficient in initiating cytotoxic events than soluble gp120 (Boutet et al., 2000).

6. Mechanisms of gp120-mediated astrocytic dysfunction Astrocytes are the most numerous cells in the brain. They form an elaborate intercellular network and have a complex and essential role in the proper functioning of the CNS. A number of HIV-1 proteins influence neuronal (van de Bovenkamp et al., 2002), nonneuronal (Brack-Werner, 1999) and astrocyte cell functions. Because of their complex role, astrocytes have the potential for both harmful and beneficial effects in the development of HAD. Some of the effects directly related to gp120 exposure are discussed below.

6.1. Alterations in astrocyte transport functions Neuronal activity and signal transmission results in extracellular pH changes, release of neurotransmitters, opening of voltage gated Kþ channels and elevation of extracellular Kþ concentration. The released neurotransmitters (e.g., glutamate, GABA) and ions (e.g. Kþ, Hþ) need to be removed from the synaptic cleft and the extracellular space in order to prevent neurotoxicity and to allow sufficient signal to noise ratio in neurotransmission during subsequent activation. Astrocytes are principally responsible for restoring the ion balance in the extracellular space and eliminating various neurotransmitters, thus providing the necessary highly regulated environment for optimal neuronal functioning. The astrocytic Naþ/Hþ exchange system (NHE) plays a role in pH regulation. The role of astrocytes in Kþ homeostasis, pH regulation, and glutamate metabolism is discussed in

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detail elsewhere in this book and will not be repeated here (see chapters by Walz; by Bevensee; and by Schousboe and Waagepetersen). Gp120 and the altered cytokine production of macrophages and microglia in HIV-1 infection affect astrocyte transport mechanisms. The resulting elevation in extracellular glutamate concentrations produces excitotoxic injury of neurons. The overactivation of glutamate-receptors (e.g., N-methyl-D -aspartate (NMDA)-receptor-coupled ion channels) on neurons in turn produce an elevation in intracellular [Ca2þ]. The increase in intracellular [Ca2þ] triggers a variety of potentially harmful enzymes, and together with nitric oxide released by activated macrophages and astrocytes and other reactive oxygen intermediates, leads to neuronal injury (Kaul et al., 2001). According to initial experiments in primary rat astrocytes, gp120 exposure and/or proinflammatory cytokines activate Naþ/Hþ exchange in astrocytes (Benos et al., 1994b, c). The resulting intracellular alkalinization influences other cellular processes. For example, glutamate uptake is inhibited and a novel, pH-sensitive Kþ channel in the astrocyte membrane is opened (Bubien et al., 1995). Similar findings were observed in human astrocytes, when whole cell currents and single-channel activity were measured by patch – clamp technique (Benos et al., 1994a; Patton et al., 2000). The single channel activity measurements also suggested that the activation of the pH sensitive Kþ channel is indirect, and must have been a consequence of cellular signaling pathway activation or changes in the intracellular environment. Because astrocytes are seldom infected by HIV-1 and gp120 has not been detected within the astrocytes, the influence of gp120 on an astrocyte surface protein, leading to an intracellular signaling cascade, was proposed. Experimental results have supported this hypothesis. Tyrosine phosphorylation of proteins (90 and 130 kDa) following gp120 administration to astrocytes has been observed; tyrosine kinase (TK) inhibitors can block this action (Benos et al., 1994b). Significant intracellular alkalization was found shortly (10 min) following administration of 25 nM gp120 to both rat and human astrocytes (Benos et al., 1994a). Human astrocyte intracellular pH (pHi) increased by 0.26 ^ 0.06; while the average increase in rat astrocyte was 0.14 ^ 0.03. The effects of gp120 on astrocytes can be blocked by amiloride or its analog dimethyl-amiloride (DMA), inhibitors of Naþ/Hþ antiport (Noel and Pouyssegur, 1995). Gp120 failed to activate pH sensitive Kþ channels in the absence of external Naþ. Replacing the external Naþ in the bathing solution with the organic monovalent cation Nmethyl-D -glucamine (NMDGA), which does not flow through ion channels and is not transported by the Naþ/Hþ exchanger, the activation of pH sensitive potassium channels can be prevented. These results suggested that Kþ channel activation is secondary to the action of gp120 on NHE. Altered Kþ conductance following gp120 administration was mediated by intracellular alkalinization. Gp120 had no effect on Kþ conductance when the intracellular pH changes were prevented. On the other hand, increasing the cytoplasmic pH with the administration of 20 mM ammonium chloride (NH4Cl) in the bathing solution resulted in a similar increase in potassium conductance as we have seen following gp120 administration. Based on results from restriction enzyme analysis, it could be concluded that one or more novel form(s) of the Naþ/Hþ exchanger became expressed in the rat and human astrocytes. Using degenerate primers made to two transmembrane regions of the entire

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family of cloned NHE isoforms, a 519 bp fragment was amplified by PCR and sequenced. Sequence analysis of the fragment demonstrated approximately 60% homology to other known NHE isoforms. However, Western blot analysis using antibodies directed against NHE1 – 4 failed to detect any of these isoforms (Patton et al., 2000). Other groups have also suggested the expression of additional NHE isoforms in astrocytes based on altered amiloride sensitivity (Pizzonia et al., 1996; Bevensee et al., 1997; Touyz et al., 1997). Our hypothesis is that the induction of activity of this currently unique NHE isoform (termed NHEX-H) is the primarily affected function of gp120 in astrocytes. The nature of this presumably new NHE isoform and the interactions between the putative astrocyte gp120 receptor and NHE needs to be addressed in future studies. The novel pH-sensitive Kþ conductance observed in human astrocytes bears striking similarities to the mSLO-3 family of Kþ channels. The mSLO-3 channels have been described in mammalian spermatocytes (Schreiber et al., 1998). The similarities are: (i) activation by alkaline pH; (ii) low sensitivity to inhibition by tetraethylammonium and barium; (iii) the apparent activation by Ca2þ. There are, however, some differences between these two channels: mSLO-3 is voltage-dependent and has a conductance of 90 picosiemens (pS) (Schreiber et al., 1998); about half of what we have observed (147 pS) (Bubien et al., 1995). The ion selectivity properties are, however, very comparable. The effect of gp120 on astrocyte glutamate transport has also been examined. The efflux rate of glutamate analog D -[3H]aspartate was measured following gp120 administration. After a 10 min exposure of rat astrocytes to 25 nM gp120, the D -[3H]aspartate efflux rate constant increased nearly 2.5-fold, from an average of 0.204 to 0.504 min21 (Benos et al., 1994a). This effect of gp120 could be abrogated by amiloride. Similar experiments were carried out on human astroglioma cells (Patton et al., 2000). 13 nM gp120 reduced [3H]glutamic acid uptake by about 30%. The gp120-mediated reduction in glutamate uptake was attenuated by dimethyl-amiloride. Glutamate uptake into astrocytes is a Naþ dependent process. The activity of glutamate co-transport protein is also reduced with cytosolic alkalinization. Since gp120 induced increase in Naþ/Hþ exchanger activity results in intracellular alkalinization, in increase of [Naþ]i and a concomitant reduction of transmembrane Naþ gradient, both these changes can have negative effect on glutamate uptake. A model that summarizes the Naþ gradient-related transport processes is shown in Fig. 2. In accordance with our findings, Volterra and colleagues observed both inhibition of glutamate accumulation and stimulation of glutamate release in primary rat astrocyte cultures treated with subnanomolar concentrations of gp120 (Vesce et al., 1997). Because the envelope protein had no detectable effect on glutamate transporters (GLT1 and GLAST) in liposomes, they concluded that the effect of the glycoprotein is not due to its direct interactions with the glutamate-transporters. In a subsequent study on rat hippocampal slices and human astrocyte cultures, Vesce and colleagues found that astrocyte glutamate release can be evoked via stimulation of the CXCR4-chemokine receptor by both gp120 and the chemokine stromal cell derived factor 1 (SDF-1). They found that the exocytic glutamate release from astrocytes is mediated by increase in [Ca2þ]i, TNF-a-dependent signaling, and prostaglandin formation (Bezzi et al., 2001b). Analyzing the time-course of the events they concluded that signaling on CXC4

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Fig. 2. Model of plasma membrane in normal astrocytes showing Naþ-coupled co-transport processes (indicated by connections between arrows for Naþ transport and transport of another compound) that control glutamate reuptake, Hþ production resulting from glucose metabolism, and the removal of cell Naþ in exchange with Kþ, as wells as channel-mediated Kþ transport. These processes must function in balance to achieve a steady state. During periods of high Naþ-coupled influx such as during glutamate reuptake, and ensuing stimulation of Naþ/Hþ countertransport, pH-sensitive Kþ channels may open to in response to intracellular alkalinization, thereby perturbing the extracellular Kþ balance. Reproduced with permission from Patton et al., 2000, Am. J. Physiol. Cell Physiol. 279, p. C706.

receptors leads to a quick release of glutamate. The inhibition of astrocyte glutamate uptake must follow the glutamate-releasing effect of the viral protein. In an earlier study, Dreyer and Lipton (1995) also found that gp120 impairs astrocyte uptake of excitatory amino acids. They suggested that at least one pathway of this inhibitory effect involves arachidonic acid (AA) released by activated macrophages (Dreyer and Lipton, 1995). In a recent study, Collins and co-workers found a robust increase (10-fold over controls) in medium glutamate levels after administration of 100 pM gp120 to organotypic rat glial cultures (Belmadani et al., 2001). Prolonged ethanol pretreatment significantly abrogated the gp120 induced decrease in astrocytic glutamate uptake and suppressed [Ca2þ]i elevations. The authors hypothesized that this neuroprotective effect of ethanol was probably caused by its inhibition of arachidonic acid secretion stimulated by gp120. Other research has only in part supported these mechanisms. Pulliam et al. (1993) did not detect changes in glutamate uptake in human fetal astrocytes following gp120 administration, but they examined the effects of long-term exposure (72 h). Kort (1998) reported no effect of recombinant gp120 on glutamate release in glial cells, although gp41 facilitated the glutamate release in a concentration-dependent manner. Several groups have reported another mechanism of gp120 and astrocyte interaction. These groups have reported elevation in [Ca2þ]i in neurons and/or astrocytes following gp120 exposure (Lannuzel et al., 1995; Medina et al., 1999). In rat cerebellar cultures, Meldolesi and co-workers observed distinct [Ca2þ]i responses in 50% of glial cells following administration of 10210 – 1029 M gp120 (Ciardo and Meldolesi, 1993; Codazzi et al., 1995). In a subsequent article, they reported tyrosine phosphorylation of a 56 kDa

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protein after 1 min of gp120 application in rat cortical astrocytes (Codazzi et al., 1996). They also observed [Ca2þ]i increase and rapid [Ca2þ]i oscillations which might convey intercellular information among astrocytes (see chapters by Cornell-Bell et al. and by Jung et al.). Geiger and colleagues registered significant, dose-related elevations in [Ca2þ]i in human fetal neurons and astrocytes following pressure-application of recombinant gp120 from concentrations as low as 5 pM (Holden et al., 1999). Amiloride significantly attenuated the increase in [Ca2þ]i in neurons and astrocytes. Because our in vitro work implicated the astrocyte as a central component of the brain’s pathological response to the HIV-1 protein product gp120, we wanted to extend these observations to a more complex situation in which the architectural relationships between astrocytes and neurons remained intact. Thus, we examined the effect of superfusing gp120 over rat brain slices on neuronal excitability. Cell excitability was assessed by measuring the number of action potentials in response to a depolarizing current pulse. Gp120 (13 nM) caused a time-dependent increase in excitability (unpublished data). These preliminary results are consistent with the hypothesis presented in Fig. 3, namely, that gp120-induced transport changes in the astrocyte converge on adjacent neurons by bringing about changes in the extracellular concentrations of glutamate and or Kþ that in turn increase neuronal excitability. Thus, the remarkably active properties of glial cells and the likely presence of rapid regulatory cross-talk between neurons and astrocytes during synaptic transmission and during nonsynaptic information transfer between the two cell types by aid of glutamate and calcium waves may be precisely those aspects of neuronal-astrocytic interactions which suffer the most in HIV-1 induced neuropsychological disease. We have extended our observations on intracellular pH changes associated with HIV to humans using Magnetic Resonance Imaging (MRI) and in vivo Magnetic Resonance Spectroscopy (MRS). MRS and MRI are useful tools in the study of brain metabolism. Data can be obtained on energy usage, metabolism, intracellular pH, and anatomical structure. We have used high field 31P-MRS to calculate intracellular pH in various brain regions in order to test our hypothesis that brain HIV-1 infection, and its sequelae, including increased levels of proinflammatory cytokines and gp120, result in an intracellular alkalinization of the astrocytes. Our preliminary data showed that a

Fig. 3. Hypothesis of HIV-1-induced changes in cytokine production and its effects on astrocytes and oligodendrocytes leading to neurotoxicity and ADC. Infected or activated macrophages/microglia release the proinflammatory cytokines Il-1, IL-6, and TNF-a, all of which affect astrocyte function, while TNF-a, together with additional TNF-a, released from astrocytes, leads to demyelination via its effect on oligodendrocytes. In addition, HIV replication causes release of HIV proteins, which together with the proinflammatory cytokines released from macrophages/microglia and interferon-g released from CD4þ cells, have detrimental effects on astrocytes. These include activation of the Naþ/Hþ exchanger, at least partly caused by induction of a new exchanger protein, leading to intracellular alkalinization and Kþ exit through pH-mediated opening of a Kþ channel. The resulting astrocytic depolarization, as well as direct cytokine effects, inhibits glutamate re-uptake in astrocytes. Increased extracellular Kþ and glutamate depolarize and activate neurons and cause neuronal Ca2þ entry. Reproduced from ‘Technical Advances in AIDS Research in the Human Nervous System’ (E.O. Major and J.A. Levy, Eds.), p 225, Plenum Press, 1995.

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significant increase in pH in neuropsychologically asymptomatic HIV-infected individuals compared to noninfected individuals occurred in the cerebellum (Patton et al., 2001). Similar changes have been found in the temporal and occipital lobes. We focused on the cerebellum in this initial study because the cerebellum contains low numbers of microglia relative to other brain regions. Additionally, astrocytes are the predominant glial cell type in this brain region (Ghez, 1998). Examination of other brain regions is currently under way. 6.2. Gp120-induced changes in astrocyte adhesion molecule expression The migration of inflammatory cells across the BBB and the invasion of brain tissue by HIV-infected immune cells are one of the important steps in HIV-1 neuropathogenesis. Adhesion molecules like intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) or E-selectin present on brain endothelial cells and astrocytes facilitate the migration of immune cells into the brain (reviewed in Lee and Benveniste, 1999; see also chapter by Couraud et al.). Adhesion molecules thus may facilitate viral entry in infected immune cells and may promote cell-to-cell adhesion. We investigated the ability of gp120 to regulate ICAM-1 expression in astrocytes and found that gp120 enhances ICAM-1 gene expression in primary rat astrocytes and microglia, primary human astrocytes, and in the human astroglioma cell line CRT (Shrikant et al., 1996). The signal transduction events involved in gp120-mediated enhancement of ICAM-1 expression in both astrocytes and microglia appears to involve activation of both protein kinase C and tyrosine kinase. Moreover, gp120 induces tyrosine phosphorylation of ‘signal transducer and activator of transcription’-1a (STAT-1a) as well as the Janus kinase-2(JAK2) in glial cells. Gp120-mediated ICAM-1 expression has functional significance, as it enhances the ability of monocytic cells to bind to gp120stimulated human astrocytes in an ICAM-1/b2 integrin-dependent fashion. The fact that these signal transduction pathways involve both protein kinase C and tyrosine kinase support the impact of gp120 on ion transport and metabolism changes in the astrocytes, suggesting a sequence of events that occur upon binding of proinflammatory cytokines and/or gp120. These results provide new insights into how gp120 can influence the involvement of glial cells and astrocytes in the pathogenesis of HAD. 6.3. Modification of cytokine and soluble mediator secretion Cytokines constitute a large family of secreted low-molecular-weight proteins that function as chemo-attractants and activators of immunologic cells. Activated astrocytes and microglia are the major sources of cytokine production in CNS diseases of inflammatory or immunologic nature. Cytokines include tumor necrosis factor-alpha (TNF-a), transforming growth factor-beta (TGF-b), interferon-gamma (IFN-g), interleukins (IL-1, IL-6, IL-8, and IL-10), colony stimulating factors (CSFs) and chemokines. Chemokines include Stromal cell derived factor-1 (SDF-1), Regulated upon Expression Normal T cell Expressed and Secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory proteins (MIP-1a and MIP-1b). These proteins

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primarily function in an autocrine or paracrine fashion, binding to specific cell-surface receptors on the target cells, eliciting a wide array of responses. They modify intracellular signaling, gene expression, protein synthesis, transport mechanisms and cellular death. A number of these proteins have been implicated in the pathogenesis of HAD (Kaul and Lipton, 1999; Benveniste et al., 1998; Kutsch et al., 2000; Allen and Attwell, 2001; Brenneman et al., 2001; Kaul et al., 2001). Gp120 induces IL-1 and TNF-a expression in rat and human brain cultures (Merrill et al., 1992; Koka et al., 1995). In another study on primary human brain cultures gp120 was found was found to be toxic due to induction of IL-6 and TNF-a (Yeung et al., 1995). Kong et al. (1996) compared the difference between various HIV-1 strains in their ability to induce soluble mediator secretion in primary mixed glial cell cultures. The application of gp120 from the HIV-1IIIB strain resulted in NO, TNF-a, and IL-1 secretion, while HIV-1SF2 increased the release of IL-6 only. The authors hypothesized that this characteristic of the viral strains is probably one of the factors underlying the strain’s neuropathogenicity. Cytokine secretion induced by gp120 has been directly correlated with neurological symptoms through the impact of gp120 on learning and memory in rats. Intracerebroventricular injection of gp120 caused memory impairment on hippocampal-dependent contextual fear conditioning and resulted in increases in Il-1b protein levels in the hippocampus and the frontal cortex. IL-1 antagonists can block memory impairment (Pugh et al., 2000). Others have found that the intrathecal injection of gp120 created exaggerated pain and hyperalgesia in rats. This effect was accompanied by time-dependent, site specific increases of TNF-a and IL-1, while IL-1 and TNF-a antagonists prevented gp120 induced pain changes (Milligan et al., 2001—see also chapter by Raghavendra and DeLeo). Yirmiya and colleagues examined the involvement of cytokines in gp120 induced sickness behavior in rats (Barak et al., 2002). Intracerebroventricular gp120 administration caused anorexia and reduction in locomotor activity associated with elevation of IL-1b mRNA expression in the hypothalamus. These effects could be partially abolished by IL-1 receptor antagonist. These observations suggest that inhibition of gp120 induced glial cytokine secretion might be a future target of HIV-1 therapy for preventing HAD. Nitric oxide (NO) has also been studied in connection to HIV-1 infection (reviewed by Mollace and Nistico, 1995; Nicotera et al., 1995). NO is a free radical that can have a wide variety of potential interactions with cellular biochemistry and has toxic effects on neurons and oligodendrocytes (Merrill et al., 1993; Mitrovic et al., 1994; Lee et al., 1995—see also chapter by Garcia and Baltrons). Astrocytes display both constitutive and inducible NO synthase (NOS) enzyme activity and are capable of producing NO (Murphy et al., 1993). HIV gp120 stimulates the inducible isoform of NOS enzyme (iNOS) and NO release, and via this mechanism it probably contributes to the neuropathological changes in HAD (Koka et al., 1995; Mollace et al., 1993; Mollace et al., 1994).

6.4. Other cellular mechanisms influenced by gp120 in astrocytes Gp120 causes additional modifications in gene expression and protein synthesis. A large body of evidence demonstrates that gp120 is able to cause cellular death in a number

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of cells, including lymphocytes (Foster et al., 1995), neurons (Scorziello et al., 1997) and astrocytes (Shi et al., 1996). Both necrosis and apoptosis occur, but the apoptotic pathway leading to programmed cell death seems to be dominant (Meucci and Miller, 1996). There are two well-described pathways for induction of apoptosis: (i) the extrinsic, or death-receptor mediated (activated by mediators like TNF-a); and (ii) the intrinsic or mitochondrial mediated (activated by increase in intracellular [Ca2þ]). Both the intrinsic and extrinsic mechanisms have been implicated in HIV neuropathogenesis (Garden et al., 2002); it is not known which pathway dominates. These two courses ultimately converge and result in increased caspase-3 proteolytic activity and mitochondrial release of cytochrome c, suggesting that the pharmacological inhibition of caspase enzymes may be used in the treatment of HAD (Garden et al., 2002). The literature consistently reports some degree of neuronal apoptosis in HAD (Yeung et al., 1995; Shi et al., 1996; Gray et al., 2000; Corasaniti et al., 2001; Garden et al., 2002; Chen et al., 2002). Whether or not astrocytes undergo apoptosis is not clear. However, various groups have reported an increase in the number of apoptotic astrocytes in autopsies of HIV positive individuals (Shi et al., 1996; Thompson et al., 2001). Researchers have described astrocytes undergoing apoptosis following administration of HIV-1 proteins or supernatants from HIV infected macrophages to cell cultures (He et al., 1997b; Mollace et al., 2002). DNA fragmentation characteristic of apoptosis and the elevated expression of the growth suppressor gene p53 have also been observed following gp120 administration in human neuroblastoma cells (Yeung et al., 1998). Gabudza and co-workers examined a panel of diverse HIV-1 primary isolates for the ability to replicate and induce neuronal and astrocyte apoptosis in primary human brain cell cultures (Ohagen et al., 1999). They discovered that HIV-1 isolates differ in their capacity in inducing apoptosis. The envelope glycoprotein was found to be the major determinant of this ability; blood-derived HIV-1 isolates induced significant levels of apoptosis in neurons and astrocytes. However, in other experiments gp120 failed to cause apoptosis in astrocytes in human CNS cell cultures (Muller et al., 1992; Lannuzel et al., 1997) and in neonatal rats (Schwartz and Nair, 1997). Because astrogliosis, a glial reaction characterized by astrocyte proliferation and activation, is considered to be one of the major characteristic of HIV-encephalopathy, Gelbard and co-workers have hypothesized that activated astrocytes may be resistant to apoptosis as they found elevated expression of anti-apoptosis genes in pediatric patients with HIV-1 encephalitis (Krajewski et al., 1997). These contradictory observations may result from the differences among HIV-strains in their capacity for apoptosis induction and/or from a number of host factors, which influence the response towards the pathogens. In a recent study, Fisher and colleagues sought to determine whether HIV-1 induces specific changes in gene expression in primary human astrocytes (Su et al., 2002). They used a new rapid hybridization method (RaSH) to identify genes showing elevated expression following HIV-1 infection or gp120 treatment. They identified 13 known and 2 novel genes that displayed elevated expression in fetal astrocytes. The authors indicated that the described astrocyte elevated genes (AEGs) may represent important genes underlying the altered physiology of astrocytes in HAD.

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7. Concluding remarks: future prospects It is important to understand the cellular and molecular mechanisms that contribute to neurodegeneration subsequent to HIV-1 infection. How can HIV-1 result in neuronal damage, especially because neurons themselves are rarely infected in vivo (Wiley et al., 1986)? How does HAD develop? Advances in our understanding of the impact of glial cells in brain development (Barres and Barde, 2000) and information processing (Bezzi and Volterra, 2001) have revolutionized research into possible mechanisms leading to the neuropathogenesis of HAD. The recognition of glia –glia and glia – neuronal cross-talk opens new perspectives for understanding the pathogenesis of diseases. Pathological processes that affect glial function have deleterious impacts on neuronal functionality and may be the primary cause of neuronal dysfunction. There is mounting evidence that astrocytes and astrocytic functions are negatively impacted by HIV-1. Thus, the effects of HIV on astrocytes needs to be addressed as new treatments for HAD are developed. Evidence has been presented that gp120 may strike close to the core of the astrocytic part(s) of some of the functionally most essential interactions between astrocytes and neurons. From better understanding of these mechanisms, new ways of treatment of HAD, and perhaps of other dementing illnesses, will undoubtedly emerge. Acknowledgements We thank Cathleen Guy for her excellent assistance in the preparation of this manuscript and figures. The preparation of this review was supported by NIH grant MH50421. References Achim, C.L., Morey, M.K., Wiley, C.A., 1991. Expression of major histocompatibility complex and HIV antigens within the brains of AIDS patients. AIDS 5, 535–541. Allen, N.J., Attwell, D., 2001. A chemokine-glutamate connection. Nat. Neurosci. 4, 676–678. Altmeyer, R., Mordelet, E., Girard, M., Vidal, C., 1999. Expression and detection of macrophage-tropic HIV-1 gp120 in the brain using conformation-dependent antibodies. Virology 259, 314 –323. Anon., 1991. Nomenclature and research case definitions for neurologic manifestations of human immunodeficiency virus-type 1 (HIV-1) infection. Report of a Working Group of the American Academy of Neurology AIDS Task Force. Neurology 41, 778–785. Atwood, W.J., Berger, J.R., Kaderman, R., Tornatore, C.S., Major, E.O., 1993. Human immunodeficiency virus type 1 infection of the brain. Clin. Microbiol. Rev. 64, 339 –366. Bagasra, O., Lavi, E., Bobroski, K., Khalili, K., Pestaner, J.P., Tawadross, R., Pomerantz, R.J., 1996. Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry. AIDS 10, 573–585. Bajetto, A., Bonavia, R., Barbero, S., Piccioli, P., Costa, A., Florio, T., Schettini, G., 1999. Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1. J. Neurochem. 73, 2348–2357. Banks, W.A., Akerstrom, V., Kastin, A.J., 1998. Adsorptive endocytosis mediates the passage of HIV-1 across the blood–brain barrier: evidence for a post-internalization coreceptor. J. Cell. Sci. 111, 533–540. Barak, O., Goshen, I., Ben-Hur, T., Weidenfeld, J., Taylor, A.N., Yirmiya, R., 2002. Involvement of brain cytokines in the neurobehavioral disturbances induced by HIV-1 glycoprotein 120. Brain Res. 933, 98 –108.

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The role of astrocytes and microglia in persistent pain Vasudeva Raghavendraa and Joyce A. DeLeob,* a

Department of Anesthesiology, Dartmouth Medical School, Dartmouth Hitchcock Medical Center, Lebanon, NH 03756, USA b Department of Anesthesiology and Pharmacology, Dartmouth Medical School, Dartmouth Hitchcock Medical Center, Lebanon, NH 03756, USA p Correspondence address: Department of Anesthesiology, HB 7125, Darmouth-Hitchcock MedicalCenter, Lebanon, NH03756, USA. Tel.: þ 1-603-650-6204; fax: þ 1-603-650-4928. E-mail: [email protected](J.A.D.)

Contents 1. 2. 3. 4. 5. 6.

7.

Introduction Glial cells: morphology and function Injury to sensory afferents activate glia: relationship to pain Glial activation enhances pain transmission Brain cytokines and pain Glia: a target for therapeutic modulation of persistent pain 6.1. Immune modulators/cytokine inhibitors 6.2. Glial-targeted metabolic inhibitors 6.3. Xanthine derivatives Concluding remarks

Over the past decade, the role of astrocytes and microglia as essential players in the induction of chronic pain has begun to be elucidated. Spinal glial activation and proinflammatory cytokine expression have been shown to mediate persistent pain, morphine tolerance and morphine withdrawal-induced hyperalgesia. Recognizing the distinct morphological and neurochemical responses of glia following stressors that induce pain, as well as their intimate interaction with neurons, implicates them in central nervous system plasticity that is the underpinning of chronic pain. Further understanding of the selective manipulation of glial action may lead to the discovery of novel, nonopioid agents for the treatment and prevention of chronic pain. Advances in Molecular and Cell Biology, Vol. 31, pages 951–966 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction The central nervous system (CNS) contains two major cell populations, neurons and glial cells. The functional role of glial cells is not as obvious as that of neurons. They were first accepted as ‘supporting cells’ of the CNS, providing both trophic and metabolic support to neurons, and as a structural matrix for the formation of synapses and synaptic plasticity. However, recent technical advances and increased interest in elucidating the role of nonneuronal cells of the CNS in both physiological and pathophysiologial processes have catapulted these cells into the forefront of neuroscience research. A wealth of data now suggests that the functional unit of the brain is not just a neuron but rather it is the neuronal – glial complex. Recent research has demonstrated that glia are intimately involved in a host of CNS pathologies. One such pathophysiological process, where the significance of glia has been implicated, is in the generation and maintenance of chronic pain. In this chapter, we will discuss the morphology of relevant glial cells, the involvement of activated glia and their secretory products (particularly cytokines) in the etiology of persistent pain states, and the clinical implications of glial and cytokine modulation for both the prevention and treatment of chronic pain, such as neuropathic and radicular pain. 2. Glial cells: morphology and function Glial cells constitute over 70% of the total cell population in the brain and spinal cord. The four main functions of glial cells are: (i) to surround neurons and provide structural support; (ii) to supply nutrients and biosynthetic products to neurons; (iii) to insulate one neuron from another; and (iv) to destroy and remove injured and dead neurons. Apart from these functions, glial cells fulfil important tasks within the neural network of the central and peripheral nervous system and play an important role in regulating the neuronal microenvironment. The three types of glial cells are astrocytes, microglia and oligodendrocytes. For the context of neuronal hypersensitivity, the pathological correlate to chronic pain, our focus will be on astrocytes and microglia. Astrocytes are star shaped glial cells, and were originally described by Virchow in 1859 as ‘nerve glue’. In response to injury, they become activated, increase in number and size, and change their pattern of gene expression such that a glial protein, glial fibrillary acidic protein (GFAP), is upregulated. Astrocytes undergo a process of proliferation, morphological changes, and enhancement of GFAP expression, termed astrogliosis (see chapter by Kalman), a common hallmark of many neurodegenerative diseases. Depending on the disease context, astrogliosis can be viewed as a beneficial event for promotion of neuronal survival by the production of growth factors and neurotrophins that support neuronal growth, or detrimental for neuronal function by formation of glial scars (Dong and Benveniste, 2001). Microglia, the CNS macrophages, are of mesodermal origin and display many similarities to cells of the monocytic lineage. As their name implies, they are the smallest of the glial cells. Neurons and the remaining types of glia differentiate from the neuroectoderm (Ling and Wong, 1993). In the CNS, microglia perform a vast number of immune-related duties (Hickey and Kimura, 1988). These ‘tiny dynamos’ underlie the

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neuroinflammatory response that occurs following damage to the CNS and the invasion of microorganisms. They seek dead and dying neurons and phagocytize them to rid the healthy tissue of such debris. In addition, there is evidence to suggest that microglia help guide neuronal changes in response to the environment, for example, dendritic branching and synaptic adaptations (Aschner et al., 1999). Microglial activation often precedes reactions of any other cell type in the brain. They respond not only to changes in the brain’s structural integrity, but also to very subtle alterations in their microenvironment, such as imbalances in ion homeostasis, which precede pathological changes that are detectable histologically. After transection of the facial nerve, it is initially microglia, not astrocytes, which proliferate, become hypertrophic and express several surface antigens (Kreutzberg, 1996). Proliferating perineuronal microglia detach afferent synaptic terminals from the motoneuron surface, beginning within a few days after facial nerve transection. Two to three weeks after axotomy, the processes of hypertrophic astrocytes take over the perineuronal position and replace the microglia. During axonal regrowth, reactive astrocytes thus maintain the state of afferent synaptic differentiation, initially accomplished by microglia (Kreutzberg, 1996). The demonstration of numerous receptors for CNS signaling molecules (adenosine triphosphate (ATP), neuropeptides, neurotransmitters) and neurotrophic factors on glia has suggested that these cells not only monitor but are also under the strict control of the neurochemical environment (see chapter by Hansson and Ro¨nnba¨ck). Given the heterogeneity of neuronal populations within distinct neuroanatomical regions, it has been proposed that the effects of the neurochemical environment on glia are sitespecific, and that this could account for differences in the degree of glia activation and inflammatory reactions in different CNS regions (Phillips and Lampson, 2000). Glial activation due to nerve injury or inflammation causes immune activation and release of different types of mediators in the CNS. Some of the mediators, such as growth factors assist in the repair mechanisms of the damaged CNS during pathological conditions. Others worsen the pathological conditions as a result of natural defense mechanisms aimed at overcoming the external stress. Glial-mediated immune activation is one of the driving forces involved in the pathophysiology of various disorders, including neurodegenerative diseases, stroke and autoimmune disorders of CNS. Activated glia are the sources of regulatory immune mediators such as cytokines, chemokines and their receptors (see chapter by Nakagawa and Schwartz). Glia also releases various lipid mediators, excitatory amino acids, nitric oxide (NO) (see chapters by Cornell-Bell and by Garcia and Baltrons), free radicals and neuroactive peptides upon their activation, which in turn modulate the neuronal responses (Kreutzberg, 1996). They may also function as antigen presenting cells (APCs), hence regulate T-cell mediated responses (Aloisi, 2001; Dong and Benveniste, 2001). From the available literature, it is evident that microglia appear to be more immunogenic (both with respect to cytokine production and antigen presentation) than other glial cells (Aloisi, 2001). On the other hand, activated astrocytes, may increase the neuronal sensitivity to glutamate and glutamate neurotoxicity during pathological conditions. This extended neurotoxicity could be due to decreased glutamate uptake or

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decreased capacity to convert glutamate to glutamine by activated astrocytes (McNaught and Jenner, 2000; Rossi et al., 2000).

3. Injury to sensory afferents activates glia: relationship to pain The conceptualization of pain processing, which began in the 17th century, when the French philosopher, Rene Descartes described the line-labeled, modality specific ‘specificity pain theory’, was stagnant until the gate control theory of pain was published in 1965 by Melzack and Wall (Melzack and Wall, 1965; Benini and DeLeo, 1999). Modern views of pain are now recognized to involve the interplay of sensory, evaluative, and affective components to create the pain experience. However, from sensation to perception, the pain pathways and the circuits that modulate the pain message are still classically viewed as entirely composed of neurons. This view is now changing. The contribution of glia in the development of synaptic plasticity, and the ability of mediators released from activated glia to induce pain, has drawn researchers to explore the possible role of glia in the pathophysiology of chronic pain. Damage to peripheral nerves and nerve roots produces intense microglial and astrocyte activation in the CNS (Fig. 1) (Gilmore and Skinner, 1979; Cova and Aldskogius, 1984; Gehrmann et al., 1991; Colburn et al., 1999; Hashizume et al., 2000a). Glial activation occurs specifically in the neuronal region containing central terminals and/or somas of the damaged nerves. The observation that glia are activated by peripheral nerve trauma is intriguing, since such injuries also cause exaggerated pain states, called neuropathic pain. As summarized in Table 1, various procedures employed in creating exaggerated pain states in animals have been reported to induce glial activation in the spinal cord. To date, immunocytochemistry (ICC) is the most widely employed method to characterize glial activation in these pathological conditions. Although numerous activation markers exist for glia, the most commonly examined are GFAP for astrocytes and complement receptor

Fig. 1. Immunocytochemistry showing microglial (OX-42) and astrocytic (GFAP) activation following L5-nerve transection and nerve root ligation at dorsal horn lumbar spinal cord of the rat.

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Table 1 Pain-enhancing procedures known to induce glial activation Procedure

Observed site of activation

Reference

Peripheral inflammation

Spinal cord

Peripheral nerve inflammation Spinal immune activation Spinal nerve transection Nerve root ligation Opioid tolerance/withdrawal-induced hyperalgesia

Spinal cord Spinal cord Spinal cord Spinal cord Spinal cord, posterior cingulate cortex and hippocampus Paraventricular and supraoptic nucleus

Meller et al., 1994; Watkins et al., 1997; Sweitzer et al., 1999; Bao et al., 2001 Watkins et al., 2001a,b Milligan et al., 2001a,b Colburn et al., 1999 Hashizume et al., 2000 Song and Zhao, 2001; Raghavendra et al., 2002

Streptozotocin-induced diabetes

Luo et al., 2002

type 3/CD11b for microglia. Fig. 2 illustrates the possible mechanisms by which glial activation leads to the development of exaggerated pain states following peripheral nerve injury or inflammation: mediators released during sensory transmission cause glial activation, which results in the release of cytokines, chemokines, excitatory amino acids, neuroactive peptides, prostaglandins and NO, which on one hand evoke pain and on the other lead to a vicious cycle by further enhancing glial activation. Many studies also demonstrated that the development of hyperalgesia (heightened response to a noxious stimulus) and allodynia (heightened response to a nonnoxious stimulus, like touch) correlated with spinal glial activation. However, the exact mechanism by which glial activation triggers the initiation and/or maintenance of behavioral hypersensitivity in these conditions is still to be answered. Using the sciatic chronic constrictive injury (CCI) neuropathic pain model in the rat, Garrison et al. (1994) observed a strong correlation between elevated spinal GFAP and the development of behavioral hypersensitivity in the same animals. Both these measures were blocked by an N-methyl-D -aspartate (NMDA) glutamate receptor antagonist, implicating a more causative vs. correlative action. Activation of glia by spinal injection of lipopolysaccharide (LPS) or subcutaneous formalin induces exaggerated pain responses, which were reduced by the functional disruption of spinal glia (Meller et al., 1994; Watkins et al., 1997). However, pharmacological studies have also shown that drugs or other procedures can block glial-induced exaggerated pain state without necessarily blocking the anatomical indices of glial activation (DeLeo and Colburn, 1999). This suggests that total inhibition of glial activation may not be an answer to overcome exaggerated pain states. Glia are indeed, intimately involved in the neuroimmune response following nerve injury or inflammation that results in persistent pain. However, the concept that all glia produce the same deleterious proteins and that ameliorating their function will have a beneficial outcome for persistent pain may be naı¨ve. The use of OX-42, an antibody that

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Fig. 2. Proposed cascade of glial events in producing behavioral and neuronal hypersensitivity following nerve injury or inflammation (SP, Substance P; EAA, Excitatory amino acids; ATP, Adenosine triphosphate; PGs, Prostaglandins; NO, Nitric oxide).

labels CR3/CD11b on microglia, is an excellent, sensitive marker of CNS trauma. However, it is not a specific marker for enhanced nociceptive transmission (Colburn et al., 1997; Sweitzer et al., 1999). With the aid of ICC, it has been demonstrated that only a subpopulation of microglia express specific surface antigens such as MHC II and CD4þ in response to peripheral nerve injury that results in persistent neuropathic pain (Sweitzer et al., 2002). Utilizing real time reverse transcriptase-polymerase chain reaction (RTPCR), we have recently demonstrated that other selective markers for microglia such as Mac-1, CD14 and Toll-like Receptor TLR4, are also upregulated in the lumbar spinal cord following spinal nerve transaction in rats. Such activation was observed 4 h after transection, and it was maintained over postoperative day 7 (unpublished data). Targeting these specific molecules may have a beneficial effect in the treatment of chronic pain following injury to peripheral nerves or nerve roots.

4. Glial activation enhances pain transmission It is of interest to know how glia are activated following peripheral nerve injury or inflammation. As summarized in Fig. 2, it is possible that neurotransmitters (e.g., substance P, excitatory amino acids, ATP, NO, prostaglandins and cytokines), released

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from activated small diameter sensory afferents upon nerve damage, infection or inflammation, activate glia which subsequently leads to the development of persistent pain state (Watkins et al., 2001a,b). Alternatively, glia may be the first cells to sense nerve damage and thus initiate the neuronal cascade (Rutkowski and DeLeo, 2002). Glial cells outnumber neurons in the CNS (see chapter by Wolff et al.), so its activation could be expected to have a significant impact on the function of neurons in the area. Activated glia release a wide range of substances that may induce pathological persistent pain. The substances released by each of these cell types (microglia or astrocytes) are not identical, as each cell type has unique characteristics and functional capabilities. Mediators released by activated glia (summarized in Fig. 3) are known to excite primary afferent and interneurons (DeLeo and Yezierski, 2001; Watkins et al., 2001a,b). Of these, pro-inflammatory cytokines will be the focus of a separate section, below. Other mediators, such as substance P, glutamate, aspartate, NO and prostaglandins released by activated glia have been the focus of intense study by pain researchers for more then two decades. The fact that glia are now known to dramatically regulate these key factors in the neuronal microenvironment suggests that at least some of the existing literature on these mediators’ involvement in exaggerated pain states may actually reflect an unrecognized contribution of glia to the effects observed. Activated glia are ideal candidates to drive exaggerated pain states. This is because microglia and astrocytes form positive feedback loops in which substances released by one cell type further activates the others, causing prolonged release of excitatory substances from these cells (Fig. 2). Further, mediators released by activated glia can act in an autocrine/paracrine fashion, and are therefore ideal for activating both glia and neurons in their general vicinity (Watkins et al., 2001a,b). Apart from releasing mediators responsible

Fig. 3. Neural and immunoregulatory mediators produced by activated glia. (IL-1ra, Interleukin-1 receptor antagonist; TNF, Tumor necrosis factor; TGF, Transforming growth factor; NOS, Nitric oxide synthase; NO, Nitric oxide; EAA, Excitatory amino acids; MCP, Monocyte chemoattractant protein; MIP, Macrophage inflammatory protein; IP-10, Interferon gamma inducible protein 10; RANTES, Regulated upon activation normal T cell expressed and secreted; MDC, Macrophage derived chemokines).

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for inducing pain states, activated glia also lose their ability to take up glutamate (McNaught and Jenner, 2000; Rossi et al., 2000), which might enhance accumulation of excess glutamate around pain transmitting neurons, which can induce exaggerated pain states. From pharmacological studies, it is now evident that glial activation is necessary and sufficient to produce enhanced pain. Disruption of spinal glial function reduces exaggerated pain created by spinal nerve transection (Sweitzer et al., 2001a,b), subcutaneous immune challenge (Meller et al., 1994), subcutaneous inflammation (Watkins et al., 1997), nerve inflammation (Watkins and Maier, 2000) and spinal immune activation (Milligan et al., 2000). In addition, the development of morphine tolerance and opioid withdrawal-induced hyperalgesia is also associated with spinal glial activation (Song and Zhao, 2001; Raghavendra et al., 2002). Here, glia were found to be activated by chronic, but not by acute morphine treatment. This suggests that glial activation due to morphine treatment is an adaptive change observed after long-term treatment. Of particular interest, the disruption of glial activation associated with chronic morphine treatment attenuated the development of opioid tolerance, and opoioid withdrawalinduced hyperalgesia (Song and Zhao, 2001; Raghavendra and DeLeo, 2002).

5. Brain cytokines and pain Increasing evidence provides support for the pivotal role of cytokines as chemical signals/neuromodulators in the CNS in normal and under pathological conditions. Besides their peripheral origins, cytokines are also produced in the brain by astrocytes, microglial cells, endothelial cells, meningeal macrophages and probably also by neurons (Koening, 1991). The synthesis of cytokines in the CNS has been documented by an increase in both their activity and gene expression following peripheral administration of endotoxin, formalin, complete Freund’s adjuvant, which occur after spinal nerve inflammation, peripheral neuropathy, central radiculopathy and during morphine withdrawal-induced hyperalgesia (Watkins and Maier, 2000; Hashizume et al., 2000a; DeLeo and Yezierski, 2001; Raghavendra et al., 2002). Receptors and binding sites for these cytokines have also been widely described in the CNS (Faggioni et al., 1995). Brain-derived cytokines cause diverse effects on the homeostatic functions controlled in the hypothalamus, such as thermoregulation, sleep, feeding, neuroendocrine secretion (see chapter by Mercier and Hatton), autonomic nervous activities and peripheral immune reactivity (Rothwell and Hopkins, 1995). These responses are almost identical to the ‘sickness symptoms’ observed during peripheral infection. Recent studies, including ours, have revealed that altering the central cytokine response, particularly IL-1b, IL-6 and tumor necrosis factor a (TNFa), modulates nociceptive responses, suggesting a seminal role of cytokines in sensitized neuronal activity. IL-1 has multiple roles in inflammation and in the immune response (see chapter by Benveniste). Of the two forms of IL-1, IL-1a and IL-1b, IL-1b is the predominant molecule in brain tissue. IL-1b has immunogenic activity, causing release of prostaglandins, IL-6, and IL-8 from monocytes and endothelial cells. IL-1 mediates its activities through receptors (R) on the cell membrane, either IL-1R type 1 (IL-1a) or type

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II (IL-1b) (DeLeo and Colburn, 1999). Enhanced production of IL-1b in the CNS was observed following nerve injury or inflammation, and enhanced pain states associated with these procedures were abolished by an IL-1 receptor antagonist (IL-1ra) (Cunha et al., 2000; Sweitzer et al., 2001a; Milligan et al., 2001a). Intracerebroventricular injection of IL-1b induces hyperalgesia and enhances nociceptive neuronal responses of trigeminal nucleus caudalis in rats (Oka et al., 1993, 1994). Relevant to nociceptive processing, IL-1b facilitates afferent sensory transmission in the somatosensory cortex (Won et al., 1995). It is also likely that certain cytokines, like IL-1b, are involved in synaptic plasticity and hyperexcitability, as suggested by their dose-dependent capacity to produce long-term potentiation in slice preparations that physiologically correlates to spinal sensitization (Rothwell, 1991; Patterson and Nawa, 1993). Recent studies have shown that the antianalgesic activity and hyperalgesic activity of dynorphin are mediated through brain IL-1b (Laughlin et al., 2000; Rady and Fujimoto, 2001). IL-1b is also responsible for the deficiency of morphine-induced analgesia in diabetic mice (Gul et al., 2000). IL-6, along with IL-1 and TNF, is one of the mediators of the acute phase response of inflammation. In addition to its role in the acute inflammatory response, it also plays an important part in host defense and chronic immune responses. Following axotomy or a freeze lesion of a spinal nerve, an increase in IL-6 mRNA is one of the earliest changes observed in the spinal cord and dorsal root ganglia (Kiefer et al., 1993; Murphy et al., 1995; Arruda et al., 1998). In addition, IL-6 message is induced in Schwann cells distal to a crush injury (Bolin et al., 1995). Our laboratory has demonstrated that an increase in spinal IL-6-like immunoreactivity expression following sciatic cryoneurolysis is closely correlated with the onset and duration of mechanical allodynia (DeLeo et al., 1996). In addition, intrathecally administered human recombinant IL-6 elicits touch evoked allodynia in normal rats (DeLeo et al., 1996), and anti-IL-6 antibody attenuates peripheral nerve injury-induced mechanical allodynia (Arruda et al., 2000). Recently, it has been demonstrated that in the chronic constriction injury model in the rat, cutaneous heat and pressure hypersensitivity, observed in the wild type control, could not be elicited in mice with a null mutation of the IL-6 gene (Murphy et al., 1999). IL-6 is also reported to modulate the analgesic actions of opioids (Bianchi et al., 1999). It has also been demonstrated that the neurotransmitter norepinephrine (NE) dose-dependently induces astrocytes to produce IL-6, an effect, which can be blocked by adrenergic receptor antagonists (Norris and Benveniste, 1993). These data have relevant implications to human neuropathic pain, one component of which is the phenomenon of sympathetically maintained pain, or Complex Regional Pain Syndromes. The tumor necrosis factors include TNFa (cachectin) and TNFb (lymphotoxin). These tumor necrosis factors produce a variety of similar, but not identical, biological effects. The TNF are ‘pro-inflammatory’ cytokines, since they have a role in initiating (along with IL-1) the cascade of other cytokines and growth factors that participate in the immune inflammatory response. TNFa is implicated in the development of persistent pain through its actions in the periphery and in the CNS (Covey et al., 2000). Like IL-1, TNFa is implicated in enhanced pain responses following administration of illness-inducing substances (Watkins et al., 1994; Raghavendra et al., 2000). Exogenous administration of TNFa to nerve roots induces abnormal discharges in dorsal horn neurons (Onda et al., 2002). Intrathecal administration of TNFa also enhances dorsal horn neuronal responses,

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including the acute responses to C-fiber stimulation, wind-up, post-discharge, and hyperalgesia (Reeve et al., 2000). We have demonstrated that glial expression of TNFa has a role in the generation of neuropathic pain using a TNFa over-expressing mouse (DeLeo et al., 2000). In addition, inhibition of endogenous TNFa by anti-TNFneutralizing antibodies, TNF-binding protein (TNF-bp) and soluble tumor necrosis factor receptor (sTNFR) attenuates hyperalgesia and allodynia-induced by peripheral inflammation and mononeuropathy (Coelho et al., 2000; Sommer et al., 2001; Sweitzer et al., 2001a). Studies have revealed that cytokines may affect neuronal excitability either directly or indirectly via an alteration of neuron –glia interaction. TNFa induces an increase in the free intracellular calcium concentration and a depolarization in astrocytes, with the consequence of disturbing voltage-dependent glial function, such as local ion concentrations and glutamate uptake (Koller et al., 1996). The neurotoxic effects of TNFa may be, in part, due to its ability to inhibit glutamate uptake by astrocytes, which in turn may result in excitotoxic concentrations of glutamate in synapses (Fine et al., 1996). This glutamate enhancing action may also be of importance in chronic pain following nerve injury. Similarly, TNFa induces substance P, a major pain mediator, in sympathetic ganglia (Ding et al., 1995). The quest to understand the neuroimmunological events that occur during persistent pain states is to determine the cell types within CNS, which are involved, and the cellular and molecular mechanisms, which are responsible for increases in spinal cytokines. In the CNS, transcriptional activation of nuclear factor kappa B (NFkB), mitogen activated protein (MAP) kinases or protein kinase C (PKC) signaling pathways are speculated to be involved in the upregulation of cytokines following peripheral nerve injury or inflammation (Sakaue et al., 2001; Ma and Quirion, 2002; Milligan et al., 2001b; Raghavendra et al., 2002). ICC and in situ hybridization (ISH) techniques allows the visualization of cytokine sources within the CNS. Activated glia, similar to other immune cells, releases the pro-inflammatory cytokines IL-1, IL-6 and TNFa. Both glia and neurons express receptors for these. Earlier, we have reported the astrocytic expression of TNFa following peripheral neuropathy (DeLeo and Colburn, 1999). IL-1 immunoreactivity is observed exclusively in astrocytes following intrathecal HIV-1 glycoprotein gp120induced exaggerated pain states (Milligan et al., 2001a). Using ISH and ICC methods, Bao et al. (2001) showed an increase in mRNA and protein levels of IL-1b, IL-6 and TNFa in the spinal cord of arthritic rats, and this increase was present in the reactive astrocytes and microglia. These studies highlight the importance of glial-released cytokines in the initiation and maintenance of persistent pain states.

6. Glia: a target for therapeutic modulation of persistent pain Chronic pain can occur after infection, inflammation or due to nerve or nerve root injury under diverse pathological conditions. Under such conditions, sensory processing in the affected body region becomes grossly abnormal. Despite decades of research, currently available drugs largely fail to control such pain. This failure may lie in the fact that such drugs were designed to target neurons rather than immune or glial cells. As previously

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discussed, glial activation and subsequent mediator release are strongly implicated in the creation and maintenance of exaggerated pain states. Hence, targeting glia under pathological conditions may culminate in the development of novel medications to treat such persistent pain states. Some of the targets and agents, which showed inhibition of glial activation and attenuated pain behaviors in animal models of persistent pain are discussed below. 6.1. Immune modulators/cytokine inhibitors One example of this class is leflunomide. Leflunomide is currently used for the treatment of rheumatoid arthritis, a systemic autoimmune inflammatory disease. The active metabolite of leflunomide (A77 1726) inhibits spinal glial activation after a peripheral nerve transection that results in mechanical allodynia. In addition, central A77 1726 administration also decreased spinal expression of MHC class II, which implicates a role of neuroimmune activation in the generation and maintenance of neuropathic pain (Sweitzer and DeLeo, 2002). Other immunomodulators such as methotrexate and cytokine antagonists attenuated behavioral hypersensitivity but at the doses studied, failed to inhibit GFAP and OX-32 expression associated with nerve injury (Hashizume et al., 2000b; Sweitzer et al., 2001a). This underscores both the importance of utilizing specific terminology, when describing glial findings and the fact that glial activation should not be defined by labeling only two surface antigen markers. 6.2. Glial-targeted metabolic inhibitors Fluorocitrate is a currently available, but quite toxic glial metabolic inhibitor. In an inflammatory pain model, fluorocitrate attenuated thermal and mechanical hyperalgesiainduced by zymosan (Meller et al., 1994). Also, the coadministration of fluorocitrate with morphine reversed the development of analgesic tolerance to the morphine (Song and Zhao, 2001). However, its action on neuronal cells at higher doses, the nonselective suppression of glial cells and the toxicity make this compound unsuitable for clinical use. 6.3. Xanthine derivatives Examples of this class of drugs include pentoxyfylline and propentofylline. These molecules suppress glial activation, inhibit release of pro-inflammatory cytokines and production of oxygen free radicals in glial cells, augment anti-inflammatory cytokine response, enhance glial release of nerve growth factors, enhance uptake of extracellular excitatory amino acids by glia, and increase extracellular adenosine content by the inhibition of glial adenosine transporters (Schubert et al., 2000). In a neuropathic pain model, centrally or systemically administered propentofylline attenuated mechanical allodynia (Sweitzer et al., 2001b) and thermal and mechanical hyperalgesia (Raghavendra and DeLeo, 2002). Chronic propentofylline treatment also spared morphine analgesia in neuropathic rats and reversed development of morphine tolerance and withdrawal-induced hyperalgesia in rats (Raghavendra and DeLeo, 2002). Attenuation of pain related

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behaviors by propentofylline is associated with the inhibition of glial activation and subsequent pro-inflammatory immune activation in the lumbar spinal cord (Sweitzer et al., 2001b; Raghavendra and DeLeo, 2002). Pentoxifylline reduces post-injury hyperalgesia in rats and post-operative pain in patients, and lowers the opioid requirement in the early post-operative period. This decrease in pain behaviors is correlated with a low level of TNFa in the serum of the pentoxyfylline treated group (Wordliczek et al., 2000). Pentoxifylline also inhibits the apoptotic death of astrocytes (Takuma et al., 2001). These compounds cross the blood – brain barrier and have a wide therapeutic window of safety, which enhance their potential clinical application. They have also successfully passed human clinical trials for use in nonpain conditions (Mielke et al., 1998). 7. Concluding remarks This review highlights the role of astrocytes and microglia as essential players in the induction of chronic pain following traumatic nerve injury, infection or peripheral inflammation. Furthermore, spinal glial activation and resultant pro-inflammatory cytokine release have been shown to mediate intriguing pain phenomena, expression of morphine tolerance and morphine withdrawal-induced hyperalgesia. Recognizing the distinct morphological and neurochemical responses of glial cells after stress stimuli, as well as their intimate interaction with neurons, directly implicates them in the CNS plasticity that is the underpinning of chronic pain. The challenge in the quest to discover novel, selective therapeutics that modulate both beneficial and detrimental glial actions will be the ability to balance the reductionist experimental approach with the understanding of the complexity of pain processing. Herein, we need to embrace mathematical concepts such as complex nonlinear systems (Seely and Christou, 2000), in which we recognize the high degree of connectivity and interdependence between variables, and resist the temptation to selectively inhibit one molecule in the dynamic interplay of the neuroimmune response of the CNS to injury. The single molecule inhibition approach has not completely eliminated behavioral sensitivity in animal studies of persistent pain and also, and perhaps more importantly, has not been fruitful in many clinical trials. For this reason, enhanced success in the prevention and potentially the treatment of refractory chronic pain conditions may depend upon a combination of selective immunomodulatory agents administered at opportune time periods after injury. References Aloisi, F., 2001. Immune function of microglia. Glia 36, 165 –179. Arruda, J.L., Colburn, R.W., Rickman, A.J., Rutkowski, M.D., DeLeo, J.A., 1998. Increase of interleukin-6 mRNA in the spinal cord following peripheral nerve injury in the rat: potential role of IL-6 in neuropathic pain. Mol. Brain Res. 62, 228 –235. Arruda, J.L., Sweitzer, S., Rutkowski, M.D., DeLeo, J.A., 2000. Intrathecal anti-IL-6 antibody and IgG attenuates peripheral nerve injury-induced mechanical allodynia in the rat: possible immune modulation in neuropathic pain. Brain Res. 879, 216–225.

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Pathogenic role of glial cells in Parkinson’s disease S. Przedborskia,* and James E. Goldmanb a

Departments of Neurology and Pathology and Center for Neurobiology and Behavior, Columbia University, New York, NY, USA p Correspondence address: Tel.: þ1-212-305-1540; fax: þ1-212 305-5450. E-mail: [email protected](S.P.) b Division of Neuropathology, Department of Pathology and Center for Neurobiology and Behavior, Columbia University, New York, NY, USA

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction Brain inflammation and neurodegenerative disorders Initiation of glial activation in PD Astrocytic and microglial alterations in PD Astrocytic and microglial alterations in parkinsonian syndromes Astrocytic and microglial alterations in experimental models of PD The protective effect of astrocytes and microglia in PD The deleterious role of astrocytes and microglia in PD Concluding remarks

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). The loss of these neurons is often associated with nonneuronal pathology such as activated microglial cells and reactive astrocytes. Although microglial and astrocytic alterations are secondary to the neuronal loss, they may still participate in the degenerative process. Indeed, glial cells may be the source of trophic factors and can protect against reactive oxygen species and glutamate.Alternatively, they can also mediate a variety of deleterious events related to the production of pro-oxidant reactive species, and proinflammatory prostaglandin and cytokines. In this chapter, we will review the microglial and astrocytic changes seen in various parkinsonian syndromes including PD per se as well as in animal models of PD. We will also discuss the question of the potentially protective and deleterious effects of glial cells in PD and discuss how those factors may contribute to the pathogenesis of this common neurodegenerative disorder. Advances in Molecular and Cell Biology, Vol. 31, pages 967–982 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disorder characterized mainly by resting tremor, slowness of movement, rigidity, and postural instability (Fahn and Przedborski, 2000) and associated with a dramatic loss of dopamine-containing neurons in the substantia nigra pars compacta (SNpc) (Hornykiewicz and Kish, 1987). Currently, the number of PD patients has been estimated at , 1,000,000 in North America with , 50,000 newly affected individuals each year (Fahn and Przedborski, 2000). To date, the most effective treatment for PD remains the administration of a precursor of dopamine, L -DOPA, which, by replenishing the brain in dopamine, alleviates almost all PD symptoms. However, the chronic administration of L -DOPA often causes motor and psychiatric side effects, which may be as debilitating as PD itself (Fahn, 1989). Moreover, as of yet there is no evidence that L -DOPA therapy can impede the neurodegenerative process in PD. There is thus an urgent need to acquire a better understanding of both etiologic (i.e., causes) and pathogenic (i.e., mechanisms of cell death) factors implicated in PD’s neurodegenerative process, not only to prevent the disease, but also to develop therapeutic strategies aimed at halting its progression. While etiological factors (e.g., mutant a-synuclein, mutant parkin, and several others that remain to be identified) are presumably pivotal in the initiation of the demise of SNpc dopaminergic neurons in PD, it has been increasingly recognized that additional factors underlie the propagation of the neurodegenerative process. To elucidate such factors, and consequently to develop new therapies, the neuropathology of PD has been revisited in search of abnormalities that could shed light on these additional pathogenic culprits. Relevant to this is the observation that the neurodegenerative process, which culminates in neuronal death, is often associated with morphological and functional alterations in glial cells in both PD and experimental models of PD (McGeer et al., 1988; Forno et al., 1992; Sheng et al., 1993; Kohutnicka et al., 1998). In this review, we will examine the role of glial cells in PD. First, we will review the microglial and astrocytic changes seen in various parkinsonian syndromes including PD per se as well as in animal models of PD. We will then discuss the question of the potentially protective and deleterious effects of glial cells in the SNpc of PD and examine how those factors may contribute to the pathogenesis of this common neurodegenerative disorder. 2. Brain inflammation and neurodegenerative disorders The inflammatory reaction of the central nervous system (CNS) consists of multiple components and should not be simply equated with the infiltration of brain tissues by blood-derived immune cells, since such infiltration only represents one particular type of inflammatory response often called ‘exudative inflammation.’ According to Wyss-Coray and Mucke (2002), triggers of inflammation recruit the innate immune system whose components in the CNS include microglia and astrocytes. Those cells, which represent the focus of this review, are strongly activated in most neurodegenerative diseases and produce a variety of inflammatory mediators and other injury response factors (Eddleston and Mucke, 1993; Mennicken et al., 1999; Nguyen et al., 2002).

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Before summarizing the changes in astrocytes and microglial cells in PD and related conditions, it may be worth discussing briefly the meaning of ‘gliosis’ sometime also called ‘reactive astrocytosis’ which the reader will often encounter in perusing the literature on inflammation in neurological diseases. Gliosis, which typically refers to scarring produced by astrocytes, is often loosely used to define no more than increased immunoreactivity for the intermediate filament, glial fibrillary acid protein (GFAP), a pathological finding common to a large number of neurological disorders. The pervasive use of this term, especially in old landmark studies, renders the interpretation of nonneuronal pathological changes challenging, as the limited range of techniques employed does not always allow an appropriate meta-analysis of the data. For instance, on many occasions it is unclear whether gliosis, evidenced by increased GFAP immunostaining, meant simply increased stainability of the tissue, increased numbers of astrocytes, increased size of astrocytes, or a combination of all of the above. Nor is it always possible to comment on the status of other glial cells such as oligodendrocytes and microglia. Therefore, while all efforts will be made to spell out what those studies actually show, investigators interested in the question of glial alterations in PD are thus encouraged to revisit it by using more modern techniques. Yet, as indicated by Wilkin and Knott (1999), oligodendrocytes, which are involved in axonal myelination, have so far not been implicated in PD, whereas both astrocytes and microglial cells have, which is why our discussion will be centered on only these latter two glial cell types. Astrocytes are essential in the normal, undamaged adult brain, for the homeostatic control of the neuronal extracellular environment (Wilkin and Knott, 1999). Conversely, little is known about microglial functions in the normal adult brain. Following an injury to the brain, astrocytes and microglial cells undergo various phenotypic changes that enable them to both respond to and to play a role in the pathological processes (Eddleston and Mucke, 1993; Gehrmann et al., 1995). Microglial activation is characterized by: (i) proliferation; (ii) increased or de novo expression of marker molecules such as major histocompatibility complex antigens; (iii) migration; and (iv) eventually transformation into a macrophage-like appearance (Banati et al., 1993). In neurodegenerative diseases, both microglia and astrocytes can become activated, producing an array of inflammatory mediators and taking on phagocytic functions (Eddleston and Mucke, 1993; Aldskogius et al., 1999; Wyss-Coray and Mucke, 2002). While some glial factors are produced specifically by reactive astrocytes or activated microglia, others, such as interleukin-1b (IL-1b) can apparently be produced by both (Rothwell, 1999). Also, some authorities suggest that microglia are responsible for more generalized phagocytosis involving activation of the complement cascade, whereas astrocytes are implicated in circumscribed phagocytic processes, such as the removal of individual synapses (Wyss-Coray and Mucke, 2002). The most efficient and aggressive phagocytes in the CNS are likely the round or ameboid microglia, which express high levels of macrophage markers, whereas ramified microglia have little phagocytic activity (Kreutzberg, 1996). In neurodegenerative diseases many microglia cells show a ramified morphology, although they express activation markers (Dickson et al., 1993), suggesting that they might be nonphagocytic (DeWitt et al., 1998). Astrocytes may also participate in

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phagocytosis, either directly (Shaffer et al., 1995) or by regulating microglial activities (DeWitt et al., 1998). 3. Initiation of glial activation in PD Most data available to date support the notion that activation of microglia and astrocytes in PD result from the perception by the defense mechanisms of ongoing neuronal perturbations, meaning that microglial and astrocytic activation within injured areas is not a primary event but rather a result of the neuronal pathology. However, the nature of the signals emanating from ‘sick’ neurons, which ultimately lead to the glial responses remain to be determined. Hypothetically, in PD, as in other neurodegenerative disorders, the functional link between neuronal perturbations and glial responses can range from subtle alterations in the microenvironment, such as imbalances in ion homeostasis, to gross leakage of the intracellular content from neighboring diseased neurons (Kreutzberg, 1996). The concept that misshaped neuronal proteins and protein aggregates could also trigger glial activation is particularly appealing in the context of PD, given the fact that mutations in the genes encoding for parkin and ubiquitin C-terminal hydrolase L1 (two enzymes of the unbiquitin/proteasome pathway) and for a-synuclein (a main component of the intraneuronal proteinacious inclusions Lewy bodies (Spillantini et al., 1998) lead to familial PD (Lim et al., 2002). Also relevant to PD is the demonstration that treatment of neurons with proteasome inhibitors elicited accumulation of ubiquinated proteins and increased neuronal production of cyclooxygenase type 2 (Cox-2) and prostaglandins (Rockwell et al., 2000), capable of activating glial cells. This suggests that glial responses can be elicited not only by extracellular protein deposits, but also by the intracellular accumulation of abnormal proteins. 4. Astrocytic and microglial alterations in PD In normal brains, resting microglia appear somewhat unevenly distributed (Lawson et al., 1990), in that the density of microglia is remarkably higher in the substantia nigra (SN) compared to other midbrain areas and brain regions such as the hippocampus (Kim et al., 2000). This observation, combined with the finding that SN neurons are much more susceptible to activated microglial-mediated injury (Kim et al., 2000) lend support to the idea that microglia may play an especially meaningful role in PD. Unlike microglia, astrocytes in undamaged brain display a fairly even distribution except in midbrain where the estimated density of GFAP-positive cells is heterogeneous, as shown by Hirsch et al. (1999). More importantly, these authors indicate that the density of GFAP-positive cells is moderate in the midbrain areas known to be most severely affected in PD (e.g., SNpc) and high in those least affected (e.g., gray substance). The nigrostriatal pathway is the dopaminergic system most affected in PD. The neurons that form this pathway have their cell bodies in the SNpc and their nerve terminals in the striatum. Of particular relevance to this review is the finding that the loss of dopaminergic neurons in postmortem parkinsonian brains is associated with marked microglial and, to a lesser extent, astrocytic alterations (McGeer et al., 1988; Forno et al., 1992;

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Banati et al., 1998; Mirza et al., 2000). However, while the damage to dopaminergic elements is consistently more severe in the striatum than in the SNpc, the described changes in microglia and astrocytes are consistently more important in the SNpc than in the striatum (McGeer et al., 1988). This discrepancy can be explained by the fact that dopaminergic structures which are degenerating are in dominance in the SNpc, whereas they are in the minority in the striatum, as dopaminergic synapses represent less than 15% of the entire pool of synapses in the striatum (Tennyson et al., 1974; Pickel et al., 1981). Aside from this topographical difference, the importance of the astrocytic and microglial changes in parkinsonian brains is also very different. The SNpc of many but not all postmortem PD cases exhibits, at best, slightly more astrocytes based on counts of GFAPand metallothionein I/II-positive cells (Mirza et al., 2000). However, the vast majority of these astrocytes exhibit a normal morphology with thin and elongated processes, and only rarely exhibit a reactive morphology with hypertrophic cell body and short processes (Forno et al., 1992; Mirza et al., 2000). Among the other astrocytic pathologic features seen in PD, the count of a-synuclein positive-inclusions within SNpc astrocytes correlates positively with the severity of SNpc dopaminergic neuronal loss (Wakabayashi et al., 2000); whether these inclusions have any pathogenic significance is still unknown. Unlike the astrocytic alterations, the microglial changes in PD are consistently more dramatic (McGeer et al., 1988; Banati et al., 1998; Mirza et al., 2000). Microscopically, several microglia exhibit robust immunoreactivity for various microglial markers and have hypertrophic elongated processes (McGeer et al., 1988; Banati et al., 1998; Mirza et al., 2000). Quantitative analysis shows that the number of SNpc activated microglia, as evidenced by CR3/43 and ferritin immunostaining, is 14– 33 times higher in PD than in controls (Mirza et al., 2000). Moreover, activated microglial cells are predominantly found in close proximity to free neuromelanin in the neuropil, and to remaining neurons, onto which they sometimes agglomerate to produce an image of neuronophagia (McGeer et al., 1988).

5. Astrocytic and microglial alterations in parkinsonian syndromes Among the various parkinsonian syndromes, PD does not have a monopoly on the association of nigrostriatal neurodegeneration and glial alterations (Oppenheimer and Esiri, 1997). In most of these syndromes, the magnitude of SNpc DA neuronal loss is variable, Lewy bodies are often lacking, and, more importantly, neurodegenerative changes extend well beyond the SNpc and the dopaminergic neurons. In addition, in most publications, authors also make mention of the presence of gliosis, usually to mean more glial fiber bundles, identified by, for example Holzer Stain; more small nonneuronal cells, visualized by hematoxylin – eosin stain; or more GFAP-positive cells. When present, these changes are noted at the level of the SNpc as well as at the level of the other affected regions of the brain. Even in the initial reports on progressive supranuclear palsy (Steel et al., 1964) and striatonigral degeneration (Adams and Salam-Adams, 1986), gliosis, as just defined, was already recognized as a prominent feature of the pathological changes seen in these syndromes. Likewise, histological examination revealed similar nonneuronal

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alterations in most of the familial forms of parkinsonian syndromes, whether they are linked to unknown (Dwork et al., 1993) or known gene defects such as mutations in parkin (Ishikawa and Takahashi, 1998; Hayashi et al., 2000) or in a-synuclein (Hishikawa et al., 2001). To date, however, while the occurrence of gliosis in all of these conditions is clearly indicated, unlike in PD, no comprehensive qualitative or quantitative analysis of these alterations is available, and simple information such as whether the reported gliosis refers to astrocytes, microglia, or to both is in most instances lacking.

6. Astrocytic and microglial alterations in experimental models of PD The neuropathological picture found in experimental models of PD is very similar to that found in PD itself. Among these models (Beal, 2001), the 6-hydroxydopamine (6OHDA) and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models are the ones that have been the most extensively used; thus, not surprisingly these are the ones for which we have the largest amount of neuropathological information. Remarkably, alteration in astrocytes and microglia found in rodents following the administration of 6OHDA (Stromberg et al., 1986; Akiyama and McGeer, 1989; Sheng et al., 1993; Przedborski et al., 1995; He et al., 1999; Nomura et al., 2000; Rodrigues et al., 2001) is fairly comparable to that seen following the administration MPTP (see below). Given this fact, and given that neuropathological data in humans are only available for the MPTP toxin, our discussion on the changes in astrocytes and microglia in experimental models of PD will be restricted to the MPTP model. Please refer to Przedborski and Vila (2003) for a comprehensive review of the MPTP model. In the few MPTP-intoxicated individuals who came to autopsy, postmortem examination revealed a paucity of SNpc dopaminergic neurons accompanied by the presence of numerous small cells intensely immunoreactive to either the astrocytic marker GFAP or to the microglial marker CR3/43 and exhibiting the morphological characteristics of reactive astrocytes and activated microglia (Langston et al., 1999); images of neuronophagia were also often seen in these SNpc specimens (Langston et al., 1999). Although no formal quantification has been performed, the authors noted that the greater the abundance of GFAP- and CR3/43-positive cells, the more profound the loss of dopaminergic neurons (Langston et al., 1999). The aforementioned findings indicate that the astrocytic and microglial changes in the SNpc are fairly similar between humans with PD and those intoxicated by MPTP, although based on the neuropathological description provided by the authors, more astrocytes appear to adopt a reactive morphology in the latter (Langston et al., 1999). From a neuropathological standpoint, microglial activation and especially neuronophagia is indicative of an active, ongoing process of cell death. While this contention is consistent with the fact that PD is a progressive condition, it challenges the notion that MPTP produces a ‘hit-and-run’ type of damage and rather suggests that a single acute insult in the SNpc could set in motion a self-sustaining cascade of events with long-lasting deleterious effects. However, neither postmortem studies in PD nor in MPTP-intoxicated individuals can provide information about the temporal relationship between the loss of dopaminergic neurons and the glial reaction in the SNpc.

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The situation is quite different in rodents. For instance, mice injected with MPTP and killed at different time points thereafter show that the appearance of reactive astrocyte parallels the destruction of dopaminergic structure in both the striatum and the SNpc, and that GFAP expression remains upregulated even after the main wave of neuronal death has passed (Czlonkowska et al., 1996; Kohutnicka et al., 1998; Liberatore et al., 1999). These findings suggest that, in the MPTP mouse model (Przedborski et al., 2000a), the astrocytic reaction is consequent to the death of neurons and not the reverse. This is supported by the demonstration that blockage of 1-methyl-4-phenylperydinium (MPPþ, the active metabolite of MPTP (Przedborski et al., 2000a)) uptake into dopaminergic neurons not only completely prevents SNpc dopaminergic neuronal death but also GFAP upregulation (O’Callaghan et al., 1990). Remarkably, activation of microglial cells, which is well documented in the MPTP mouse model (Czlonkowska et al., 1996; Kohutnicka et al., 1998; Liberatore et al., 1999; Dehmer et al., 2000), occurs much earlier than that of astrocytes, and more importantly reaches a maximum before the peak of dopaminergic neurodegeneration (Liberatore et al., 1999). In light of the MPTP data presented above, it can be surmised that the response of both astrocytes and microglial cells to the demise of SNpc dopaminergic neurons clearly occurs within a time frame allowing these glial cells to participate in the neurodegeneration of the nigrostriatal pathway in the MPTP mouse model and possibly in PD. In the following sections, we will examine through which beneficial or detrimental mechanisms astrocytes and microglia can possibly play a role in the neurodegenerative process in PD.

7. The protective effect of astrocytes and microglia in PD As mentioned above, the astrocytic and microglial response to injury may in fact have beneficial effects, which, in the case of PD, could attenuate neurodegeneration. Among the different mechanisms by which glial-derived neuroprotection could be mediated, the first that comes to mind involves the production of trophic factors. It is well recognized that various types of glial cells in mature and, to a greater extent, in immature tissues can provide a host of trophic factors that are essential for the survival of dopaminergic neurons. Among these, glial-derived neurotrophic factor (GDNF), which is released by reactive astrocytes (Schaar et al., 1993) and by activated microglia following a mechanical lesion of the striatum (Batchelor et al., 2000) seems to be the most potent factor supporting SNpc dopaminergic neurons during their period of natural, developmental death in postnatal ventral midbrain cultures (Burke et al., 1998). It is also worth emphasizing that GDNF induces dopaminergic nerve fiber sprouting in the injured rodent striatum (Batchelor et al., 1999), and that this effect is markedly decreased when GDNF expression is inhibited by intrastriatal infusion of antisense oligonucleotides (Batchelor et al., 2000). Furthermore, GDNF, delivered either by infusion of the recombinant protein or by viral vectors, has been shown to attenuate dopaminergic neuronal death and to boost dopaminergic function within injured neurons in various animal species exposed to the neurotoxins 6-OHDA or MPTP (Gash et al., 1996; Choi-Lundberg et al., 1997; Kordower et al., 2000; Eberhardt et al., 2000). Unfortunately, in humans with PD, much less enthusiastic results have been obtained thus far, in that repetitive intraventricular

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injections of recombinant GDNF to one advanced parkinsonian patient were poorly tolerated and failed to halt the progression of the disease (Kordower et al., 1999). Brainderived neurotrophic factor (BDNF) is another trophic factor that can also be released by reactive astrocytes (Rubio, 1997; Stadelmann et al., 2002) and by activated microglia (Batchelor et al., 1999; Stadelmann et al., 2002) and that can support the survival and process outgrowth of dopaminergic structures in the striatum (Batchelor et al., 1999). The usefulness of BDNF as a potential neuroprotective factor has been well documented in several experimental models of PD (Spina et al., 1993; Frim et al., 1994; Levivier et al., 1995), but whether it can produce similar beneficial effects in human PD remains to be demonstrated. Although we did not discuss the question of oligodendrocytes in PD in this review, it is important to emphasize that this specific type of glial cell has emerged as a provider of various potent trophic factors (Du and Dreyfus, 2002). Even though it remains unclear how trophic factor production is regulated in these cells, especially in pathological situations (Du and Dreyfus, 2002), it is relevant to this review to report the observation that striatal oligodendrocytes greatly improve the survival and phenotype expression of mesencephalic dopaminergic neurons in culture, while simultaneously decreasing the apoptotic demise of these neurons (Sortwell et al., 2000). Glial cells may also protect dopaminergic neurons against degeneration by scavenging toxic compounds released by the dying neurons. Dopamine can produce reactive oxygen species (ROS) through different routes (Przedborski and Jackson-Lewis, 2000). Along this line, glial cells may protect remaining neurons against the resulting oxidative stress by metabolizing dopamine via monoamine oxidase-B and catechol-O-methyl transferase present in astrocytes, and by detoxifying ROS through the enzyme glutathione peroxidase, which is detected almost exclusively in astrocytes (Hirsch et al., 1999). Astrocytes, which can avidly take up extracellular glutamate via the glutamate transporters GLT1 and GLAST (see chapter by Schousboe and Waagepetersen), may mitigate the presumed harmful effects of the subthalamic excitotoxic input to the SN (Benazzouz et al., 2000), which is hyperactive in PD (DeLong, 1990). Taken together, the data reviewed here support the contention that glial cells, and especially astrocytes, could have neuroprotective roles in PD. Whether any of those actually dampen the neurodegenerative process in parkinsonian patients remains to be demonstrated.

8. The deleterious role of astrocytes and microglia in PD As we will see now, there are also many compelling findings, which support the contention that both astrocytes and microglia could be harmful in PD. In this context, the spotlight appears to be more on activated microglial cells and less on reactive astrocytes. The importance of activated microglial cells in the neurodegenerative process is underscored by the following demonstrations in rats (Liu et al., 2000): (i) the stereotaxic injection of bacterial endotoxin lipopolysaccharide (LPS) into the SNpc causes a strong activation of microglia throughout the SN followed by a marked degeneration of dopaminergic neurons; and (ii) the pharmacological inhibition of microglial activation prevents LPS-induced SNpc neuronal death. Similarly, LPS-induced microglial activation

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leads to injury of dopaminergic cell line MES 23.5 and dopaminergic primary ventral midbrain neurons in culture (Le et al., 2001). Activated microglial cells can produce a variety of noxious compounds including ROS, reactive nitrogen species (RNS), pro-inflammatory prostaglandins and cytokines. Among the array of reactive species, lately the lion’s share of attention has been given to RNS due to the prevalent idea that nitric oxide (NO)-mediated nitrating stress could be pivotal in the pathogenesis of PD (Przedborski et al., 1996; Ara et al., 1998; Pennathur et al., 1999; Giasson et al., 2000; Przedborski et al., 2001). So far, however, none of the characterized isoforms of NO synthase (NOS) (see chapter by Garcia and Baltrons) have been identified within SNpc dopaminergic neurons; hence, NO involved in the nitrating stress of PD most likely originates from other neurons, astrocytes, and microglial cells, as we hypothesized previously (Przedborski et al., 1996). It is particularly relevant to mention that numerous astrocytes in the SNpc of PD patients (Hunot et al., 1996) and microglia in the SNpc of MPTP-intoxicated mice (Liberatore et al., 1999; Dehmer et al., 2000), but not of controls, are immunoreactive for inducible NOS (iNOS). Upon its induction, this NOS isoform produces high amounts of NO (Nathan and Xie, 1994) as well as superoxide radicals (Xia and Zweier, 1997)—two reactive species that can either directly or indirectly promote neuronal death by inflicting oxidative damage. It should also be mentioned that a main source of glial-derived ROS emanates from the microglial enzymatic complex NADPHoxidase, which upon its induction and activation can produce large amounts of superoxide radicals (Colton et al., 1996). Although so far little information is available on NADPHoxidase in PD, it has been demonstrated that it is activated in the SNpc of mice after MPTP administration (Wu et al., 2002) and that NADPH-oxidase deficiency mitigates MPTP neurotoxicity (Wu et al., 2003). Prostaglandins and their synthesizing enzymes, such as Cox-2, constitute a second group of potential culprits. Indeed, Cox-2 has emerged as an important determinant of cytotoxicity associated with inflammation (Seibert et al., 1995; O’Banion, 1999). In the normal brain, Cox-2 is significantly expressed only in specific subsets of forebrain neurons that are primarily glutamatergic in nature (Kaufmann et al., 1996), suggesting a role for Cox-2 in the postsynaptic signaling of excitatory neurons. However, under pathological conditions, especially those associated with inflammation, Cox-2 expression in the brain can increase significantly, as do the levels of its products (e.g., prostaglandin E2), which are responsible for many of the Cox-2 cytotoxic effects. Interestingly, the Cox-2 promoter shares many features with the iNOS promoter (Nathan and Xie, 1994) and thus, these two enzymes are often coexpressed in disease states associated with inflammation. Therefore, it is not surprising to find Cox-2 and iNOS expressed in SNpc astrocytes and microglia of postmortem PD samples (Knott et al., 2000); PGE2 content is also elevated in the SNpc from PD patients (Mattammal et al., 1995). Of relevance to the potential role of prostaglandin in the pathogenesis of PD is the demonstration that pharmacological inhibition of both Cox-2 and Cox-1 attenuates MPTP toxicity in mice (Teismann and Ferger, 2001; Teismann et al., 2003). A third group of glial-derived compounds that can inflict damage in PD is the pro-inflammatory cytokines. Several among these, including tumor necrosis factor-a (TNF-a) and IL-1b, are increased in both SNpc tissues and cerebrospinal fluids of PD patients (Mogi et al., 1994, 1996, 2000), although some of the reported alterations may be

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related to the chronic use of the anti-PD therapy L -DOPA (Bessler et al., 1999). Nevertheless, at autopsy, convincing immunostaining for TNF-a, IL-1b and interferon-g (IFN-g) is observed in SNpc astrocytes from PD patients (Hunot et al., 1999). These cytokines may act in PD on at least two levels. First, while they are produced by reactive astrocytes, they can stimulate other astrocytes and even microglia not yet activated, thereby amplifying and propagating the astrocytic and microglial response and consequently the glial-related injury to neurons. Relevant to this scenario are the following demonstrations (Hunot et al., 1999): astrocytic-derived TNF-a, IL-1b and IFNg activate microglial cells, which start to express the macrophage cell surface antigen Fc [ R11 (CD23). Activation of CD-23 on these newly activated microglial cells induces iNOS expression and the subsequent production of NO by activated microglia which, in turn, can amplify the production of cytokines by astrocytes (e.g., TNF-a) and can diffuse to neighboring neurons. Second, astrocytic and microglial-derived cytokines may also act directly on dopaminergic neurons by binding to specific cell surface cytokine receptors (e.g., TNF-a receptor). Once activated, these cytokine receptors trigger intracellular death-related signaling pathways, whose molecular correlates include translocation of the transcription nuclear factor-k-B (NF-k-B) from the cytoplasm to the nucleus and activation of the apoptotic machinery. In connection with this, PD patients exhibit a 70fold increase in the proportion of dopaminergic neurons with NF-k-B immunoreactivity in their nuclei compared to control subjects (Hunot et al., 1997). In relation to apoptosis, Bax, a potent pro-apoptotic protein, is upregulated after MPTP administration and its ablation prevents the loss of SNpc dopaminergic neurons in this experimental model (Vila et al., 2001); also, caspase-3, a key agent of apoptosis, is activated in postmortem PD samples (Hartmann et al., 2000). Finally, it is worth mentioning the possible dual role of astrocytes in glutamate cytotoxicity. Astrocytes, which are intimately entangled with synapses (Hama et al., 1994; Ventura and Harris, 1999), take up extracellular glutamate, as noted above, whereby they can abate glutamate excitotoxicity. On the other hand, astrocytes can also release glutamate, as well as ATP and D -serine (Haydon, 2001). Glutamate release is stimulated by increases in intracellular free calcium, itself increased by the binding of a variety of neurotransmitters or ATP to the astrocyte or by gap junctions between astrocytes, which allow for calcium passage from cell to cell (see chapters by Hansson and Ro¨nnba¨ck and by Cornell-Bell). Indeed, glutamate release from astrocytes can increase calcium levels in adjacent neurons, through binding to neuronal NMDA receptors. Thus, astrocytes can modulate synaptic activity by releasing compounds that interact with neuronal NMDA receptors (Pasti et al., 1997; Kang et al., 1998; Araque et al., 1998; Haydon, 2001). It may be that under pathological conditions, the astrocytic release of glutamate is toxic to neurons, but proof of such toxicity is not yet at hand.

9. Concluding remarks In this review, we have tried to succinctly discuss the issue of astrocytic and microglial alterations in PD and how this cellular component of PD neuropathology, which has been neglected far too long, plays out in the overall neurodegenerative process. Accordingly,

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key findings, and, as often as possible, recent studies were included in our discussion to give an up-to-date look at this question. Although we have tried to provide the reader with a balanced view on the issue, it is our opinion that given the available evidence to date, those supporting a detrimental role of the astrocytes and microglia in PD outweighs that supporting a beneficial role. We also believe that should astrocytes and microglia indeed be implicated in the neurodegenerative process in PD, it is unlikely that these cells initiate the death of SNpc dopaminergic neurons, but may quite possibly propagate the neurodegenerative process. This view, if confirmed, could have far-reaching therapeutic implications, since targeting specific aspects of the glial-related cascade of deleterious events may prove successful in slowing or even halting further neurodegeneration in PD (Przedborski et al., 2000b). Acknowledgements The authors wish to thank Mr Brian Jones for his help in the manuscript preparation. The authors wish also to acknowledge the support of the NIH/NINDS Grants R29 NS37345, RO1 NS38586 and NS42269, NS17125, P50 NS38370, and P01 NS11766-271 the US Department of Defense Grant (DAMD 17-99-1-9471 and DAMD 17-03-1), the Lowenstein Foundation, the Lillian Goldman Charitable Trust, the Parkinson’s Disease Foundation, the Muscular Dystrophy Association, and the ALS Association.

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Upregulation of peripheral-type (mitochondrial) benzodiazepine receptors in hyperammonemic syndromes: consequences for neuronal excitability Mireille Be´langer, Samir Ahboucha, Paul Desjardins and Roger F. Butterworthp Neuroscience Research Unit, Hoˆpital Saint-Luc, University of Montreal, Montreal, Quebec, Canada p Correspondence address: Neuroscience Research Unit, C.H.U.M./Campus Saint-Luc, 1058 St-Denis Street, Montreal, Quebec, Canada H2X 3J4. Tel.: þ514-890-8000 ext. 5759; fax: þ514-412-7314. E-mail: [email protected](R.F.B.)

Contents 1. 2. 3.

4. 5. 6.

7.

Introduction PTBR expression in human hyperammonemic disorders PTBR expression in experimental hyperammonemic syndromes 3.1. Liver failure 3.2. Congenital hyperammonemia PTBR in ammonia-exposed neural cells Endogenous PTBR ligands in hyperammonemia Consequences of brain PTBR changes in hyperammonemia 6.1. Mitochondrial function 6.2. Neurosteroid synthesis Concluding remarks

Hyperammonemia resulting from liver failure, Reye Syndrome or congenital urea cycle disorders leads invariably to severe neurological impairment. Neuropathologic studies in hyperammonemia reveal selective changes of astrocyte morphology consisting of brain edema (acute hyperammonemia) or Alzheimer Type II astrocytosis (chronic hyperammonemia). In addition, exposure of astrocytes to increased concentrations of ammonia results in alteration in expression of genes coding for key astrocytic proteins including glial fibrillary acidic protein, glucose and amino acid transporters and the peripheral-type (mitochondrial) benzodiazepine receptor (PTBR). A selective increase in expression of Advances in Molecular and Cell Biology, Vol. 31, pages 983–997 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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the isoquinoline binding subunit protein of PTBR together with increased binding of the isoquinoline ligand PK 11195 are consistently observed in brain in both human and experimental hyperammonemic conditions. In addition, brain concentrations of putative endogenous ligands for PTBR, including diazepam binding inhibitor (DBI) and its processing product octadecaneuropeptide, are increased in these disorders. Activation of PTBR in brain by neuronally released DBI, resulting in the formation of neurosteroids with potent neuroactive properties, represents a further example of altered astrocyte – neuronal trafficking, a phenomenon which is consistently encountered in hyperammonemic disorders. Pharmacological manipulation of PTBR and/or its associated neurosteroid synthesis pathways may afford novel therapeutic approaches to the management and treatment of the cerebral consequences of hyperammonemia.

1. Introduction Hyperammonemia is a consistent finding in patients with acute or chronic liver failure, Reye Syndrome or congenital urea cycle disorders. Clinical symptoms of hyperammonemia in the adult include personality changes, sleep disturbances and deficits of attention, progressing to stupor and coma. Seizures are not uncommon in patients with acute liver failure or congenital hyperammonemias. Neuropathological findings in hyperammonemic disorders depend upon the severity and duration of hyperammonemia and on the age of the patient. Developing brain is exquisitely sensitive to increased concentrations of ammonia. For example, congenital deficits of the urea cycle enzyme ornithine transcarbamylase (OTC) results in brain atrophy, ventricular enlargement and significant neuronal cell death, some of which occurs in utero (Michalak and Butterworth, 1997). In contrast, exposure of the adult brain to ammonia results in more subtle changes in morphology and function of astrocytes. Acute hyperammonemia associated with acute liver failure or Reye Syndrome results in brain edema which, if sufficiently severe, leads to intracranial hypertension and death due to brain herniation. The prevalence of brain herniation in patients with acute liver failure is correlated with arterial ammonia concentrations (Clemmesen et al., 1999). Brain edema in acute liver failure patients is primarily cytotoxic in nature (Kato et al., 1992) and the astrocyte is the cell that uniquely manifests swelling. On the other hand, chronic hyperammonemia results in a characteristic alteration of astrocyte morphology known as Alzheimer Type II astrocytosis. First described by Alzheimer in 1912, these changes consist of an enlarged astrocytic nucleus, prominent nucleolus and margination of the normal chromatin pattern (Adams and Foley, 1953). Alzheimer Type II changes are encountered in both experimental and human chronic hyperammonemic conditions resulting from end-stage chronic liver failure as well as in urea cycle enzymopathies (Harper and Butterworth, 2002). Importantly, exposure of primary cultures of astrocytes to serum samples from patients with end-stage chronic liver failure results in classical Alzheimer Type II changes (Mossakowski et al., 1970), suggesting the presence of a blood –brain barrier permeant circulating factor in liver failure, which has generally been considered to be ammonia.

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The selective vulnerability of the astrocyte to increased concentrations of ammonia has resulted in the systematic study of astrocytic integrity and function in hyperammonemia. Results of these investigations reveal that important astrocytic functions such as the maintenance of the extracellular environment and cellular water homeostasis are severely impaired in hyperammonemia. In particular, the expression of several genes coding for key astrocytic proteins is altered in hyperammonemia (Table 1). Such alterations include increased expression of the astrocytic neurofilament protein glial fibrillary acidic protein (GFAP) (Sobel et al., 1981; Be´langer et al., 2002) and altered expression of astrocytic transporters for glucose (GLUT-1; Desjardins et al., 2001), water (aquaporin IV; Margulies et al., 1999), glutamate (EAAT-2; Knecht et al., 1997) and glycine (GLYT-1; Zwingmann et al., 2002). A highly consistent finding in brain in both acute and chronic hyperammonemic disorders is increased expression of the peripheral-type benzodiazepine receptor (PTBR). PTBR, also referred to as the mitochondrial benzodiazepine receptor or the v3 receptor, is a heteromeric complex of three subunit proteins including an isoquinoline binding protein (IBP, 18 kDa), a voltage-dependent anion channel (VDAC, 32 kDa), and an adenine nucleotide carrier (ANC, 30 kDa) (McEnery et al., 1992) (Fig. 1). The IBP subunit of PTBR is organized in clusters of four to six associated with one VDAC (Papadopoulos et al., 1994, 1997). The PTBR is expressed not only in peripheral tissues such as adrenals, kidney and testis but also in brain, where it is localized in high concentrations in astrocytes. PTBR sites are also expressed to some extent by other neural cell types including neurons, choroid plexus, ependymal linings of the ventricles as well as oligodendrocytes and microglia (Casellas et al., 2002; Jayakumar et al., 2002). Selective radioligands for PTBR have been identified and include the isoquinoline carboximide derivative PK11195, the benzodiazepine 40 -chlorodiazepam (Ro5-4864) and 2-aryl3-indoleacetamide (FGIN-1). Binding of these ligands has been studied in several species. In human brain, both PK11195 and Ro5-4864 binding are saturable, single sites with high (nanomolar) affinity. However, heterogeneity of these two sites is observed with regard to their susceptibility to detergents, histidine residue modifications and pharmacological characteristics. For example, FGIN-1 displaces only PK11195 binding, but not Ro5-4864, Table 1 Alterations in expression of genes coding for key astrocytic proteins in hyperammonemic disorders Hyperammonemia

Gene

Alteration

Reference

Belanger et al., 2002; Sobel et al., 1981 Desjardins and Butterworth, 2002; Kadota et al., 1996 Desjardins et al., 2001 Knecht et al., 1997 Zwingmann et al., 2002 Margulies et al., 1999

Acute

Chronic

X

X

Glial fibrillary acidic protein (GFAP)

#

X

X

Peripheral-type benzodiazepine receptor (PTBR) Glucose transporter (GLUT-1) Glutamate transporter (EAAT-2) Glycine transporter (GLYT-1) Water Channel Protein (Aquaporin IV)

"

X X X X

" # # "

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Fig. 1. Schematic representation of the molecular components of the PTBR complex. PTBRs are localized on the outer mitochondrial membrane and consist of four to six isoquinoline carboxamide binding protein (IBP) subunits associated with a voltage-dependent anion channel (VDAC) and an adenine nucleotide carrier (ANC). PTBRs regulate the transport of cholesterol across the inner mitochondrial membrane where it is subsequently converted to pregnenolone (see Fig. 4) by the cytochrome P450 cholesterol side chain cleavage enzyme (P450ssc).

whereas the isoquinoline and benzodiazepine sites are mutually displaceable with nanomolar affinity suggesting allosteric interactions between these sites (Raghavendra Rao and Butterworth, 1997). In addition to binding to this receptor, benzodiazepines have also been found to bind to a site on cultured astrocytes associated with calcium channel activity (see chapter by Peng et al.). Alterations in expression of PTBR or one of its subunits have consistently been reported following exposure of brain to increased concentrations of ammonia both in vitro and in vivo (for review, see Desjardins and Butterworth, (2002)). 2. PTBR expression in human hyperammonemic disorders End-stage chronic liver failure in humans results in increased blood and brain ammonia concentrations and in a severe neuropsychiatric syndrome known as hepatic encephalopathy (HE). In a study in autopsied brain tissue from cirrhotic patients who died in end-stage liver failure, increased densities of [3H]PK11195 binding sites were observed in both frontal cortex and caudate nucleus (Lavoie et al., 1990). Increased binding site densities were correlated with the presence of HE and of Alzheimer-type II astrocytosis in this material. Previous studies in this same autopsy material had failed to show any significant changes in binding site densities for the GABA-A receptor ligand [3H]muscimol or the GABA-related central benzodiazepine receptor ligands

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[3H]flunitrazepam or [3H]Ro15-1788 (flumazenil) (Butterworth et al., 1988), confirming a selective effect on PTBR. In a follow-up study, expression of the IBP subunit of PTBR was investigated in autopsied brain tissue from chronic liver failure patients by immunoblotting, using a monoclonal antibody to PTBR. Increased IBP expression was confirmed and again, as was the case with the [3H]PK11195 binding site increase, shown to correlate with the presence of Alzheimer type II changes (Fig. 2). No significant alterations of IBP mRNA were observed compared to material from agematched controls suggesting that the mechanism responsible for altered PTBRs in brain in human chronic liver failure are post-transcriptional in nature. Furthermore, the findings of a significant correlation between the increased PTBR expression, HE and Alzheimer type II changes suggest that PTBR increases are intimately involved in the pathogenesis of the central nervous system dysfunction in human chronic liver failure. Further support for this notion appeared in a recent communication in which Positron Emission Tomography was used with [11C]PK11195 as ligand to examine PTBR sites in vivo in patients with chronic liver failure (Cagnin et al., 2001). Increased binding sites were observed bilaterally in right dorsolateral prefrontal cortex, pallidum and putamen and the magnitude of the increase was negatively correlated with the degree of cognitive impairment in these patients as assessed by psychometric testing. It should be borne in mind, however, that chronic liver failure leads to the deposition of manganese in pallidum and other basal ganglia structures (Spahr et al., 1996), and that manganese exposure also results in increased PTBR expression and in Alzheimer Type II changes in astrocytes (Hazell et al., 1999). Consequently, PTBR increases in basal ganglia in chronic liver failure could be the result of manganese (in addition to ammonia)

Fig. 2. Increased expression of the PTBR isoquinoline binding protein (IBP) in autopsied human brain tissue. (a) representative immunoblots (Western blots) using a monoclonal antibody against IBP. Lane 1: normal subject; lane 2: HE patient without Alzheimer type-II astrocytosis; lane 3: HE patient with Alzheimer type-II astrocytosis (b) Densitometric determination of IBP immunoblots. Values represent mean ^SD ðn ¼ 4Þ: Values significantly different from controls (by ANOVA) are indicated by p ðp , 0:05Þ:

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toxicity. Increases in densities of binding sites for [3H]PK11195 have also of been described in a wide range of neurodegenerative disorders, including Alzheimer’s disease (Diorio et al., 1991), cerebral ischemia (Stevensen et al., 1995) and temporal lobe epilepsy (Sauvageau et al., 2002). However, in contrast to the hyperammonemic conditions, these disorders are associated with significant damage or loss of neurons and the increased PTBR signal has generally been attributed to microglial activation and/or reactive gliosis in these conditions. Such glial reactions are not a major feature of hyperammonemic syndromes in adults but could contribute to the increased PTBR signal in brain in congenital hyperammonemic disorders in which there is significant neuronal cell loss. 3. PTBR expression in experimental hyperammonemic syndromes 3.1. Liver failure The surgical construction of an end-to-side portacaval anastomosis in the rat results in chronic hyperammonemia and mild HE. Studies using [3H]PK11195 and an in vitro binding assay reveal increases of PTBR sites in brain (cerebral cortex), and kidney preparations (Raghavendra Rao et al., 1994). [3H]PK11195 binding sites in heart are unaltered following portacaval shunting, whereas those in testis are significantly reduced. These differences in the response of PTBRs in different organs suggest that PTBR is regulated by distinct mechanisms. Increased PTBR site densities in cerebral cortex become apparent as early as 24 h following portacaval anastomosis in the rat (Leong et al., 1994), and quantitative receptor autoradiographic studies reveal region-selective increases in [3H]PK11195 binding sites in cerebellum, pons . thalamus, cerebral cortex . hippocampus . striatum of portacaval-shunted animals (Gigue`re et al., 1992; Leong et al., 1994) (Fig. 3A). This pattern of change parallels the loss of activity of the astrocytic marker enzyme glutamine synthetase (uniquely responsible for ammonia removal by brain (Norenberg and Martinez-Hernandez, 1979; Cooper and Plum, 1987) and suggests regional vulnerability of astrocytes to chronic liver failure and hyperammonemia (Girard et al., 1993). In a subsequent study, IBP mRNA measured by reverse transcription polymerase chain reaction (RT-PCR) was found to be increased 2.5 fold in cerebral cortex and 1.7 fold in kidney following portacaval anastomosis in the rat (Desjardins et al., 1999). However, in contrast to the IBP subunit, expression of the VDAC subunit was not altered in brain following portacaval anastomosis (Fig. 3B). Densities of [3H]PK11195 binding sites were concomitantly increased demonstrating that PTBR induction in chronic liver failure is the result of selective increases in IBP subunit gene expression. It has recently been demonstrated that the magnitude of the increase of IBP expression was positively correlated with the degree of portal-systemic shunting following portacaval anastomosis (Belanger et al., 2001). PTBR binding sites have also been investigated in brain in experimental acute liver failure. Thioacetamide is a potent hepatotoxin, which causes acute liver failure, hyperammonemia and brain edema following intraperitoneal administration in both mice and rats. Thioacetamide-induced acute liver failure in the mouse leads to

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Fig. 3. (A) Representative autoradiograms of [3H]PK11195 binding to rat brain sections following end-to-side portacaval anastomosis (c and d) or sham-operation (a and b). Sections were incubated with 1 nM [3H]PK11195 and densities of specific binding sites were determined by densitometry. Portacaval anastomosis results in increases in [3H]PK11195 binding site densities in cortex and striatum (a, c) and hippocampus and thalamus (b, d). Modified from Gigue`re et al. (1992). (B) Expression of IBP and VDAC subunits of PTBR in rat frontal cortex following portacaval anastomosis (shunt; lanes 4, 6 and 8) or sham operation (sham; lanes 3, 5 and 7). b-Actin (347 bp), used as a housekeeping gene, VDAC (309 bp) and IBP (234 bp) were reversed transcribed and amplified by PCR for 20, 22 and 29 cycles, respectively. Lane 1: molecular weight standard; AMV reverse transcriptase was omitted from the reaction as a negative control (lane 2).

increased IBP mRNA and increased densities of binding sites for the benzodiazepine ligand [3H]Ro5-4864 (Kadota et al., 1996). Increases of binding sites for the isoquinoline ligand [3H]PK11195 were also reported in brain in this animal model, and in the brains of normal rats with acute hyperammonemia resulting from the administration of ammonium acetate (Itzhak et al., 1995). Acute liver failure in the rat resulting from hepatic devascularization (portacaval anastomosis followed by hepatic artery ligation) also results in increased expression of IBP mRNA in brain (Desjardins et al., 2002).

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3.2. Congenital hyperammonemia [3H]PK11195 binding sites were studied in the brain and peripheral organs of mice with congenital hyperammonemia resulting from an X-chromosomal defect of the urea cycle enzyme OTC. OTC deficiency led to increased densities of binding sites for [3H]PK11195 in brainstem . cerebellum . cerebral cortex as well as in kidney, testis and liver of OTC-deficient animals (Raghavendra Rao et al., 1993). Neuropathological evaluations in OTC-deficiency reveal the presence of Alzheimer-type II astrocytosis (Michalak and Butterworth, 1997). These findings again support the notion of a key role for PTBR in the pathogenesis of the central nervous system complications of hyperammonemia.

4. PTBR in ammonia-exposed neural cells Exposure of primary cultures of rat cortical astrocytes to millimolar concentrations of ammonia results in increased densities of binding sites for [3H]PK11195 (Itzhak and Norenberg, 1994). Cells exposed in this manner exhibit frank degenerative changes and swelling. However, in contrast to the in vivo situation, Alzheimer type II changes are rarely observed (Gregorios et al., 1985).

5. Endogenous PTBR ligands in hyperammonemia A wide range of endogenous molecules with high affinity for the PTBR have been identified. One such ligand is diazepam binding inhibitor (DBI), an 11 kDa polypeptide. DBI-immunolabeling has been reported to be concentrated to some extent in neurons (Alho et al., 1988) but mainly in glial cells (Alho et al., 1991) in the brain. Furthermore, DBI is localized in cells (both in brain and in the periphery) known to express large amounts of PTBR. Cerebrospinal fluid concentrations of DBI are elevated up to five-fold in patients with chronic liver failure, and the magnitude of the increase of DBI is significantly correlated with the clinical staging of HE in these patients (Rothstein et al., 1989). Octadecaneuropeptide (ODN) is an important processing product of DBI. ODN displaces benzodiazepine ligands from the PTBR (Bender and Hertz, 1986; Guidotti et al., 1988) in cultured astrocytes. Using an immunocytochemical approach and an antibody of high specific activity to synthetic ODN, the effects of experimental chronic liver failure on ODN distribution were studied. Four weeks after portacaval shunting in the rat, ODNimmunolabeling of nonneuronal elements (astrocytes, ependymal cells) was selectively increased in cerebral cortex and mesencephalic structures (Butterworth et al., 1991). Other putative endogenous ligands for PTBR include porphyrins, many of which exhibit very high affinities for PTBR (Verma et al., 1987) and it has been suggested that these compounds may represent the true endogenous ligands for the receptor (Casellas et al., 2002). However, no studies so far have addressed the effects of hyperammonia on brain porphyrins.

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6. Consequences of brain PTBR changes in hyperammonemia PTBRs are involved in the regulation of a wide range of cellular functions including cell proliferation, immunomodulation, porphyrin transport, apoptosis and steroidogenesis (see review by Casellas et al., 2002). 6.1. Mitochondrial function Mitochondrial fractions purified from brain homogenates are enriched in PTBRs (Anholt et al., 1986). Furthermore, separation of the mitochondrial inner and outer membranes by digitonin treatment showed that the binding site for [3H]PK11195 was released together with the outer mitochondrial marker protein monoamine oxidase, confirming that PTBR is preferentially located on the outer mitochondrial membrane. Alterations of mitochondrial morphology (Kato et al., 1992; Norenberg and Lapham, 1974) and mitochondrial dysfunction (Felipo and Butterworth, 2002) are consistent findings in brain in hyperammonemic disorders, and there is evidence to suggest that changes in PTBR expression are implicated in these changes. In support of this possibility, exposure of cultured glioma cells to PTBR agonists results in proliferation and swelling of mitochondria (Shiraishi et al., 1991), phenomena that are characteristic of hyperammonemic disorders (Kato et al., 1992; Norenberg and Lapham, 1974). In addition to the regulation of mitochondrial swelling, activation of the PTBR decreases the cellular respiratory control ratio, and is involved in the regulation of succinate-cytochrome C oxidoreductase (site II) activity (Casellas et al., 2002). Furthermore, both PK11195 and Ro5-4864 have been shown to modify mitochondrial respiration with potencies that are correlated with their affinities for the PTBR (Hirsch et al., 1998). It has been proposed that PTBR plays an important role in the mediation of oxygen-dependent signal transduction by acting as an oxygen sensor (Casellas et al., 2002). Additional evidence for a role of PTBR in oxidative processes is provided by the report of a significant protective effect of PTBR of certain cell lines against oxidative damage (Carayon et al., 1996). Superoxide radicals are produced in brain in acute hyperammonemia (Kosenko et al., 1997) and following exposure of neural cells to PTBR ligands (Jayakumar et al., 2002). 6.2. Neurosteroid synthesis PTBR is a vital component of the steroidogenic synthetic process. It mediates cholesterol transport from the outer to the inner mitochondrial membrane and, in this regard, an amino acid sequence in the PTBR which recognizes cholesterol has recently been identified (Li and Papadopoulos, 1998). Moreover, a steroidogenic acute regulatory protein involved in cholesterol transport across the mitochondrial membrane has been shown to be closely associated with PTBR in situ (West et al., 2001). Following cholesterol uptake, enzymes of the P450 group catalyze the conversion of cholesterol to pregnenolone, the precursor of a novel class of compounds known as neurosteroids (Fig. 4) (see review by Mellon and Griffin (2002) and chapter by Melcangi et al.).

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Fig. 4. Stimulation of PTBR results in cholesterol uptake by the mitochondrion, the first step in the synthesis of neurosteroids. One such neurosteroid, allopregnenolone (shown here) has potent neuromodulatory properties. See text for explanation of abbreviations.

Evidence for neurosteroid synthesis in brain derives from experiments demonstrating their formation from cholesterol in glial cells (Rupprecht, 1997). Furthermore, de novo synthesis of neurosteroids in brain has been demonstrated following PTBR activation (Korneyev et al., 1993), and the enzymes responsible (localized in both astrocytes and neurons) have been identified (Mellon and Griffin, 2002). Progesterone is formed from pregnenolone by the action of K5 –K4-isomerase/3b-dehydrogenase (Fig. 4). Reduction of progesterone is catalyzed by 5a-reductase to form 5a-dihydroprogesterone (5a-DHP) which is further reduced to allopregnenolone by the action of 3a-hydroxysteroid dehydrogenase (3a-HSD). Allopregnenolone is a very potent positive allosteric modulator of the GABA-A receptor. Not only does allopregnenolone displace t-butylcyclophosphorothionate (TBPS) from the GABA-A receptor-associated chloride channel, it also enhances GABA-elicited chloride currents and modulates the binding of both muscimol and benzodiazepine to the GABA receptor complex (Paul and Purdy, 1992). Allopregnenolone is also a negative allosteric modulator of the serotonin 5-HT3 receptor, albeit at somewhat higher concentrations (Mellon and Griffin, 2002). ODN stimulates neurosteroid biosynthesis through activation of both PTBR (Guidotti et al., 1988) and the central-type benzodiazepine receptor associated with the GABA-A complex (Do-Rego et al., 2001). Brain concentrations of pregnenolone are increased in hyperammonemic mice in parallel with increased binding sites for [3H]PK11195 (Itzhak et al., 1995), suggesting that

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ammonia-induced upregulation of PTBR has the potential to lead to increased neurosteroid synthesis in brain. Furthermore, a preliminary study in autopsied brain tissue from patients who died in hepatic coma reveals increased concentration of substances with neurosteroid-like properties shown to cause enhancement of the binding of both [3H]muscimol and [3H]flunitrazepam to the GABA-A receptor complex (Ahboucha et al., 2002). Activation of astrocytic PTBRs resulting in the production of neurosteroids with potent neuroactive properties represents a further example of altered astrocyte – neuronal trafficking in hyperammonemia. A second example of loss of trafficking between these two cell types is provided by the findings of a loss of capacity of the astrocyte to uptake neuronally released amino acids (Butterworth, 1993; Knecht et al., 1997). Evidence for impaired astrocyte – neuronal trafficking in hyperammonemia has also been described using 13C-nuclear magnetic resonance spectroscopy (Sonnewald et al., 1996; Zwingmann et al., 2003). A major interest in the role of neurosteroids in relation to hyperammonemic syndromes results from the ability of these compounds to recapitulate many of the central nervous system symptoms (such as sedation, and alteration of sleep rhythms) that are characteristic of human hyperammonemic disorders. Future studies using pharmacologic agents targeted specifically at neurosteroid synthetic enzymes or the neurosteroid modulatory site on the GABA-A receptor could offer potential new therapeutic approaches in these disorders (Fig. 5).

Fig. 5. Neurosteroids produced as a result of increased PTBR activation have modulatory effects on both GABAA and 5HT3 receptors. SER: smooth endoplasmic reticulum.

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7. Concluding remarks In summary, there is a consistent body of evidence demonstrating that PTBR sites are upregulated in both human and experimental hyperammonemic conditions. In both situations, upregulation of PTBR sites appears to result from a selective effect of ammonia on expression of the IBP subunit gene on the PTBR complex. However, the precise molecular mechanism responsible for this increase is unknown. Studies in cultured astrocytes reveal that PTBR upregulation by ammonia exposure also occurs but whether or not similar changes occur in other neural cell types under conditions of high ammonia has not been systematically investigated. Hyperammonemic states resulting from liver failure are associated with increased brain concentrations of the putative PTBR endogenous peptide ligands DBI and ODN. These findings of concomitant upregulation of PTBR sites together with increased brain concentrations of DBI/ODN suggest that activation of the PTBR occurs in brain in hyperammonemia. Activation of PTBRs in brain could have serious consequences for neuronal excitability given the wealth of data demonstrating that such activation results in the synthesis of neurosteroids, many of which are potent modulators of GABAergic and serotoninergic neurotransmission. In particular, given the suggested role of neurosteroids in the regulation of sleep patterns and their sedative-hypnotic properties, increased synthesis of these compounds following PTBR activation could be implicated in the pathogenesis of HE. Further studies are required in order to assess this possibility.

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Butterworth, R.F., Lavoie, J., Giguere, J.F., Pomier-Layrargues, G., 1988. Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. Hepatology 8, 1084–1088. Butterworth, R.F., Tonon, M.-C., De´sy, L., Gigue`re, J.-F., Vaudry, H., Pelletier, G., 1991. Increased brain content of the endogenous benzodiazepine receptor ligand, octadecaneuropeptide (ODN), following portacaval anastomosis in the rat. Peptides 12, 119– 125. Cagnin, A., Taylor-Robinson, S.D., Forton, D.M., Banati, R.B., 2001. In vivo quantification of cerebral “peripheral benzodiazepine binding site” in minimal Hepatic Encephalopathy: a [11C]R-PK11195 Positron Emission Tomography study. J. Hepatol. 34, 58. Carayon, P., Portier, M., Dussossoy, D., Bord, A., Petitpretre, G., Canat, X., Le Fur, G., Casellas, P., 1996. Involvement of peripheral benzodiazepine receptors in the protection of hematopoietic cells against oxygen radical damage. Blood 87, 3170–3178. Casellas, P., Galiegue, S., Basile, A.S., 2002. Peripheral benzodiazepine receptors and mitochondrial function. Neurochem. Int. 40, 475 –486. Clemmesen, J.O., Larsen, F.S., Kondrup, J., Hansen, B.A., Ott, P., 1999. Cerebral herniation in patients with acute liver failure is correlated with arterial ammonia concentration. Hepatology 29, 648–653. Cooper, A.J., Plum, F., 1987. Biochemistry and physiology of brain ammonia. Physiol. Rev. 67, 440–519. Desjardins, P., Butterworth, R.F., 2002. The “peripheral-type” benzodiazepine (omega 3) receptor in hyperammonemic disorders. Neurochem. Int. 41, 109– 114. Desjardins, P., Bandeira, P., Raghavendra Rao, V.L., Butterworth, R.F., 1999. Portacaval anastomosis causes selective alterations of peripheral-type benzodiazepine receptor expression in rat brain and peripheral tissues. Neurochem. Int. 35, 293 –299. Desjardins, P., Michalak, A., Therrien, G., Chatauret, N., Butterworth, R.F., 2001. Increased expression of the astrocytic/endothelial cell glucose transporter (and water channel) protein GLUT-1 in relation to brain glucose metabolism and edema in acute liver failure. Hepatology 34, 254. Diorio, D., Welner, S.A., Butterworth, R.F., Meaney, M.J., Suranyi-Cadotte, B.E., 1991. Peripheral benzodiazepine binding sites in Alzheimer’s disease frontal and temporal cortex. Neurobiol. Aging 12, 255–258. Do-Rego, J.-L., Mensah-Nyagan, A.G., Beaujean, D., Leprince, J., Tonon, M.-C., Luu-The, V., Pelletier, G., Vaudry, H., 2001. The octadecaneuropeptide ODN stimunates neurosteroid biosynthesis through activation of central-type benzodiazepine receptors. J. Neurochem. 76, 128–138. Felipo, V., Butterworth, R.F., 2002. Mitochondrial dysfunction in acute hyperammonemia. Neurochem. Int. 40, 487–491. Gigue`re, J.-F., Hamel, E., Butterworth, R.F., 1992. Increased densities of binding sites for the “peripheral-type” benzodiazepine receptor ligand [3H]PK11195 in rat brain following portacaval anastomosis. Brain Res. 585, 295–298. Girard, G., Gigue`re, J.-F., Butterworth, R.F., 1993. Region-selective reductions in activities of glutamine synthetase in rat brain following portacaval anastomosis. Metab. Brain Dis. 8, 207 –215. Gregorios, J.B., Mozes, L.W., Norenberg, L.-O.B., Norenberg, M.D., 1985. Morphologic effects of ammonia on primary astrocyte cultures. I. Light microscopic studies. J. Neuropathol. Exp. Neurol. 44, 397 –403. Guidotti, A., Berkovich, A., Ferrarese, C., Santi, M.R., Costa, E., 1988. Neuronal-glial diffential processing of DBI to yield ligands to central or peripheral benzodiazepine recognition sites. In: Sauvenet, P., Langer, S.Z., Morselli, P.L. (Eds.), Imidazopyridines in Sleep Disorders. Raven Press, New York, pp. 25–38. Harper, C.G., Butterworth, R.F., 2002. Nutritional and metabolic disorders. In: Graham, D.I., Lantos, P.L. (Eds.), Greenfield’s Neuropathology. Arnold, London, pp. 607–652. Hazell, A.S., Desjardins, P., Butterworth, R.F., 1999. Chronic exposure of rat primary astrocyte cultures to manganese results in increased binding sites for the ‘peripheral-type’ benzodiazepine receptor ligand 3 H-PK11195. Neurosci. Lett. 271, 5–8. Hirsch, T., Decaudin, D., Susin, S.A., Marchetti, P., Larochette, N., Resche-Rigon, M., Kroemer, G., 1998. PK11195, a ligand of the mitochondrial benzodiazepine receptor, facilitates the induction of apoptosis and reverses Bcl-2-mediated cytoprotection. Exp. Cell. Res. 241, 426–434. Itzhak, Y., Norenberg, M.D., 1994. Ammonia-induced upregulation of peripheral-type benzodiazepine receptors in cultured astrocytes labelled with [3H]PK11195. Neurosci. Lett. 177, 35 –38.

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Role of the cytokine network in major psychoses Norbert Mu¨llerp and Markus J. Schwarz Hospital of Psychiatry and Psychotherapy, Ludwig-Maximilians-Universita¨t, Nußbaumstr. 7, 80336 Mu¨nchen, Germany p Correspondence address: Tel.: þ 89-5160-3397; fax: þ 89-5160-4548. E-mail: [email protected](N.M.)

Contents 1. 2. 3.

4.

5.

6.

7.

Introduction Major psychoses—disorders with a neurobiological background The cytokine network in the CNS 3.1. Source of CNS cytokines 3.2. IL-6 in the central nervous system Immune systems 4.1. Innate and adaptive immunity in humans 4.2. Polarized type-1 and type-2 immune responses Schizophrenia 5.1. Relationship between clinical symptoms and immune alterations 5.2. Viral hypothesis of schizophrenia 5.3. Neurodevelopmental hypothesis 5.4. Blood – brain barrier impairment 5.5. Activation of the innate unspecific immune system 5.6. IL-6 and schizophrenia 5.7. Type-2 immune response activation in schizophrenia 5.8. Type-1 immune response and schizophrenia 5.9. Glia cells, type-1 –type-2 immune response and the tryptophan – kynurenine metabolism 5.10. B cells and anti-psychotic treatment 5.11. Implications for therapy Major depression 6.1. Similarity between major depression and ‘sickness behavior’ 6.2. Immune activation in MD 6.3. Depression: a syndrome with different immune pathologies? 6.4. Cytokines and effects of anti-depressants Conclusion

Advances in Molecular and Cell Biology, Vol. 31, pages 999–1031 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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Immune alterations in major psychoses, i.e., in schizophrenia and in major depression have been described since decades. However, modern immunological methods and new insights into the highly developed and functionally differentiated immune system allows an integrative view of both, the older and also recent findings of immunological abnormalities in major psychoses. The conceptual advances in immunology require the reevaluation of older immunological findings in schizophrenia. In this overview, recent advances in immunological research regarding the differentiation between the type-1 and type-2 immune response and the specific and unspecific arms of the immune system are discussed. The unspecific ’innate’ immune system shows signs of an overactivation in unmedicated schizophrenic patients. Increased levels of Interleukin-6 (IL-6) and the activation of the IL-6 system in schizophrenia might be the result of the activation of type2 monocytes/macrophages, too. On the contrary, several parameters of the specific cellular immune system are blunted, e.g., the decreased type-1 related immune parameters in schizophrenic patients both, in vitro and in vivo. It seems that a type-1/type-2 imbalance with a shift to the type-2 immune response is associated with schizophrenia. There are indications that the type-1/type-2 imbalance is reflected in differential activation of astrocytes and microglial cells in the CNS which is associated with a different activation of the tryptophane – kynurenine metabolism. During therapy with anti-psychotics, the specific type-1 related immune answer becomes activated, but also the B cell system and the antibody production. In major depression, the activation of the type-1 cellular immune system dominates the immune response. This is the case especially in nonmelancholic depressed patients and during suicidality. Anti-depressant medication seems to influence the immune response, too. 1. Introduction Major psychoses—affective disorders and schizophrenia—are diseases with a high impact on the quality of life and on social functioning. The risk of suicide is about 20– 50 times higher than in the normal population. The direct and indirect costs in the US for schizophrenia have already 1991 been estimated to US$ 65,000,000,000 per year (Wyatt et al., 1995). Schizophrenia has a world-wide prevalence of about 1%. The prevalence of major depression (MD) is about ten times higher, but depression shows a more favorable course with a lower risk for a chronic course. The costs for manicdepressive illness in the US for 1991 have been estimated to US$ 45,000,000,000 (Wyatt and Henter, 1995). 2. Major psychoses—disorders with a neurobiological background The etiology of both types of major psychoses is unknown, but there is no doubt that disturbances in the neurotransmission are involved in the pathophysiology. The vulnerability-stress model has been proposed for both disorders (Zubin and Spring, 1977), i.e., patients with an increased biological (genetically inherited) risk for schizophrenia or affective disorder develop symptoms of the disease during increased stress. In depression, a deficiency of serotonergic and/or noradrenergic neurotransmission

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Table 1 Examples for schizophrenic positive and negative symptoms Positive symptoms

Negative symptoms

Delusions Hallucinations Thinking disturbance Tension Motor symptoms

Lack of dive Blunted affect Abulia Social withdrawal Cognitive disturbances

in the CNS has been suggested. In schizophrenia a disturbance of the dopaminergic neurotransmission plays a major role. There is evidence that overactivity of the dopaminergic neurotransmission is responsible for schizophrenic positive symptoms, while underactivity of doparminergic neurotransmission is involved in schizophrenic negative symptoms (Table 1). An increased risk for both types of major psychoses is genetically inherited, although no major gene has been identified until now. A polygenic transmission seems to be responsible for both schizophrenia and affective disorder. The more it became evident that the noradrenaline (Schildkraut, 1965; Matussek, 1966) and serotonin hypotheses (Coppen and Swade, 1988) of depression were not sufficient to explain depressive disorders, the more other pathological agents came into the focus of interest. It has been shown that immune processes can mediate severe depressive syndromes and/or schizophreniform psychoses. Autoimmune disorders, such as lupus erythematodes (Kru¨ger, 1984), scleroderma (Mu¨ller et al., 1992), Sjo¨gren syndrome (Raps et al., 1986), and anti-phospholipid syndrome (Kurtz and Mu¨ller, 1994) have been reported to cause symptoms of schizophrenia or even induce severe depressive states (in the case of lupus erythematodes). Thus, the influence of an immune process—possibly triggered by an infectious agent as environmental stressor—on the pathogenesis of psychoses has been under discussion for a long time (see also below). Moreover, from a clinical point of view major psychoses show several parallels to autoimmune disorders. These parallels include early onset in many cases, genetic vulnerability, waxing and waning course, and the preponderance of females. Cytokines mediate information between cells of the peripheral immune system and the CNS during infection and autoimmune disorders of the CNS, but also during health (see chapter by Mercier and Hatton). Recent findings show that cytokines are important in psychiatric disorders, possibly mainly due to their influence on neurotransmission. 3. The cytokine network in the CNS 3.1. Source of CNS cytokines Cytokines interact with CNS cells in different ways and they originate from different sources. First, several cytokines such as Interleukin (IL)-1 (Banks and Kastin, 1992), IL-2 (Banks et al., 1993) and TNF-a (Gutierrez et al., 1993), can be transported from the blood into the CNS by active transport mechanisms, albeit only slowly. Second, glia cells secrete cytokines after activation by an antigenic challenge. Finally, Norris and Benveniste (1993)

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reported that the cytokine secretion in the CNS can be stimulated by neurotransmitters. They showed that noradrenaline stimulates the release of IL-6 from astrocytes in vitro in a dose dependent manner, an effect that can be antagonized by blockade of the adrenergic receptors. 3.2. IL-6 in the central nervous system IL-6 is a pleiotropic cytokine, which is released from different cell types in the blood (macrophages, monocytes, T and B cells). One function of IL-6 is to activate B cells to synthesize antibodies (Plata-Salaman, 1991). However, like several other cytokines, IL-6 is not only synthesized and released in immune cells of the peripheral blood, IL-6 is also produced by activated astrocytes and microglia cells in the CNS. Several findings suggest that IL-6 may mediate exacerbation of autoimmune disorders in the CNS (Dunn, 1992), e.g., the observations that IL-6 supports the differentiation of B cells, stimulates the local IgG synthesis in the CNS, and causes a blood – brain barrier disturbance (Frei et al., 1989; Muraguchi et al., 1988). In the hypothalamus, IL-6 can induce the release of growth hormone releasing hormone and thyrotropin releasing hormone, and it stimulates in vitro the secretion of prolactin and growth hormone from pituitary cells (Spangelo et al., 1989). The influence of IL-6 on neurotransmitters is discussed in this paragraph as an example, other cytokines are known to influence neurotransmitters, too. IL-6 can stimulate neurons in vitro to secrete dopamine and probably also other catecholamines (Hama et al., 1991). Peripheral application of IL-6 in animal experiments enhanced dopaminergic and serotonergic turnover in the hippocampus and frontal cortex, without affecting noradrenaline (Zalcman et al., 1994). Conversely, noradrenaline can stimulate astrocytes to release IL-6 (Dunn, 1992). Both observations point to a direct influence of activation of cytokines, especially IL-6, on catecholaminergic neurotransmission. Since IL-6 is closely linked to the function of other cytokines e.g., IL-1, IL-2, and TNFa, this finding indicates that neurotransmitters can activate the cascade of cytokines (Ransohoff and Benveniste, 1996). This represents an important psychoneuroimmunological regulatory mechanism affecting immune disorders (including autoimmune disorders), susceptibility to infections, and psychiatric disorders. With regard to the vulnerability-stress model, noradrenaline released during stress (Engel et al., 1980) can act as a cytokine-activating stimulus, which activates the cytokine cascade, possibly interfering with the balance of the immune response in the CNS. 4. Immune systems 4.1. Innate and adaptive immunity in humans The immune system has been developed during evolution over millions of years. A highly differentiated system, consisting of different lines of defense, was established in order to guarantee successful defense against numerous different invading, life threatening microorganisms like bacteria, viruses, or parasites. A widespread heterogeneity was the

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Table 2 Components of the unspecific ‘innate’ and the more specific cellular ‘adaptive’ immune systems in humans Components

Innate

Adaptive

Cellular

Monocytes, Makrophages, Granulocytes, NK-cells, g/d-cells Complement, APP, Mannose binding Lectin (MBL)

T and B cells

Humoral

Antibodies

consequence: two functionally different immune systems, both representing different types of barriers, and each consisting of cellular and humoral immune components (Table 2). The ‘innate’ immune system is the phylogenetically older, ‘primitive’ one. Its cellular arm is represented by monocytes/macrophages, granulocytes, and natural killer (NK) cells. The humoral arm consists of acute phase proteins (APPs) and the complement system. This ‘unspecific‘ immune system represents the first line of defense. The specific part of the immune system of higher organisms, including humans, is the ‘adaptive’ immune system, consisting of the cellular arm of the T and B cells and the humoral arm of specific antibodies. This system includes higher functions like immunological memory, and it can be conditioned. In case of a reexposition to a specific antigen, this system can recognize the enemy and initiate a specific immune answer. The innate and the adaptive immune systems are functionally balanced. Within the adaptive immune system there is another balance with respect to the activation of the cellular and the humoral immune system.

4.2. Polarized type-1 and type-2 immune responses The cellular arm of the adaptive immune system is mainly operating though cytokines, which have been defined in the mouse model as the T-helper-1 (TH-1) system—helper cells, which produce the activating ‘immunotransmitters’ IL-2 and Interferon-g (IFN-g). Since not only T-helper cells (CD4þ cells) but also monocytes/macrophages and other cell types produce these cytokines (IL-12 is mainly produced by macrophages, activating TH-1 cells), this immune response is named Type-1 immune response, while the humoral arm of the adaptive immune system is mainly activated via the Type-2 immune response— T-helper cells (TH-2) or monocytes/macrophages (M2), which produce mainly IL-4, IL-10, and IL-13 (Table 3). The different types of immune response are polarized, i.e., there is a reciprocal inhibition of the other type of immune response, while each response activates a cascade of cytokines. Cytokines such as Tumor-necrosis-factor-a (TNF-a and IL-6 are mainly secreted from monocytes and macrophages. While TNF-a is an ubiquitous cytokine, which mainly activates the type-1 response, IL-6 activates the type-2 response including the antibody production (Table 4). In parallel to the polarized TH-1 and TH-2 immune response it is known today that also macrophages show a type-1 and type-2

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Table 3 The polarized immune response

Cytokines

Type 1

Type 2

IL-2 IL-12 IFN-g IL-18

IL-4 IL-13 IL-10

Table 4 Cellular source of the polarized immune response

Blood and lymphatic organs Source T-Helper cells CNS

Type 1

Type 2

Macrophages type-1 (M1)

Macrophages type-2 (M2)

CD4þ(TH-1) [Microglia]

CD4þ (TH-2) [Astrocytes]

polarized cytokine response, the M1 macrophages mainly producing IL-2, IL-12, and INFg, the M2 macrophages producing IL-4, IL-10, and IL-13 (Mills et al., 2000). The type-1 system promotes cell-mediated immune responses against intracellular pathogens, whereas the type-2 response helps B cell maturation and promotes humoral immune responses against extracellular pathogens. Type-1 and type-2 cytokines antagonize each other in promoting their own type of response, while suppressing the other type of immune response. Which system will dominate over the other one, depends on the relative timing and ratio between IL-4 and IFN-g together with IL-12 (Seder and Paul, 1994; Romagnani, 1995; Paludan, 1998). Such a polarized immune response happens not only at the peripheral level but also in the CNS. Although initiation of T cell responses is unlikely to occur within the CNS, T cells and monocytes will be massively recruited, if pathogens are placed into the cerebral ventricles (Aloisi et al., 2000). Perivascular macrophages, owing to their location close to the blood – brain barrier, can stimulate cells within brain parenchyma to proliferate and secrete Type-1 cytokines (Ford et al., 1996). Following extravasation into the CNS parenchyma, T cells also interact with intrinsic CNS cells, particularly microglia and astrocytes (Aloisi et al., 2000). Microglia progressively acquire a clear-cut macrophage phenotype in response to CNS injuries (Kreutzberg, 1996) and the production of the Type1 cytokine IL-12 (Krakowski and Owens, 1997; Stalder et al., 1997) and of Type-2 cytokines such as IL-10 and TGF-b (Aloisi et al., 2000). Astrocytes are also potential sources of TGF-b, which inhibits MHC II and ICAM-1 expression in macrophages/ microglia (Hailer et al., 1998). Microglia and astrocytes in addition secrete chemokines (see chapter by Nakagawa and Schwartz) that may affect the recruitment of T-helper cells and macrophages. In sum, a complex network between microglia, astrocytes, and T cells is

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Fig. 1. Dysbalance of the immune system in schizophrenia. Insufficient activation of the type-1 immune response and relative over-activation of the type-2 response.

involved in the balance between the type-1 and type-2 system, which in turn might have impact on immune responses within the CNS (Fig. 1). Further studies have to elucidate whether the polarization of macrophages into M1 and M2 macrophages have a functional consequence for the recruitment of macrophages from the periphery into the CNS, or for the function of microglial cells. Other data suggest that a functional polarization between the brain immune cells is related to astrocytes and microglial cells. Microglial cells, deriving from peripheral macrophages, secrete preferably type 1 cytokines such as IL-12, while astrocytes inhibit the production of IL12 and ICAM-1, which both are part of the type-1 system, while they secrete the type-2 cytokine IL-10 (Aloisi et al., 1997; Aloisi et al., 2000; Xiao and Link, 1999).

5. Schizophrenia 5.1. Relationship between clinical symptoms and immune alterations Immunological alterations in schizophrenia have been described in the international literature since the beginning of the last century (Bruce and Peebles, 1903; Dameshek, 1930; Lehmann-Facius, 1939). Signs of an inflammatory disease process in schizophrenia have been found in a subgroup of schizophrenic patients (Ko¨rschenhausen et al., 1996). Clinical features of the subgroup showing signs of an immunological or inflammatory disease have been studied by several groups of researchers. It was observed that the immunological parameters are influenced by the type of symptomatology, e.g., paranoid or negative symptoms (Cazzullo et al., 1998; Mu¨ller and Ackenheil, 1995), the acuity of the disease (Korte et al., 1998; Sperner-Unterweger et al., 1992; Wilke et al., 1996), and the ¨ zek et al., 1971; Saunders and Muchmore, 1964; Maes et al., 1995a; drug treatment (O

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Masserini et al., 1990; Mu¨ller et al., 1991; Mu¨ller et al., 1997b,c; Mu¨ller et al., 1999; Pollma¨cher et al., 1996). Also, the clinical response to treatment with neuroleptics seems to be related to immune parameters (Mu¨ller et al., 1993a). Our own data show that the IgG content of CSF is especially high in patients with pronounced schizophrenic negative symptoms, which are associated with an unfavorable course of the disorder and often with treatment resistance (Mu¨ller and Ackenheil, 1995). These findings are consistent with the suggestion that the inflammatory response system is activated in patients which are resistant to treatment with anti-psychotic medication (Maes et al., 2000). A clearcut differentiation between immunological subgroups in schizophrenia has not yet been established. However, the relationship between clinical characteristics of schizophrenia and parameters of the immune system may help to define subgroups with specific disturbances of the immune system.

5.2. Viral hypothesis of schizophrenia Different case reports of patients presenting psychiatric symptoms during viral infections with herpes-simplex encephalitis, varicella-zoster encephalitis, or subacute sclerosing panencephalitis led to the suggestion that viral infections may play a role in the pathogenesis of schizophrenia. Epidemiologic data showing an increased number of manifestations of schizophrenia in winter-born individuals (Torrey et al., 1977), and geographic variability (Torrey, 1987), as well as an increased incidence of schizophrenia in children of mothers infected during pregnancy in an influenza epidemic in England 1957 (O’Callaghan et al., 1991) point to a virus-pathogenesis (Yolken and Torrey, 1995). Numerous studies have detected raised titers of antibodies against certain viruses (herpessimplex-, measles-, cytomegalo-, varicella-, borna-virus) in schizophrenic patients as compared to controls, and/or increased CSF/serum ratios of viral antibodies, indicating a local synthesis of antibodies in the brain or CSF (Hoechtlen and Mu¨ller, 1992; Torrey et al., 1977; Albrecht et al., 1980; Bechter et al., 1999; Gottlieb-Stematsky et al., 1981). However, the meaning of these findings is controversial, and most authors did not observe viruses or virus genomes in CSF or brain tissue of schizophrenic patients (Stevens et al., 1984; Taylor and Crow, 1986; Alexander et al., 1992). It may be assumed that viruses could be detectable only during certain states of virusinduced diseases (‘hit and quit‘). Thus, the fact that viruses or a virus genome could not be isolated does not exclude the involvement of viruses in the pathogenesis of the disease. The virus hypothesis and the autoimmune hypothesis of schizophrenia may be connected with regard to the potential role of viruses in the molecular mimicry: viruses induce cells to change their identity—probably by expressing surface antigens—thus, these cells are no longer recognized as ‘self’, but as ‘non-self’, and they are therefore are attacked by the individual’s own immune cells. From that point of view, our findings of an immune activation in schizophrenia are compatible with the virus hypothesis of schizophrenia. Recent studies indicate that schizophrenia is associated with upregulation of a number of different RNA species (Yolken et al., 1999). These RNAs are supposed to represent genomic or messenger RNA derived from novel viral agents. In this regard, it is of interest that several of the (predicted) proteins are homologous to viruses of non-human origin.

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The authors discuss that possible unidentified human retroviruses together with genetic determinants may be responsible for the development of schizophrenia—viral infection of neural cells can, in combination with genetic determinants of susceptibility, result in the profound alteration of brain function typical of schizophrenia (Yolken et al., 1999). The finding that a certain polymorphism in the TNF-a gene may protect from cytomegalovirus infection in schizophrenia (Schwarz et al., 2001) and that high antibody titers against cytomegalovirus in schizophrenia seem to be associated with a better outcome to anti-inflammatory therapy (Mu¨ller et al., 2001) point not only to the link of a viral infection with an increased (immuno-)genetic vulnerability but also to an influence on the therapeutic response.

5.3. Neurodevelopmental hypothesis The excess of late winter/early spring births of patients with schizophrenia has been attributed to infectious, nutritional, or other environmental factors (Torrey et al., 1997; Mortensen et al., 1999). The hypothesis that an infection of pregnant mothers is related to an increased risk for schizophrenia in the offspring implicates sequelae for the development of the unborn offspring. The pathological mechanisms underlying prenatal and perinatal risk factors for schizophrenia, including infection, remain largely unstudied. Infection during pregnancy is one of different factors defined as obstetrical complications. Cytokines are known to regulate normal brain development and have been implicated in abnormal brain development (Merill, 1992; Mehler et al., 1995; Mehler and Kessler, 1995, 1997). It has been hypothesized that proinflammatory cytokines generated by the immune system are important mediators of the association between maternal infection, abnormal brain development, and increased risk for schizophrenia and other neurodevelopmental disorders (Leviton, 1993; Jarskog et al., 1997). There is no doubt about the presence of a significant association between obstetrical complications and the ultimate development of schizophrenia (Geddes and Lawrie, 1995; Jones et al., 1998; Dalman et al., 1999; Hultman et al., 1999). It is likely, that the association between schizophrenia in the offspring and exposure in utero or early postnatally to obstetrical complications is a more general phenomenon, not limited to a single etiologic viral agent such as influenza. Not only infectious processes in general, but also other pathological factors such as hypoxia are known to lead to an altered cytokine production in the brain. Proinflammatory cytokines are neurotoxic to a variety of developing neurons in vitro. For example, IL-1b, IL-6, and TNF-a decrease survival of fetal dopaminergic and serotonergic neurons in vitro (Jarskog et al., 1997). Moreover, IL-1b decreases neuron survival in primary cultures of embryonic rat hippocampus (Arajujo and Cotman, 1995), and TNF-a potentiates glutamate excitotoxicity in cultures of fetal cortical neurons (Chao and Hu, 1994). In an animal model in pregnant rats of prenatal exposure to infection it could be shown that Escheria coli lipopolysaccharides (LPS) in different doses lead to alterations in cytokine levels. Low dose LPS was associated with significant increase of IL-1b, IL-6, and TNF-a in the placenta, and IL-6 in the amniotic fluid. High dose LPS was associated with a significant elevation of IL-6 and TNF-a in the placenta, and of TNF-a in

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the amniotic fluid, but with a significant decrease of TNF-a in the fetal brain. These observations show that maternal exposure to infection alters proinflammatory cytokine levels in the fetal environment, which may have a significant impact on the developing brain (Urakubo et al., 2001). Some authors postulate that microglial dysfunction initiated by early CNS viral exposure results in the abnormal neural development and neurotransmitter dysfunction seen in schizophrenia (Munn, 2000). This theory is supported by findings of abnormally high microglial activation in a subgroup of schizophrenic patients (Bayer et al., 1999). An increase in the numerical density of HLA-DR-positive microglial cells (HLA-DR is the marker of microglia activation), which is not related to aging, has recently been described in brains from chronic schizophrenics, especially in the temporal and frontal cortex (Radewicz et al., 2000). 5.4. Blood – brain barrier impairment Abnormalities of the CSF are regularly reported in about 20– 30% of psychiatric patients (Kirch et al., 1985; Naber et al., 1986). The authors’ investigations of schizophrenic patients have shown increased total protein content (. 45 mg%) or a BBB impairment in about one third, and an intrathecal IgG production in 15% of the patients (Mu¨ller and Ackenheil, 1995). The increased immunoglobulins and the BBB-disturbance are likely to be part of an immune process, whose pathophysiological relevance is not yet clear. Nevertheless, a correlation of schizophrenic psychopathology with IgG content in CSF suggests a close relationship between an immune process and the pathophysiology of schizophrenia in those patients who show negative symptoms (Mu¨ller and Ackenheil, 1995). A local CNS immune activation may be controlled insufficiently due to a deficient communication between the peripheral immune system and the CNS. A penetration of evading cells through the BBB is necessary for effective control of several inflammatory processes (Reich et al., 1992; Shankar et al., 1992). The authors’ investigations have shown that anti-psychotic treatment and psychopathological improvement are associated with a significant increase in adhesion molecule expressing T-lymphocytes in the blood; moreover, a strong correlation between adhesion molecule expressing lymphocytes and both BBB disturbance and CSF parameters (total protein, albumin, IgG) was observed (Mu¨ller et al., 1997c). Adhesion molecules are surface markers expressed on lymphocytes in order to mediate adhesion of lymphocytes to endothelial cells and penetration into tissues (Fabry et al., 1992, 1994; OppenheimerMarks et al., 1991). One speculative explanation of these findings is the involvement of adhesion molecules in an immunoregulatory mechanism taking place between astrocytes, microglia cells and lymphocytes from the blood. Such a mechanism may be facilitated by the action of neuroleptics (Mu¨ller et al., 1997c, 1999). IL-2 and IL-6 play a pivotal role in the BBB disturbance. Peripheral application of IL-2 can cause an alteration of the BBB (Rosenstein et al., 1986; Ellison et al., 1987, 1990; Saris et al., 1988), and a disturbance of the BBB seems to be involved in the CNS effects of peripherally applied IL-2. The alteration of the BBB by intrathecal application of IL-2 (Watts et al., 1989) suggests that an increased intracerebral IL-2 production may contribute to the opening of the BBB.

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5.5. Activation of the innate unspecific immune system Although systematic investigations of the innate immune system are lacking in schizophrenia, there are several hints that this part of the immune system may be more activated in schizophrenic patients than in controls. There is a report that monocytes are increased in schizophrenic patients compared to controls (Wilke et al., 1996), and our own investigations of unmedicated schizophrenic patients also showed higher amounts of monocytes in schizophrenia than in controls (unpublished results). An increase of cells of the ’first immune barrier’ was also found in gdþCD8þ cells in unmedicated schizophrenics (Mu¨ller et al., 1998). A significantly higher proportion of mononuclear phagocytes/macrophages in the CSF of schizophrenics compared to neurological controls has also been reported in a small subgroup of patients, and it was followed by a normalization of the cytological picture at a follow-up investigation during anti-psychotic treatment (Nikkila¨ et al., 1999). Thus, it seems that similar immunological processes take place in the peripheral and the CNS immune systems. 5.6. IL-6 and schizophrenia 5.6.1. Increase in the IL-6 system in schizophrenia Several reports showed increased serum IL-6 levels in schizophrenia (Ganguli et al., 1994; Maes et al., 1995a; Frommberger et al., 1997; Lin et al., 1998). Two of the reports described a relationship between increased IL-6 levels and clinical features of schizophrenia: high serum IL-6 levels were related to the duration of the disorder (Ganguli et al., 1994) and to treatment resistance (Lin et al., 1998). These findings suggest that IL-6 serum levels might be especially high in patients with an unfavorable course of the disease. On the other hand, methodological concerns have to be taken into consideration. IL-6 is a cytokine, which is mainly released paracrinely, and the function of serum-levels is not yet completely understood. Moreover, the cytokine IL-6—as IL-2, IL-1 and other cytokines—is not stable and a multitude of methodological pitfalls have to be considered. The soluble IL-6 receptor (sIL-6R) is a more stable marker of the IL-6 system, both in the blood and in the CSF. Investigations of the sIL-6R levels in the CSF showed that high levels of sIL-6R can be found especially in schizophrenic patients with a more marked paranoid-hallucinatory syndrome (Mu¨ller et al., 1997a). These investigations also point to a more altered IL-6 system in patients with an unfavorable course of the disease: longer duration of illness, treatment resistance, or more marked paranoid-hallucinatory symptomatology. Another study found reduced levels of sgp130 in the CSF of schizophrenic patients compared to depressed patients and to psychiatric healthy controls (Schwarz et al., 2001). This result supports the view of a disturbance in the IL-6 system in schizophrenia, because gp130 is part of the IL-6 system. The soluble protein spg130 is acting as an antagonist to the gp130 receptor, and it mediates inhibition of the IL-6 system (Narazaki et al., 1993). Functionally, decreased sgp130 levels in the CSF point to a decrease in the inhibition of the IL-6 system, i.e., a functional increase in the activation.

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5.6.2. Anti-psychotic therapy and the IL-6 system There are several observations that anti-psychotic therapy with neuroleptics is accompanied by a functional decrease of the IL-6 system. A significant decrease of IL-6 during therapy with neuroleptics was described by Maes et al. (1995a). Two studies found a significant decrease of sIL-6R levels during anti-psychotic therapy with neuroleptics (Maes et al., 1995a; Mu¨ller et al., 1997b). Studies from human CNS cell cultures also showed an inhibitory effect of different neuroleptics on the production of IL-6 after stimulation with LPS, more marked for phenothiazines compared to butyrophenones (unpublished results). Similar observations have also been reported by other authors (Lin et al., 1998). There are indications suggesting a time-dependent effect of anti-psychotic treatment on IL-6: one study reported that short term treatment with clozapine (median 12 days) induced an increase in IL-6 levels (Maes et al., 1997b) and another group found an increase of IL-6 after two weeks of clozapine treatment, but a decrease after an additional four weeks of treatment (Pollma¨cher et al., 1996). 5.7. Type-2 immune response activation in schizophrenia IL-6 is a product of the activation of the type-2 immune response. Moreover, several other results are also pointing to an activation of the type-2 immune response in schizophrenia. IL-10 is a characteristic cytokine of the type-2 immune response. An increase of IL-10 in schizophrenic patients compared to healthy controls was reported (Cazzullo et al., 1998). Another study observed a strong correlation between IL-10 levels in CSF and schizophrenic negative symptoms in 62 unmedicated schizophrenics (Van Kammen et al., 1997). In medicated schizophrenics, treated with haloperidol, a significant correlation exists between CSF IL-10 levels and the severity of schizophrenic psychosis—measured by Bunney-Hamburg psychosis rating scale (Van Kammen et al., 1997). These findings point out, that IL-10 levels in the CSF are related to the severity of the psychosis, especially to the negative symptoms. Another characteristic cytokine that is produced during a type-2 immune response is IL-4. An increase of IL-4 levels in the CSF of juvenile schizophrenic patients has recently been reported (Mittleman et al., 1997). This finding points out that the probable enhancement of the type-2 immune response in schizophrenia is not only a phenomenon of the peripheral immune system, but that it also seems to play a role in the CNS immune system. The production of IgE is also a sign of an activation of the type-2 immune response. Increased levels of IgE in schizophrenic patients compared to controls have been observed (Ramchand et al., 1994). 5.8. Type-1 immune response and schizophrenia 5.8.1. Interleukins The key characteristics of the type-1 immune system are the production of IFN-g, IL-2, and IL-12. One of the often replicated findings in schizophrenia is the decreased in vitro production of IL-2 (Villemain et al., 1989; Ganguli et al., 1995; Hornberg et al., 1995;

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Bessler et al., 1995; Cazzullo et al., 1998). This phenomenon has often been interpreted as the consequence of an exhaustion of the lymphocytes after overproduction of IL-2. However, it may equally well reflect a reduced capacity of lymphocytes to produce IL-2. The observation of a decreased production of IL-2 fits well with another finding: The decreased production of IFN-g (Wilke et al., 1996; Rothermund et al., 1996). Both findings point to a blunted production of type-1 related cytokines in schizophrenia. A lack of activation of the type-1 arm of the cellular immune system has also been postulated by other researchers (Sperner-Unterweger et al., 1999). Our own findings of a decreased production of lymphocytes after stimulation with different specific antigens might also reflect a reduced capacity for a type-1 immune response in schizophrenia. Especially after stimulation with tuberculin, which provokes a type-1 mediated immune response, the reaction was blunted (Mu¨ller et al., 1991).

5.8.2. Decreased levels of sICAM-1 in unmedicated schizophrenics Recently, decreased levels of the soluble intercellular adhesion molecule 1 (sICAM-1) in the serum of schizophrenic patients have been described (G. Wieselmann, personal communication; Schwarz et al., 2000). ICAM-1 is a molecule that mediates the adhesion of lymphocytes to other lymphocytes, to endothelial cells and to parenchymal cells. Moreover, it also mediates the activation of the cellular immune system. ICAM-1 is part of the TH-1 immune response (Van Seventer et al., 1990; Kuhlman et al., 1991). Therefore, decreased levels of the soluble form of ICAM-1, which is shedded from lymphocytes, seems to represent a state of activation of the type-1 immune system. However, reduced sICAM-1 levels have recently been found not only in serum, but also in CSF of schizophrenic patients (Schwarz et al., 2002). The latter finding points out that the blunted activation of the type-1 immune system may not be restricted to the peripheral immune system, because CSF parameters reflect more directly the immune pathology of the CNS. One of the ‘classical’ epidemiological findings in schizophrenia research is the negative association between schizophrenia and rheumatoid arthritis (Vinogradov et al., 1991). This negative association can be interpreted as two sides of the type-1/type-2-balance coin—represented by increased sICAM-1 levels in rheumatoid arthritis and decreased sICAM-1 levels in schizophrenia. sICAM-1 is also a key-molecule that mediates the inflammatory reaction in rheumatoid arthritis, where increased sICAM-1 levels are regularly found (Neidhart et al., 1995). Rheumatoid arthritis is a disorder that is primarily mediated by the cellular type-1-related immune system. Fig. 2 shows blunted type-1 immune response of the ‘cellular’ immune system, which is represented by decreased secretion of IFN-g, IL-2, and sICAM-1, while preferentially type-2 cytokines are secreted.

5.8.3. Type-1 immune response and anti-psychotic therapy in schizophrenia Recent findings point out that neuroleptics may have type-1 immune response stimulating effects. In vitro studies show that the blunted IFN-g production becomes normalized after therapy with neuroleptics (Wilke et al., 1996). An increase of soluble IL-2

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Fig. 2. Cytokines and the cellular immune system in major depression-focus on IL-6 and acute phase proteins.

receptors has been described by several groups (Maes et al., 1994; Pollma¨cher et al., 1995; Mu¨ller et al., 1997b). Since sIL-2R are shedded from activated T cells, the increase might reflect an increase of activated, IL-2 bearing T cells. An increase of CD4þCD45ROþ cells during therapy with neuroleptics was observed by different groups (Cazzullo et al., 1998; Mu¨ller et al., 1997c). CD4þCD45ROþ cells, ‘memory cells’ are one of the main sources of IFN-g production. The increase of this subpopulation during therapy may contribute to an increase of the IFN-g production. The reduced sICAM-1 levels in the serum of schizophrenics do not normalize during short term anti-psychotic therapy, but a statistically not significant tendency to an increase of sICAM-1 could be observed (Schwarz et al., 2000). On the other hand, the leucocyte function antigen 1 (LFA-1) molecule on CD4þ cells shows a significantly increased expression during anti-psychotic therapy (Mu¨ller et al., 1999). LFA-1 is the ligand of ICAM-1. Moreover, a blunted reaction to vaccination with salmonella was not observed in ¨ zek et al., 1971). These studies point out, patients that were treated with neuroleptics (O that the type-1 immune response increases during anti-psychotic therapy. Recently, an elevation of IL-18 serum levels has been described in medicated schizophrenics (Tanaka et al., 2000). Since IL-18 plays a pivotal role in the type-1 immune response, this finding is consistent with other descriptions of type-1 activation during anti-psychotic treatment. Interestingly, there is a recent report that described a correlation between the dose of short term anti-psychotic treatment, the mononuclear cell count in the CSF, the CSF macrophage count, and the total lymphocyte count (Wahlbeck et al., 2000). The correlation was especially found in patients treated with chlorpromazine-like low-potency phenothiazines. This is the first report that describes a relationship between anti-psychotic treatment and cellular immunity in the CSF; further investigations are required to elucidate whether lymphocytes and macrophages are activated—an activation of the cellular immune system also within the CNS could reflect an activation of the peripheral immune system. Moreover, it would be interesting to know whether a special subgroup of schizophrenics show an increase of immune cells in the CSF during therapy—the total cell count was within the normal range.

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5.9. Glia cells, type-1 – type-2 immune response and the tryptophan– kynurenine metabolism As mentioned above, there is no doubt that schizophrenia is a disorder of the dopaminergic neurotransmission. As indicated by the pharmacological profile of atypical anti-psychotics such as clozapine, the serotonergic system appears to be also involved in the pathophysiology of schizophrenia (Carlsson et al., 1999). Based on the experiences with the drug phencyclidine (PCP)—an NMDA glutamate receptor antagonist – the third mentioned neurotransmitter system is the glutamatergic system (Olney and Farber, 1995; Olney et al., 1999). Immunological mechanisms have been proposed to be the underlying pathological factor for the disturbance of the neurotransmitter systems. Although it has been shown that cytokines such as IL-1, IL-2, and IL-6 influence the dopaminergic and serotonergic neurotransmission (Ransohoff and Benveniste, 1996; Zalcman et al., 1994), the exact mechanism still needs to be elucidated. The polarized immune response may play a key role within this mechanism. Data suggest that the polarization between type-1 and type-2 immune response in the CNS takes place between microglial cells and astrocytes. Microglial cells secrete preferably type-1 cytokines such as IL-12. On the other hand, astrocytes inhibit the production of IL-12 and ICAM-1, which both are part of the type-1 system, while they secrete the type-2 cytokine IL-10 (Aloisi et al., 1997; Aloisi et al., 2000; Xiao and Link, 1999). The concept of underactivation of the type-1 immune response in schizophrenia implies that within the CNS there might be a dysbalance of activation between astrocytes and microglial cells with an overweight of astrocyte activation. This dysbalance may also affect the metabolism of the amino acid tryptophan, the precursor of serotonin and kynurenine. The tryptophan – kynurenine pathway metabolism is located in microglial cells and in astrocytes. However, in microglial cells the complete metabolism from tryptophan via kynurenine to quinolinic acid and picolinic acid takes place while in astrocytes a key enzyme is lacking that degradates kynurenine to 3-hydroxy-kynurenine (Guillemain et al., 2001). In other words, an overweight of astrocyte activation may be responsible for an accumulation of kynurenic acid in the CNS which cannot further be metabolized to 3hydroxy-kynurenine (Schwarcz and Pelliciari, 2002). A second key-player in metabolizing of 3-hydroxy-kynurenine are monocytic cells infiltrating the CNS. They help astrocytes in the further metabolism to quinolinic acid (Guillemain et al., 2001). However the low levels of sICAM-1 (ICAM-1 is the molecule that mainly mediates the penetration of peripheral monocytes and lymphocytes into the CNS) in the serum and in the CSF of unmedicated schizophrenic patients (Mu¨ller et al., 2000; Mu¨ller et al., 2003) and the increase of adhesion molecules during anti-psychotic therapy (Mu¨ller et al., 1999) indicate that the penetration of monocytes and lymphocytes may be reduced in unmedicated schizophrenic patients. These two factors—the polarized immune response in the CNS and the disturbance in penetration of monocytes through the BBB—may contribute to an accumulation of kynurenic acid in the CSF and certain CNS regions of schizophrenic patients. Indeed, an accumulation of kynurenic acid in schizophrenic patients has been described in the CSF

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(Erhardt et al., 2001a) and in certain CNS areas, e.g., the prefrontal cortex (Schwarcz et al., 2001) which plays a pivotal role in schizophrenia. This accumulation of kynurenic acid in certain CNS regions might be responsible for changes in the dopaminergic and glutamatergic neurotransmission. Kynurenic acid acts as a NMDA-receptor agonist and NMDA receptor agonists are associated with schizophrenic symptomatology in animals and in humans (Carlsson, 1998). On the other hand, increased kynurenic acid levels lead to increased dopaminergic activity in the nigro-striatal and in the mesocorticolimbic dopaminergic systems (Erhardt et al., 2001b; Erhardt and Engbert, 2002), which are also known to play a key role in the pathophysiology of schizophrenia (Carlsson, 1998). Also the increase of dopaminergic activity seems to be mediated via astrocytic dopamine-receptors (Wu et al., 2002). Vice versa, peripheral administration of L -DOPA, amphetamines or substances such as methylphenidate cause a functionally significant decrease in the concentration of kynurenic acid (Wu et al., 2002). Dopaminergic antagonists such as the anti-psychotic haloperidole downregulate kynurenic acid (Schwarcz et al., 2001). This hypothesized close functional relationship between the type-1/type-2 immune response, inflammation and disturbed dopaminergic and glutamatergic neurotransmission mediated by the tryptophane – kynurenine system in schizophrenia is to the authors’ opinion a speculative, but convincing concept for the pathogenesis of schizophrenia.

5.10. B cells and anti-psychotic treatment Activated B cells are antibody producing cells. Several publications describe an activation of B cells with increased antibody production during anti-psychotic treatment. Already during the 1970s, in vitro studies showed an increase of antibody production after stimulation with phenothiazines (Gallien et al., 1977; Zarrabi et al., 1979). Many studies described an increased antibody production in schizophrenic patients, leading to the discussion of an autoimmune etiology of schizophrenia (Ganguli et al., 1987). Nevertheless, the role of anti-psychotic treatment has not been regarded in several of these studies. Although findings have repeatedly reported that about 20– 35% of schizophrenic patients show features of an autoimmune process (Mu¨ller and Ackenheil, 1998), the role of present or former therapy with neuroleptics may not have been taken into consideration. An increase of IgG—the most important antibody class—in the CSF has been described especially in patients with predominant negative symptoms (Mu¨ller and Ackenheil, 1995). Increased antibody titers against 60 and 70 kDa heat shock proteins is one of the recent interesting findings in schizophrenia, because it may reflect a mechanism of loss of neuronal protection (Schwarz et al., 1998; Schwarz et al., 1999; Kilidireas et al., 1992). However, antibodies against heat shock protein 60 are especially found in patients during anti-psychotic therapy. An increased number of patients with activated B cells (CD5þCD19þ cells), compared to healthy controls, has been described in schizophrenic patients treated with antipsychotics. Our own study shows an increase of activated B cells during anti-psychotic therapy (Mu¨ller et al., 1997c). Thus, it seems that not only the type-1 immune response

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is activated during anti-psychotic therapy, but also the antibody production by activated B cells. During anti-psychotic therapy, both arms of the specific adaptive immune system seem to become activated: the specific cellular immunity of the type-1 system, but also the B cell system with its humoral arm, the antibody production. An activation of the unspecific innate immune system can be found mainly in unmedicated schizophrenic patients, suggesting that this may reflect the disease process and not the result of anti-psychotic treatment. 5.11. Implications for therapy Recently, we conducted a therapeutic study with the anti-inflammatory agent celecoxib in schizophrenia. The cyclooxygenase 2 inhibitor celecoxib is known to upregulate the type-1 immune response and to downregulate the type-2 immune response. This study was planned as a double blind randomized add-on study of celecoxib versus placebo to the anti-psychotic risperidone. The celecoxib add-on therapy group showed a significantly better outcome with regard to the schizophrenic symptomatology, compared to the risperidone and placebo group. This result showed that additional treatment with celecoxib had significant positive effects on the therapeutic action of risperidone with regard to the total schizophrenic psychopathology (Mu¨ller et al., 2002). Moreover, the fact that treatment with an immunomodulatory drug shows beneficial effects on the symptomatology of schizophrenia, indicates that immune dysfunction in schizophrenia is not just an epi-phenomenon, but related to the pathomechanism of the disorder. Interestingly, several cases of a successful use of the cerebrospinal fluid filtration method in psychiatric patients have been described recently. The filtration is an experimental therapeutic method for treatment of therapy-resistant patients suffering from Guillain-Barre´-syndrome. In some of the patients the schizophrenic negative symptoms improved rapidly, as did the neurological ‘soft’ signs and the cognitive function. The authors suppose that toxic factors are removed by the filtration (Bechter et al., 1999). From a scientific point of view, those therapeutic approaches are interesting, but they need of course further evaluation. 6. Major depression 6.1. Similarity between major depression and ‘sickness behavior’ An immunological model of MD is the sickness behavior, the non-specific reaction of the organism to infection and inflammation. Sickness behavior is characterized by weakness, malaise, listlessness, inability to concentrate, lethargy, decreased interest in the surroundings, and reduced food intake—all of which are depression-like symptoms. The sickness-related psychopathological symptomatology during infection and inflammation is mediated by cytokines such as IL-1, IL-6, TNF-a, and IFN-g. Their active pathway from the peripheral immune system to the brain is via afferent neurons and through direct targeting at amygdala and other brain regions after diffusion at the circumventricular

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organs and choroid plexus (Dantzer, 2001) and perhaps also via cytokine-induced release of cytokines from the abluminal surface of brain capillary endothelial cells (see chapter by Mercier and Hatton). Undoubtedly, there is a strong relationship between the cytokine system and the neurotransmitter system, but a more differentiated analysis may be required to understand the specific mechanisms underlying the heterogeneous disease entity of MD. Blood levels of several cytokines have already been investigated in MD and their interpretation based on the type-1/type-2 immune response concept may help to generate hypotheses for discrimination between subgroups of MD. 6.2. Immune activation in MD 6.2.1. Immune cells Older studies showed an increase of T-helper cells (CD4þ cells) and an increased CD4þ/CD8þ ratio in depressive disorders (Maes et al., 1992; Mu¨ller et al., 1993b; Syva¨lathi et al., 1985). This finding points to an immune activation and was the starting position for a series of further studies. Further investigations of the cellular components of the immune system focused on monocytes and macrophages. Increased numbers of peripheral mononuclear cells have been described by different groups of researchers (Herbert and Cohen, 1993; Seidel et al., 1996a; Rothermundt et al., 2001a). Neopterin is a sensitive marker of cell-mediated immunity. The main source of neopterin is monocytes/ macrophages. In agreement with the findings of increased monocytes/macrophages, an increased secretion of neopterin has been described in patients suffering from MD by several groups of researchers (Duch et al., 1984; Dunbar et al., 1992; Maes et al., 1994; Bonaccorso et al., 1998). 6.2.2. IL-6 As a product of monocytes and macrophages, IL-6 is one of the most frequently investigated immune parameters in MD patients. Most publications report of a marked increase of in vitro IL-6 production (Maes et al., 1993) or serum IL-6 levels in depressed patients (Maes et al., 1995b). Most of these studies also report elevated plasma levels of APPs—markers of the unspecific (innate) immune system. Contradictory results are very few, indicating reduced (Katila et al., 1989), or unchanged serum IL-6 levels (Maes et al., 1995c; Brambilla and Maggioni, 1998). An age-related increase of IL-6 serum values was reported in patients with MD (Ershler et al., 1993). From a methodological point of view, the potential influence of possibly interfering variables such as smoking, gender, recent infections and prior medication on IL-6 release and concentration must be considered (Haack et al., 1999). Prostaglandin E2 (PGE2) stimulates IL-6. Therefore an increased secretion of PGE2 would be expected in depressive disorders, too. Older studies described increased PGE2 both in the cerebrospinal fluid and in the serum of depressed patients (Linnoila et al., 1983; Calabrese et al., 1986). Increased concentrations of PGE2 in saliva of depressed patients have been repeatedly described (Ohishi et al., 1988; Nishino et al., 1989). Moreover, in vitro studies show an increased PGE2 secretion from lymphocytes of depressed patients compared to healthy controls (Song et al., 1998).

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Data of IL-6 and the IL-6 system in the CSF are still rare. The only available data on CSF IL-6 have been published by our group. Herein, we found markedly decreased levels of IL-6 and its soluble receptor subunit IL-6Ra in elderly patients with MD compared to matched healthy control persons (Stu¨bner et al., 1999). As stated above, IL-6 is a highly important inducer of antibody production (Th2 immune response) and indeed, some data show increased antibody titers in MD. As in schizophrenia, a great heterogeneity of antigen-specificity of the antibodies, such as anti-nuclear anti-phospholipid, anti-thyroidal or anti-viral antibodies was found (Amsterdam and Hernz, 1993; Maes et al., 1991; Haggerty et al., 1987). There is no doubt that IL-6 is involved in modulation of the hypothalamic –pituitary – adrenal (HPA) axis (Plata-Salaman, 1991). Activation of the HPA axis is one of the bestdocumented changes in MD (Roy et al., 1987). Furthermore, the relationship between psychological or physical stress and an enhanced IL-6 secretion in the peripheral immune system seems to be well established (Salas et al., 1990; LeMay et al., 1990). An impaired ability to cope with stress is often observed in depressed patients. Thus, the high number of data showing elevated peripheral IL-6 levels in MD patients may be related to psychological stress. On the other hand, there is evidence for a relationship between high peripheral IL-6 levels and elevated central nervous system serotonin availability. Intravenous or intraperitoneal administration of IL-6 in an animal model induced not only an activation of the HPA axis, but also an increase in brain tryptophan and serotonin metabolism, whereas norepinephrine metabolism was unaffected (Wang and Dunn, 1998). Accordingly, IL-6 may be a mediator of activation of the HPA axis and of the central nervous serotonin system after administration of the endotoxin LPS (Wang and Dunn, 1999). Thus, elevated plasma levels of IL-6 do not fit with the hypothesis of a serotonin deficiency in MD. On the other hand, there is also a report showing a correlation between increased IL-6 production in culture supernatants of mitogen-stimulated peripheral leukocytes in vitro and decreased tryptophan levels in depressed patients that emphasizes the influence of IL-6 on serotonin metabolism (Maes et al., 1993). Serotonin synthesis in the CNS is at least partly dependent on the availability of tryptophan in the blood (Fernstrom and Faller, 1977). It should be recognized that an inherent heterogeneity exists in the etiology of depression and different neurotransmitter systems may be disturbed. Norepinephrine is the second major biogenic amine, which has been proposed to be causally involved in the pathophysiology of MD (Schildkraut, 1965; Bunney and Davis, 1965) and in the mechanism of anti-depressant drug action (Mo¨ller, 2000). It has to be investigated, if MD patients with elevated peripheral IL-6 are primarily suffering from a central nervous norepinephrine deficiency—in contrast to the suicidal patients with marked serotonin deficiency and possible type-1-dominated immune response. However, studies on other type-2 cytokines are missing, and the current data on IL-6 alone are insufficient to establish any type-2 hypothesis in MD. Nevertheless, the question arises, if an overactive type-2 immune response would be the common immunological hallmark of both non-suicidal depression and negative schizophrenia—two different disease concepts, which show similarities in the psychopathology.

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6.3. Depression: a syndrome with different immune pathologies? 6.3.1. The type-1-serotonin-link as biological basis for suicidality? Data on IL-2 in MD are mainly restricted to measurements of its soluble receptor in peripheral blood. The blood levels of sIL-2R were repeatedly found to be increased in MD patients (Sluzewska et al., 1996; Maes et al., 1995b,c). One single study dealt with CSF measurement of sIL-2R levels and reported a highly significant reduction in MD patients compared to healthy control subjects (Levine et al., 1999). Production of both IL-2 and IFN-g is the typical marker of a type-1 immune response. IFN-g is produced in higher amounts by lymphocytes of patients with MD than in healthy controls (Seidel et al., 1996a,b). Higher plasma levels of IFN-g in depressed patients, accompanied by lower plasma tryptophan availability were described in 1994 (Maes et al., 1994). Mendlovic and colleagues discriminated between suicidal and non-suicidal MD patients in a small study. They found distinct associations between suicidality and type-1 immune response on the one hand and a predominance of type-2 immune parameters in non-suicidal patients on the other hand (Mendlovic et al., 1999). This combination of psychopathological symptoms and their immunological correlates may represent a very successful strategy in immunological research of MD. In the following we want to introduce a line of evidence for a relationship between the type-1 cytokine IFN-g, tryptophan metabolism, and suicidality. The essential amino acid tryptophan is the precursor of two distinct metabolic pathways, leading to the products serotonin or kynurenine. The enzyme indoleamine 2,3dioxygenase (IDO) metabolizes tryptophan to kynurenine, which is then converted to quinolinic acid by the enzyme kynurenine hydroxylase. Both IDO and kynurenine hydroxylase are induced by IFN-g. The activity of IDO is an important regulatory component in the control of lymphocyte proliferation (Mellor and Munn, 1999). It induces a halt in the lymphocyte cell cycle due to the catabolism of tryptophan (Munn et al., 1999). The type-2 cytokines IL-4 and IL-10 inhibit the IFN-g-induced tryptophan catabolism by IDO (Weiss et al., 1999). The enzyme IDO is located in several cell types including monocytes, microglial cells and astrocytes (Alberati et al., 1996). An IFN-g-induced, IDO-mediated decrease of central nervous tryptophan availability may lead to a serotonergic deficiency. While in schizophrenia the further metabolism of kynurenine to kynurenic acid may be an important factor influencing the dopaminergic neurotransmission via type-2 immune response (see above), in depression the deficiency of serotonin via activation of the type-1 immune response, especially INF-g, seems to be the link between immunity, inflammation, and serotonergic or dopaminergic neurotransmission, respectively. One of the most consistent findings in biochemical research dealing with mental disorders is that some patients with low 5-hydroxyindoleacetic acid (5-HIAA)—the metabolite of serotonin—in CSF are prone to commit suicide (Lidberg et al., 2000). This gives additional evidence for a possible link between the type-1 cytokine IFN-g and the IDO-related reduction of serotonin availability in the CNS in suicidal patients. On the basis of epidemiological data it has also been hypothesized that high IL-2 levels are associated with suicidality (Penttinen, 1995). Thus, clinical studies have observed an activation of the type-1 immune response that might be related to suicidality. There have been descriptions

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of increased levels of serum sIL-2R in medication-free suicide attempters irrespective of the psychiatric diagnosis (Na¨ssberger and Tra¨ksman-Bendz, 1993), and treatment with high-dose IL-2 has been associated with suicide in a case report (Baron et al., 1993). Recently a small study showed that T cells of suicidal patients with MD have type-1 characteristics, while T cells of non-suicidal depressed patients showed type-2 characteristics (Mendlovic et al., 1999). We propose that the possible involvement of IFN-g induced IDO activity in the pathophysiology of suicidality in MD should be considered in future studies. 6.3.2. Melancholic versus non-melancholic depression From the point of view of many researchers the diagnosis MD reflects different etiological and pathophysiological subgroups as older diagnostic manuals differentiated in entities such as endogenous depression, neurotic depression, or psychoreactive depression. Moreover, different etiologies have been suggested for monopolar and bipolar depression. Therefore it could be expected that different immunological patterns are found in different types of depression. Immunological investigations have only recently been performed in different types of MD. A group of patients suffering from MD was investigated, a part of them fulfilling the criteria of melancholic type of depression. Melancholic patients had normal counts of leucocytes, lymphocytes, and NK-cells, while the non-melancholic depressed patients showed increased leukocytes, lymphocytes, and NK-cells. On the other hand, the melancholic patients showed a decreased production of IL-2, IFN-g and IL-10, while the non-melancholic patients had a cytokine production comparable to the healthy controls (Rothermundt et al., 2001a). During the anti-depressive treatment, the monocyte count stayed higher in the non-melancholic patients also after two and four weeks of treatment. Accordingly, alpha-2 Macroglobulin, another marker of inflammation, was increased in non-melancholic patients both before therapy and after two and four weeks of treatment. There were no differences between healthy controls and patients suffering from either melancholic and non-melancholic depression with respect to C-reactive protein, haptoglobin, and in vitro production of IL-1b (Rothermundt et al., 2001b). 6.3.3. Conclusion These results show that different types of depression—melancholic versus nonmelancholic—are associated with different immune states. Moreover, suicidality seems to show a further distinct immune pattern. This, however, points to a methodological pitfall regarding studies of immunity and depression: The heterogeneity of the results of immunological studies might at least in part due be to different types of depression including suicidality. Maybe the finding of increased sIL-2R and IL-1 concentrations in depression applies to a patient group showing more often the melancholic type of depression (Maes et al., 1991). Considering this caveat, it is doubtful whether the pleiotropic cytokine IL-6 and the other components of the IL-6 system such as the IL-6 receptor and the signal transducing molecule gp130 are specifically and universally altered in MD. Moreover, increased IL-6 levels have also been observed in patients suffering from

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Table 5 Markers of the type-1/type-2 immune responses in major depression Site of cytokine expression

Type-1

Type-2

In vitro production Peripheral

IFN-g " sIL-2R h , IFN-g " , IFN-g " ) TRP # sIL-2R " Type-1-serotonin-link in suicidal MD?

IL-6 h IL-6 h

CSF Hypothesis

IL-6 # , sIL-6R # Type-2-dominance or an overactivation of monocyte/macrophage system in non-suicidal MD?

h or i means repeated consistent data for elevation or decrease, respectively; " or # means less consistent data; $ means no changes; g means controversial results.

schizophrenia, but this was associated with the duration of the disease and the paranoid psychopathology (Ganguli et al., 1994; Mu¨ller et al., 1997c). Taken together, there might be a relationship between immunological, neurochemical and clinical variables in MD patients: suicidality in MD may be related to a central nervous serotonin deficiency, possibly induced by an IFN-g (type-1 immune response) mediated IDO activation, whereas elevated levels of IL-6 (type-2 immune response) might indicate a distinct group of MD patients without deficiency of the serotonergic system. The possible predictive value of immunological parameters on anti-depressant therapy regimen has to be unraveled in future studies. However, future immunological studies on MD should consider clinical and neurochemical variables to a greater extent (Table 5).

6.4. Cytokines and effects of anti-depressants Studies of the immune effects of anti-depressants still show conflicting results (Miller and Lackner, 1989; Kenis and Maes, 2002). However, attention has focused on the interaction of the serotonergic system and the immune system. It has been suggested that serotonin has an inhibitory action on antibody production, since an inverse relationship has been demonstrated between brain serotonin concentration and antibody synthesis in previous studies (Devoino et al., 1970). The suggested role of the cytokines in depression leads on to expectation that anti-depressants would have an inhibitory effect on certain activating cytokines. An inhibitory function of the serotonin system on the IFN-g induced MHC expression (Sternberg et al., 1987) and on mitogen induced T cell proliferation (Bonnet et al., 1984) was observed in studies using modern immunological methods. During the last several years a modulatory, mostly inhibitory effect of serotonin reuptake inhibiting drugs on activation of immune parameters has been demonstrated in animal experiments (Bengtsson et al., 1992; Zhu et al., 1994). Inhibitory effects of selective serotonin reuptake inhibitors on APPs were also observed in animal

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investigations (Song and Leonard, 1994), and there are several hints that the severe side effects of zimelidine are due to immunological mechanisms of the 5-HT system (Thomas, 1989). From an immunological point of view, some anti-depressants seem to be able to induce a shift from a type-1 to a type-2 immune response, since Maes and colleagues demonstrated the ability of three different anti-depressants (sertraline, clomipramine, and trazodone) to significantly reduce the IFN-g/IL-10 ratio in vitro. All three drugs reduced IFN-g production significantly, sertraline and clomipramine in addition increased the IL-10 production significantly (Maes et al., 1999). Regarding other in vitro studies, a significantly reduced production of IFN-g, IL-2, sIL-2R, and IL-10 was found after six weeks of anti-depressant treatment compared to pretreatment values (Seidel et al., 1995, 1996a). The type of treatment, however, was not specified. There are some other studies, showing no effect of anti-depressants to the in vitro stimulation of cytokines (overview: Kenis and Maes, 2002), but methodological issues have to be taken into account. Cum grano salis it can be concluded that anti-depressants of different classes show a downregulation of the type-1 cytokine production in vitro (Kenis and Maes, 2002, Song and Leonard, 2000). An interesting study showed a relationship between IL-6 production by lymphocytes from depressed patients and the treatment response: patients producing low levels of IL-6 showed a better response to treatment with amitriptyline over a six-week period than patients showing a high IL-6 production. During treatment, however, both groups turned to a normal IL-6 production. The production of TNF-a was high in responders to amitriptyline and turned to normal during therapy (Lanquillon et al., 2000). Regarding the serum levels, several researchers observed a decrease of IL-6 during treatment with the serotonin reuptake inhibitor fluoxetine (Sluzewska et al., 1995). A decrease of IL-6 serum levels during therapy with different anti-depressants has been observed by other researchers, too (Frommberger et al., 1997). Other groups, however, did not find any effect of certain anti-depressants on serum levels of different cytokines (Maes et al., 1995b, 1997a,b; Landmann et al., 1997). However, methodological concerns regarding serum levels of cytokines have to be taken into account. Regarding the relationship between IL-6 and PGE2 as mentioned above, an inhibiting action of anti-depressants on PGE2 would be expected (Pollack and Yirmiya, 2002). Over twenty years ago it has been suggested that anti-depressants inhibit PGE2 (Mtabaji et al., 1977). A recent in vitro study has shown that both tricyclic anti-depressants and selective serotonin inhibitors attenuated cytokine-induced PGE2 and nitric oxide production by inflammatory cells from synovial tissue (Yaron et al., 1999). The downregulating effects of anti-depressants on inflammatory cytokines, associated with the findings of increased proinflammatory cytokines and cellular immune markers, which were found mainly in non-suicidal, non-melancholic patients suffering from MD point out that this immune effect might be related to the therapeutic effects of antidepressants. Further therapeutic strategies for MD should take these immunological findings into account, including the different observations in the different clinical subgroups. From this point of view, development of immunotherapeutic strategies for drug treatment in MD will be a future research goal.

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7. Conclusion The current state regarding the correlation between the immune system and major psychoses reflects a controversial picture. In depression many psychoneuroimmunological studies have been performed by several groups especially during the last years. The findings point to different immunological/inflammatory abnormalities in different types of depression, such as non-melancholic versus melancholic symptomatology, and suicidality. In schizophrenia, the disease process seems to be related to an underactivation of the type-1 immune response and an overactivation of the type-2 response, and some antipsychotics counterregulate this effect by activating the type-1 response. A subgroup of schizophrenic patients, however, shows zsigns of an autoimmune process. The more conclusive results of immunological research in schizophrenia led to preliminary therapeutic studies with immunomodulatory or anti-inflammatory agents, which show encouraging results.

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Watts, R.G., Wright, J.L., Atkinson, L.L., Merchant, R.E., 1989. Histopathological and blood–brain barrier changes in rats induced by an intracerebral injection of human recombinant interleukin-2. Neurosurgery 25, 202–208. Weiss, G., Murr, C., Zoller, H., Haun, M., Widner, B., Ludescher, C., Fuchs, D., 1999. Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells. Clin. Exp. Immunol. 116, 435 –440. Wilke, I., Arolt, V., Rothermundt, M., Weitzsch, Ch., Hornberg, M., Kirchner, H., 1996. Investigations of cytokine production in whole blood cultures of paranoid and residual schizophrenic patients. Eur. Arch. Psychiatry Clin. Neurosci. 246, 279–284. Wu, H.Q., Rassoulpour, A., Schwarcz, R., 2002. Effect of systemic L -Dopa administration on extracellular kynurenate levels in the rat striatum. J. Neural Transm. 109, 239–249. Wyatt, R.J., Henter, I., 1995. An economic evaluation of manic-depressive illness—1991. Soc. Psychiatry Psychiatr. Epidemiol. 30, 213 –219. Wyatt, R.J., Henter, I., Leary, M.C., Taylor, E., 1995. An economic evaluation of schizophrenia—1991. Soc. Psychiatry Psychiatr. Epidemiol. 30, 196 –205. Xiao, B.G., Link, H., 1999. Is there a balance between microglia and astrocytes in regulating Th1/Th2-cell responses and neuropathologies? Immunol. Today 20, 477 –479. Yaron, I., Shirazi, I., Judovich, R., Levartovsky, D., Caspi, D., Yaron, M., 1999. Fluoxetine and amitriptylin inhibit nitric oxide, prostaglandin E2, and hyaluronic acid production in human synovial cells and synovial tissue cultures. Arthritis Rheum. 42, 2561– 2568. Yolken, R.H., Johnston, N., Leister, F., Torrey, E.F., 1999. Stanley Neuropathology Consortium (1999). The use of substraction libraries for the identification of RNA species upregulated in the brains of individuals with schizophrenia. In: Mu¨ller, N. (Ed.). Psychiatry, Psychoimmunology, and Viruses. Spinger, Wien, NY, pp. 7–17. Yolken, R.H., Torrey, E.F., 1995. Viruses, schizophrenia, and bipolar disorder. Clin. Microbiol. Rev. 8, 131 –145. Zalcman, S., Green-Johnson, J.M., Murray, L., Nance, D.M., Dyck, D., Anisman, H., Greenberg, A.H., 1994. Cytokine-specific central monoamine alterations induced by interleukin-1, -2 and -6. Brain Res. 643, 40–49. Zarrabi, M.H., Zucker, S., Miller, F., Derman, R.M., Romeno, G.S., Hartnett, J.A., Varma, A.O., 1979. Immunologic and coagulation disorders in chlorpromazine-treated patients. Ann. Int. Med. 91, 194–199. Zhu, J., Bengtsson, B.O., Mix, E., Thorell, L.H., Olsson, T., Link, H., 1994. Effect of monoamine reuptake inhibiting antidepressants on major istocompatibility complex expression on macrophages in normal rats and rats with experimental allergic neuritis (EAN). Immunopharmacology 27, 225– 244. Zubin, J., Spring, B., 1977. Vulnerability—a new view of schizophrenia. J. Abnorm. Psychol. 86, 103–126.

Shared effects of all three conventional anti-bipolar drugs on the phosphoinositide system in astrocytes Leif Hertz,a,b,* Ye Chen,c Yuly Bersudskyb and Marina Wolfsona a

Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beersheba, Israel p Correspondence address: RR 2, Box 245, Gilmour, Ont., Canada K0L 1W0 E-mail: [email protected] b Stanley Center for Bipolar Disorders, Faculty of Health Sciences, Ben Gurion University of the Negev, Beersheba, Israel c Department of Operational and Undersea Medicine, Naval Medical Research Center, Silver Spring, MD 20910, USA

Contents 1. 2. 3.

4.

5.

Introduction The phosphatidylinositide – inositolphosphate cycle Inositol 3.1. Inositol turnover and inositol pools 3.2. Effects of mood-stabilizing drugs on inositol uptake and pool size Effect of chronic treatment with mood-stabilizing drugs on signaling 4.1. Down-regulation of transmitter-induced changes in [Ca2þ]i 4.2. Effect of inositol uptake inhibitors in the lithium– pilocarpine test Concluding remarks

Chronic, but not acute treatment with any of the three conventional anti-bipolar drugs (lithium, valproic acid and carbamazepine) have recently been found to inhibit inositol uptake into primary cultures of astrocytes at high extracellular inositol concentrations, but to enhance it at low inositol concentrations. The possible importance of this effect for the therapeutic effects of these drugs in mood disorders is reviewed in relation to the phosphatidylinositide – inositolphosphate cycle, the turnover of inositol between its cellular pools and the extracellular fluid, and the effect of lithium on signaling in astrocytes and of inositol uptake inhibitors in the lithium – pilocarpine test. Advances in Molecular and Cell Biology, Vol. 31, pages 1033–1048 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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1. Introduction The term ‘phosphoinositide system’ will be used in this review as encompassing not only the phosphoinositide second messenger cascade, operating via the two second messengers inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), but also uptake and metabolism of the precursor myo-inositol (in the following referred to as inositol). There is evidence that uptake and metabolism of inositol are of major importance for the provision of phosphatidyl 4,5-bisphosphate (PIP2), the precursor for IP3 and DAG. In addition to being a precursor in the phosphoinositide second messenger system, inositol is also an important osmolyte, used to regulate intracellular tonicity during exposure to hypotonic or hypertonic surroundings. This is achieved by inositol release to the extracellular space during hypotonia and enhanced inositol accumulation during hypertonia. This aspect of inositol functioning will only be discussed in passing. Different facets of the phosphoinositide system are altered by chronic, but not by acute, treatment with drugs that have therapeutic effect in bipolar disorder. Chronic, but not acute, administration of lithium salts (in the following referred to as ‘lithium’) is effective for mood-stabilization in bipolar (manic-depressive) patients with little effect on diseasefree controls (Schou, 1968, 2001). Lithium treatment may reduce the efficacy of the phosphoinositide second messenger system by interfering with inositol availability. Direct effects on some components of this system have also been described after chronic treatment with lithium, and some of these may be secondary to inositol depletion (Manji et al., 1996). A crucial question has been whether newer mood-stabilizing drugs, such as valproic acid and carbamazepine (Post et al., 1992), share the ability to affect the phosphoinositide system. In some, but not all, cases this seems to be the case. Many pharmacological effects of inositol and lithium have been described using primary cultures of astrocytes or glioma cells. Moreover, noradrenaline and serotonin, two transmitters acting via the phosphoinositide system, have receptors on astrocytes (see chapter by Hansson and Ro¨nnba¨ck), and affect crucial aspects of astrocytic function (Chen et al., 1995; Chen and Hertz, 1999—see also chapter by Peng et al.). Although this by no means is an indication that anti-bipolar drugs may not exert direct effects on neurons, it is a distinct possibility that these drugs primarily target astrocytes. This is especially the case, since IP3 is an important second messenger in astrocytes, where it contributes to the propagation of transcellular Ca2þ waves, which in turn influence neuronal activity (see chapter by Cornell-Bell et al. and by Shuai et al.). 2. The phosphatidylinositide –inositolphosphate cycle The biochemical core of the phosphoinositide second messenger system is the phosphatidylinositide –inositolphosphate cycle illustrated in Fig. 1. In this cycle different transmitters (e.g., noradrenaline, serotonin, acetylcholine) activate phospholipase C to hydrolyze PIP2, a component of the plasma membrane, localized at the inner leaflet of the phospholipid bilayer. The hydrolysis of PIP2 produces the two second messengers, cytosolic IP3 and membrane-associated DAG, each of which triggers ‘down-stream’ reactions, DAG by activating protein kinase C and IP3 by causing a release of Ca2þ from intracellular stores (see chapter by Scapagnini et al.). Subsequently IP3 is degraded to

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Fig. 1. The phosphatidylinositide–inositolphosphate cycle, in which receptor stimulation leads to conversion of phosphatidylinositide-4,5-bisphosphate (PIP2), a minor component of the cell membrane, to the two second messengers inositoltrisphosphate (IP3), which is released to the cytosol (within the oval), and DAG, which remains membrane-associated (between the oval and the octagonal). Subsequent metabolism of IP3 produces inositol bisphosphate (IP2), inositol monophoshate (IP) and myo-inositol (inositol), and the generated inositol condenses with cytidine monophosphoryl-phosphatidate (CMP-PA), a metabolite of DAG to form phosphatidylinositol (PI), from which PIP2 is regenerated via PIP. Tear and wear of cycle constituents as well as release (efflux) of inositol are compensated for by de novo synthesis of inositol from glucose and by uptake of inositol from the extracellular fluid, mediated by two different inositol transporters: a high-affinity Naþdependent myo-inositol transporter (SMIT) and a lower-affinity HMIT. Inhibition of inositol monophosphatase by lithium (Li), indicated by vertically striped bar, interferes with the formation of inositol from IP and thus with regeneration of PIP2, and may thereby jeopardize formation of IP3 and DAG upon renewed receptor stimulation. However, valproic acid (VPA) and carbamazepine (Cbz) do not inhibit inositol monophosphatase, but VPA inhibits inositol formation from glucose (vertically striped bar). Chronic (but not acute) treatment with either Li, VPA, or Cbz inhibits one astrocytic inositol transporter (vertically striped bar) but stimulates another astrocytic inositol transporter (vertically striped arrow), which may account for often elusive effects of lithium treatment on inositol pool size in brain.

inositol bisphosphate (IP2) and inositol monophosphate (IP) by inositol polyphosphate monophosphatases. IP is further hydrolyzed by inositol monophosphatase to inositol. DAG is converted to phosphatidate and condensed with cytidine triphosphate to form cytidine monophosphoryl-phosphatidate (CMP-PA), which reacts with inositol to generate new PIP2 via phosphatidyl monophosphate (PI) and phosphatidyl bisphosphate (PIP). The rate of turnover in the phosphatidylinositide – inositolphosphate cycle depends upon the extent to which the phosphoinositide second messenger cascade is activated, but based upon accumulation of IP during inhibition of inositol monophosphatase with lithium (Sherman et al., 1985), it can be estimated to exceed 1 mmol/h per kg wet wt in the normal ‘resting’ brain (Hertz et al., 1997). The formation of inositol from IP is uncompetitively inhibited by lithium (Fig. 1; Hallcher and Sherman, 1980), leading to a lithium-induced increase in IP both in the brain in vivo (Sherman et al., 1981) and in astrocytoma cells (Batty and Downes, 1995). Due to the uncompetitive nature of the inhibition this increase will not overcome the inhibition of the enzyme. From the IP concentrations in astrocytoma reported by Batty and Downes in the presence and absence of lithium (at the therapeutically relevant concentration of 1 mM) and the uncompetitive character of the inhibition we have previously calculated that 1 mM lithium decreases the inositol monophosphatase-mediated flux by 50% (Hertz et al., 1997).

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As suggested by Berridge et al. (1982, 1989) the inhibition of inositol monophosphatase by lithium and a possible resulting shortage of inositol for re-synthesis of PI may reduce the availability of PIP and PIP2 and thus lead to impairment of the continued ability to generate IP3 and DAG from PIP2 upon renewed receptor stimulation (the inositol depletion theory). A small, but significant decrease in PI has recently been demonstrated in rat brain during chronic treatment with lithium at pharmacologically relevant doses (Pettegrew et al., 2001), but it takes extreme reduction of inositol supply in cultured 1321N1 astrocytoma cells to evoke a decrease in PI by lithium (Batty and Downes, 1994). Some authors have observed a decrease in brain inositol levels during prolonged treatment with lithium, but the decrease is often limited to a few brain regions (Allison and Stewart, 1971; Moore et al., 1999a; Manji and Lenox, 1999), and other authors found no decrease at all (e.g., Jope and Williams, 1994; Pettegrew et al., 2001). Moreover, in contrast to the requirement for chronic treatment to achieve lithium’s therapeutic effect, the inhibition of inositol monophosphatase activity is immediate (Hallcher and Sherman, 1980; Sherman et al., 1985), and the increase in inositol in human brain becomes apparent earlier than the therapeutic effect of lithium (Moore et al., 1999a). Newer anti-bipolar anti-convulsants, such as carbamazepine and valproic acid, do not share the ability if lithium to inhibit inositol monophosphatase (Vadnal and Parthasarathy, 1995). However, an ability of valproic acid to inhibit inositol synthesis from glucose has been observed in yeast (Vaden et al., 2001), suggesting that this drug may also lead to inositol depletion (Fig. 1), and valproic acid has recently been found to decrease inositol levels in brain (O’Donnell et al., 2000). Since de novo synthesis of inositol proceeds via IP, it is also likely to be inhibited by lithium. However, carbamazepine has not been reported to inhibit the formation of inositol from glucose. There are presently two sets of observations describing similar effects by all three conventional mood-stabilizing drugs: lithium, valproic acid and carbamazepine. One of these is the ability to affect inositol uptake in glioma cells after chronic, but not acute treatment with therapeutically relevant concentrations of either lithium, carbamazepine or valproic acid (Lubrich and van Calker, 1999; Wolfson et al., 2000a,b), which will be discussed below in some detail. The second is an induction of morphological changes in growth cones of dorsal root ganglion neurons from newborn rats, an effect, which can be counteracted by inositol (Williams et al., 2002). In contrast to the effects on inositol uptake, which require treatment for more than one week, the growth cone effects appear already after a few hours of drug treatment (A. Mudge, personal communication). 3. Inositol 3.1. Inositol turnover and inositol pools 3.1.1. Inositol content and formation Inositol is a 6-carbon cyclic polyol chemically closely related to glucose, from which it is synthesized in plants and several mammalian organs (Aukema and Holub, 1994). Inositol is accordingly present in the diet, and it can enter cerebrospinal fluid via the choroid plexus and brain parenchyma across the blood –brain barrier, albeit slowly

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(Spector and Lorenzo, 1975). It is also synthesized within the brain (Hauser, 1963; Novak et al., 1999), and it has been calculated that normally inositol uptake across the blood – brain barrier and de novo synthesis of inositol in brain each provide approximately similar amounts of inositol to the brain (Hertz et al., 1997). However, this calculation does not consider possible efflux of inositol across the blood –brain barrier, and it may accordingly overestimate the contribution of blood-borne inositol. Nevertheless, the ability of inositol to cross the blood –brain barrier means that systemic administration of inositol can increase the inositol pool in brain (Patishi et al., 1996); however, the increase is only transient (Moore et al., 1999b). The inositol concentration in plasma is around 60 mM, that in cerebrospinal fluid and probably also in brain extracellular fluid is a few times higher, and the intracellular content is in the low millimolar range (Hertz et al., 1997; Fisher et al., 2002). Early evidence indicated that neurons contain very little free inositol (Glanville et al., 1989; Brand et al., 1993), but newer measurements of inositol content in cultured cerebellar granule neurons and especially NT2-N cells, a cell line model of differentiated neurons, suggest that the pool sizes of free inositol in neurons and astrocytes may be rather similar (Novak et al., 1999). However, there are pronounced differences, e.g., between different types of neurons and to a lesser extent between astrocytes from different regions (Lubrich et al., 2000). The Purkinje cell layer in the cerebellum appears to have higher inositol content than most other brain regions (Fisher et al., 2002). Immunohistochemical staining has shown that myo-inositol 1-phosphate synthase, a key enzyme for the production of inositol via IP, is confined to the vascular elements, an observation that was corroborated by the demonstration that enzyme activity was displayed only by isolated microvessels and not by other cell fractions (Wong et al., 1987). However, based on these studies it may not be possible to exclude that synthesis of inositol could occur in astrocytic endfeet closely associated with the microvessels. Since inositol synthesis only occurs in or close to the microvessels, all brain cells and cellular constituents, with the possible exception of astrocytic endfeet, must accumulate inositol from the extracellular space in order to compensate for tear and wear in the phosphatidylinositide –inositolphosphate cycle and inositol release (efflux) from the cells. Therefore uptake kinetics for inositol in different cell types and possible interference of inositol uptake by anti-bipolar drugs are of major importance for the availability of inositol within cells and thus for the maintenance of the phosphoinositide system. 3.1.2. Inositol transporters Up till recently it was supposed that only one inositol transporter, the Naþ-dependent myo-inositol transporter (SMIT) existed in mammalian cells. It is consistent with this point of view that, with the exception of Wolfson et al. (1998), all authors had reported relatively similar kinetics (Vmax ; Km ) for inositol uptake into primary cultures of astrocytes, astrocytoma cells and neuroblastoma cells. As can be seen from Table 1, Km has consistently been found to be between 12 and 50 mM and Vmax to be 0.1 –0.5 nmol/min per mg protein, with the exception of 10 times lower value in some of the neuroblastoma cell lines. Moreover, inositol uptake in cerebral cortical neurons in primary cultures at an inositol concentration of 135 mM (which probably yields an uptake relatively close to the

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Vmax value of this uptake system) amounts to 0.23 nmol/min per mg protein (Acevedo et al., 1997). Based upon a Lineweaver – Burk covering the entire concentration range between 3.6 mM and 10 mM inositol, Wolfson et al. (1998) determined the Km for inositol uptake into primary cultures of mouse astrocytes to be , 500 mM, and the Vmax to be 1.5 nmol/ min per mg protein (Table 1). This difference from other results shown in Table 1 is due to an additional low-affinity, high-capacity uptake system called into action at sufficiently high extracellular inositol concentrations. This uptake system is functionally relevant, since the observed Km value is close to the inositol concentration in cerebrospinal fluid (, 400 mM). However, when the concentration range up to 10 mM is depicted in the Lineweaver –Burk plot, a possible contribution from a high-affinity, lower capacity uptake will be obscured. We have therefore now re-plotted the original values from Wolfson et al. (1998) within the range 3.6– 100 mM, and identified an additional high-affinity uptake with similar kinetics as those described by other authors (Table 1). Thus, two different uptake systems, one with a Km of , 40 mM, and the second with a Km of , 500 mM exist in mouse astrocytes in primary cultures. Also, Lubrich et al. (1999) pointed out that an additional, apparently non-saturable uptake system operated in their primary cultures of rat astrocytes at inositol concentrations above 1 mM. Batty et al. (1993) found no evidence for a low-affinity inositol uptake system in their 1321 N1 astrocytoma cells, but a preliminary kinetic analysis of inositol uptake in U251-MG astrocytoma cells (M. Wolfson, Y. Bersudsly and L. Hertz, unpublished experiments) showed the presence of two kinetically different compartments, and it allowed a reasonably accurate determination of Km (, 40 mM) and Vmax (0.2 nmol/min per mg protein) for the high-affinity system together with a more uncertain estimate of a Km of . 200 mM and a Vmax of at least 1.5 nmol/min per mg protein for the low-affinity uptake system (last line of Table 1). The demonstration of two uptake systems for inositol in astrocytes has become very pertinent with the recent demonstration of an additional adenosine transporter, the Hþdependent myo-inositol transporter (HMIT), which is abundant in brain and is mainly, although not exclusively present in glial cells (Uldry et al., 2001). Since HMIT has a relatively high Km (, 100 mM) it is likely that the low-affinity inositol uptake shown in both primary cultures of astrocytes and at least one astrocytoma cell line represents HMITmediated uptake. However, this needs to be verified experimentally, and one possible way to distinguish between SMIT- and HMIT-mediated inositol uptake is that the former is inhibited by a lowering of pH, whereas HMIT is stimulated at low pH (Uldry et al., 2001). However studies of inositol uptake at different pH values remain to be performed. 3.1.3. Inositol pools Studies of efflux of previously accumulated radioactive inositol from primary cultures of astrocytes or from astrocytoma cells into a non-radioactive medium have consistently led to the conclusion that inositol in astrocytes is present in several, kinetically different compartments (Isaacks et al., 1999; Wolfson et al., 2000a). In Table 2 the characteristics observed by Wolfson et al. (1998; 2000a) are shown. It can be seen that inositol in one pool is rapidly released from the cells (half-life 15 min); that another pool turns over somewhat more slowly (half-life 45 min); and that there is a large pool, which turns over quite slowly

Cell type

Primary cortical interneurons Neuroblastoma cells Primary rat astrocytes Primary mouse astrocytes Primary mouse astrocytes C-6 glioma cells 1321 N1 astrocytoma cells U251-MG astrocytoma cellsa a

High-affinity uptake Km (mM)

Vmax (nmol min21 mg21)

12– 46 13– 50 25 ,40 15 40 ,40

.0.23 0.01– 0.15 0.16– 0.47 0.06 0.4 0.12 0.18 0.2

Low-affinity uptake Km (mM)

Vmax (nmol min21 mg21)

500

1.5

.200

.1

A human malignant glial cell line (Ponte´n and Macintyre, 1968). Calculated from inositol uptake rates of 10, 25, 37.5, 50 and 100 mM inositol, using 8 –12 individual cultures at each concentration.

b

Reference

Acevedo et al., 1997 Fisher et al., 2002 Fisher et al., 2002 Wiesinger, 1991 Wolfson et al., 1998 Paredes et al., 1992 Batty et al., 1993 Present workb

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Table 1 Kinetics (Km ; Vmax ) for high- and low-affinity uptake of myo-inositol into preparations of isolated neurons or astrocytes

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Table 2 Pool sizes (nmol/mg protein), half-lives ðt1=2 Þ; rate constants ð0:693=t1=2 Þ and efflux (nmol/(min mg protein)) from kinetically determined inositol pools in primary cultures of mouse astrocytes Pool

Pool sizea (nmol mg21)

Half-lifeb (min)

Rate constantc (min21)

Effluxd (nmol min21 mg21)

Rapidly exchanging Intermediate Slowly exchanging

5.1 6.9 22.3

15.4 46 533

0.045 0.015 0.0013

0.23 0.10 0.03

a

Determined from equilibrated inositol space in Wolfson et al. (1998) and efflux curves in Wolfson et al. (2000a). Determined from efflux curves in Wolfson et al. (2000a). c Calculated from half-lives ðt1=2 Þ as 0:693=t1=2 : d Calculated by multiplication of pool sizes with rate constants. b

(half-life 9 h). Similar compartmentation, but somewhat longer half-lives can be deduced for astrocytoma cells from the efflux data presented by Glanville et al. (1989). In the neuronal NT2-N cells there is, in contrast, only one compartment that turns over with a half-life, which can be calculated to amount to more than 24 h (Novak et al., 1999), i.e., three times more slowly than the most slowly exchanging compartment in astrocytes. The magnitude and the release rate of the most rapidly exchanging compartment in Table 2 are similar to those reported by Isaacks et al. (1999). They found that inositol efflux from this compartment is greatly increased during incubation in hypotonic medium as a means of volume regulation by reduction of the content of inositol and other intracellular components functioning as osmolytes. This compartment is different from the inositol pool(s) participating in the phosphatidylinositide – inositolphosphate cycle. This is shown by the observation by Bersudsky et al. (1994) that a small reduction in the total pool of inositol in the rat brain during joint exposure to lithium (inhibiting inositol monophosphatase activity) and pilocarpine (stimulating PIP2 degradation) led to seizures, that could be counteracted by additional administration of inositol, whereas chronic hyponatremia did not induce seizure activity in spite of a much larger reduction in inositol pool size due to inositol efflux. The inositol pool from which PI is re-synthesized is therefore probably correlated with either the most slowly exchanging compartment shown in Table 2 or perhaps rather with the intermediate compartment. This inositol pool may be larger than generally envisaged, since Batty et al. (1998) have obtained evidence suggesting that PIP2 in glioma cells is re-splenished from PI synthesized from inositol in large cellular domains, possibly distributed between different membrane fractions. 3.2. Effects of mood-stabilizing drugs on inositol uptake and pool size 3.2.1. Uptake It was shown by Lubrich and van Calker (1999) that chronic, but not acute treatment with 1 mM lithium inhibits inositol uptake in human astrocytoma cells and in primary cultures of astrocytes from all regions studied, including cerebral cortex as well as hippocampus. At the same time there was a reduction in the expression of SMIT.

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Intriguingly, therapeutic concentrations of valproic acid or carbamazepine had a similar effect. This was the first observation that all three conventional anti-bipolar drugs share a common effect on any aspect of the phosphoinositide second messenger system in any brain preparation. However, this finding does not explain why chronic administration of lithium in several brain regions does not decrease inositol pool size and why demonstration of a reduction in inositol content in many cases has been elusive. This may be understandable in the light of a subsequent study by Wolfson et al. (2000b), in which cells from a different astrocytoma cell line were treated chronically with either 0.5– 2 mM lithium, 25 – 100 mM carbamazepine or 0.3– 1.2 mM valproic acid (one half to twice usual therapeutic blood levels). Wolfson et al. (2000b) treated the cells at an inositol concentration of 40 mM (the standard inositol concentration of the medium), and they measured inositol uptake at 25, 40, or 50 mM inositol. The uptake of 50 mM inositol was reduced by one half after two weeks of treatment with 1 mM lithium (Fig. 2), confirming the observation by Lubrich and van Calker (1999). A similar reduction of inositol uptake at concentrations of 50 mM (and higher) can be demonstrated for valproic acid and carbamazepine (Wolfson et al., 2000a,c; M. Wolfson, Y. Bersudsky and L. Hertz, unpublished experiments). Surprisingly, the uptake at 25 mM inositol was, however, significantly enhanced by chronic lithium treatment, i.e., almost doubled. A quantitatively similar stimulation (91%) was observed at 25 mM inositol after treatment with 0.6 mM valproic acid for 2 weeks, whereas the response to treatment with 50 mM carbamazepine was much more pronounced and quite variable in magnitude (401 þ 109%). This might suggest that the concentration of carbamazepine was relatively larger (compared to its effect) than that of the two other drugs, since similar treatment with 25 mM carbamazepine caused a stimulation (121%), which was similar to that seen with 1 mM lithium and 0.6 mM valproic acid. In contrast, treatment for 2 weeks with 0.5 mM lithium or 0.3 mM valproic acid (one half of therapeutically relevant concentration) had no significant effect on uptake of 25 mM

Fig. 2. Uptake of [3H]myo-inositol during 60 min in U251 MG astrocytoma cells treated with 1 mM lithium chloride for 2 weeks before the uptake experiment as well as during the uptake (filled columns) and in untreated control cultures (open columns). Uptakes were measured at 25, 40 and 50 mM inositol in the medium. SEM values are shown by vertical bars. At both 25 and 50 mM the difference between control cultures and lithiumtreated cultures was statistically significant (P , 0.05). From Wolfson et al. (2000b).

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inositol. Conversely, a doubling of the concentration of lithium and valproic acid led to a stimulation, which was of the same magnitude as that seen with 50 mM carbamazepine. Treatment for only one week with either of the drugs had no significant stimulatory effect. The anti-depressant amitriptyline (Lubrich and van Calker, 1999) and the anti-psychotic haloperidol (M. Wolfson, Y. Bersudsky and L. Hertz, unpublished experiments) have no similar effects. However, previous exposure to elevated inositol concentrations downregulates the uptake (Wolfson et al., 1998; Lubrich and van Calker, 1999), possibly explaining why dietary inositol supplementation causes only a transient increase in the inositol content in brain (Moore et al., 1999b). With the kinetics illustrated in Table 1 the high affinity system must account for most of the uptake at 25 mM inositol, but progressively smaller fractions, when the concentration exceeds its Km value of , 40 mM. Accordingly it must be the high-affinity system, which is enhanced after chronic treatment with lithium, carbamazepine or valproic acid. Based on the Km values for SMIT and HMIT this would suggest a stimulation of SMIT, but the inhibition of SMIT expression after chronic treatment with any of the three anti-bipolar drugs (van Calker and Lubrich, 1999) does not support this hypothesis. However, the reason for the altered uptakes is not necessarily a change in transporter expression, but it could be intracellular pH changes, e.g., a gradually developing intracellular alkalinization, which would reduce HMIT activity and stimulate SMIT activity. In other cell types there is evidence that chronic treatment with lithium may change pH-regulating mechanisms (Bitran et al., 1990). 3.2.2. Equilibrated inositol space Changes in uptake rate of inositol are likely to affect the efficacy of the phosphoinositide second messenger system only if they change the pool size of available inositol. This pool size may be estimated by determination of ‘equilibrated inositol space’, i.e., the total amount of accumulated labeled inositol after it has achieved equilibrium with all exchangeable intracellular inositol. The effect of chronic treatment with lithium for two weeks on the equilibrated inositol space is shown in Fig. 3. It can be seen that this pool changes in parallel with inositol uptake: an increase at low inositol concentrations, no effect at intermediate inositol concentrations (25 and 50 mM), and a decrease at elevated inositol concentrations. A similar effect could be demonstrated for valproic acid, whereas only a decrease could be found in the case of carbamazepine (results not shown). This might be related to the fact that the relative concentration of carbamazepine may have been higher than those of the two other drugs. 4. Effect of chronic treatment with mood-stabilizing drugs on signaling 4.1. Down-regulation of transmitter-induced changes in [Ca2þ]i It is a pertinent question whether the changes in inositol uptake and pool size affect the responsiveness of astrocytes to transmitters acting via the phosphoinositide second messenger system. Astrocytes in primary cultures and in the brain in vivo express receptors for noradrenaline (see chapter by Hansson and Ro¨nnba¨ck), which increases

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Fig. 3. Effect of treatment with 1 mM lithium for 2 weeks on equilibrated spaces of [3H]myo-inositol after 24 h exposure to the isotope in U251 MG astrocytoma cells. All results for the lithium-treated cells (filled squares) are shown as percentages of the corresponding spaces in untreated control cultures (open squares). The [3H]myo-inositol spaces were measured at 10, 25, 50, 100, 500 and 1000 mM inositol in the medium, and the concentrations are plotted logarithmically. SEM values are indicated by vertical bars (if exceeding beyond the symbols), and statistically significant differences between control cultures and lithium-treated cultures (P , 0:05 or better) are indicated by asterisks. Results are means of four individual experiments for both control and treated cultures at each concentration and are previously unpublished experiments by M. Wolfson, Y. Bersudsky and L. Hertz.

phospholipid hydrolysis (Ritchie et al., 1987), raises the concentration of free cytosolic Ca2þ ([Ca2þ]i) (Chen and Hertz, 1999) and stimulates energy metabolism (see chapter by Hertz, Peng et al.). As illustrated in Fig. 4, chronic exposure to 1 mM lithium during 7– 14 days greatly attenuates the noradrenaline-induced increase in [Ca2þ]i in primary cultures of astrocytes, maintained at an inositol concentration of 40 mM (Chen and Hertz, 1996). At the same time there is a small but statistically significant decrease in the non-stimulated [Ca2þ]i. Unfortunately it is not known whether identical responses can be evoked by chronic administration of valproic acid or carbamazepine. Nor is it known whether a similar effect of noradrenaline might be elicited in any type of neuronal preparation. Although noradrenaline does stimulate the phosphoinositide second messenger system in cultured neurons (Weiss et al., 1988), there is little, if any, evidence of noradrenalinemediated increases in [Ca2þ]i. Carbachol does increase [Ca2þ]i in human neuroblastoma SH-SY5Y cells, but pretreatment for 7 days with lithium has no significant effect on the increase (Pacheco and Jope, 1999). A reduction of transmitter-induced increase in [Ca2þ]i in cultured astrocytes by chronic exposure to lithium has also been observed in C-6 glioma cells exposed to serotonin (Yamaji et al., 1997) and in primary cultures of astrocytes stimulated with thrombin (Kagaya et al., 2000), which likewise causes an increase in astrocytic [Ca2þ]i by activation of the phosphoinositide second messenger system (see chapter by Hansson and Ro¨nnba¨ck).

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Fig. 4. Free cytosolic calcium concentration ([Ca2þ]i) in primary cultures of mouse astrocytes during basal conditions (left part) and during exposure to 1 mM noradrenaline (right part) in untreated control cultures (open columns) and in sister cultures (filled columns) from the same batches, which were studied in parallel and had been treated with 1 mM lithium chloride (at 40 mM inositol) for 7–14 days before the experiment as well as during the measurements. SEM values are shown by vertical bars, and statistically significant differences between treated and untreated cultures are indicated by asterisks. From Chen and Hertz (1996).

4.2. Effect of inositol uptake inhibitors in the lithium – pilocarpine test 4.2.1. The lithium –pilocarpine test A quasi-behavioral indication of altered signaling mechanisms in the brain after treatment with lithium (although generally only during a relatively short period) is the seizure-provoking effect of joint administration of lithium and pilocarpine (the lithium – pilocarpine test). As indicated by the finding that the induced seizures can be attenuated by administration of inositol (Tricklebank et al., 1991; Kofman et al., 1993), the seizures may be secondary to a longer than usual increase in DAG on account of the reduced ability to synthesize IP from the DAG metabolite CMP-PA, when inositol supply is deficient (Fig. 1). If the effects of lithium, carbamazepine and valproic acid on inositol availability and thus on the phosphatidylinositide – inositolphosphate cycle are functionally meaningful, one would therefore expect that lithium effects could be mimicked, or at the very least accentuated, by simultaneous administration of an inositol uptake inhibitor.

4.2.2. Inositol uptake inhibitors Fucose and nordidemnin are known to inhibit inositol uptake in other cell types, and both compounds also inhibit inositol uptake in astrocytes (Wolfson et al., 2000a,c). Fucose has been found to inhibit inositol uptake in neuroblastoma cells (Yorek et al., 1992), but in preliminary studies using a different neuroblastoma cell line, we observed no inhibition (M. Wolfson, Y. Bersudsky and L. Hertz, unpublished experiments). It would be of importance to study the effect of inositol uptake inhibitors in additional neuronal preparations, before any safe conclusions can be drawn whether their effects in vivo are due to inhibition of inositol uptake in both neurons and astrocytes or to a selective inhibition of astrocytic uptake.

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4.2.3. Effects of inositol uptake inhibitors in the lithium – pilocarpine test Two inositol uptake inhibitors, fucose (Einat et al., 1998) and nordidemnin (Wolfson et al., 2000a – c), have been tested for their possible seizure-provoking effect in the lithium – pilocarpine test. Both inhibitors enhanced the effect of lithium together with pilocarpine by reducing the latency from their administration till the onset of seizures for nordidemnin. However, it has not been possible to replace lithium with an inositol uptake inhibitor, possibly because both fucose and nordidemnin have to be given intracerebroventricularly, and it may be difficult to maintain effective concentrations over sufficiently long time periods.

5. Concluding remarks The present chapter has compiled evidence in favor of an ‘expanded inositol depletion hypothesis’ according to which interference with inositol uptake by anti-bipolar drugs in astrocytes (without excluding related effects in neurons) may lead to down-regulation (and in some cases perhaps up-regulation) of the phosphoinositide second messenger system. An advantage over the ‘classical’ lithium depletion theory is that (i) the proposed hypothesis accounts for the effects of all three mood-stabilizing drugs studied; (ii) it is consistent with the need for chronic treatment; and (iii) it may explain the limitation of lithium effects on brain inositol pool size to specific brain regions, depending upon extracellular inositol concentrations. Also, the dual effects of chronic treatment with antibipolar drugs on inositol uptake at different inositol concentrations appears intuitively to be more in agreement with the peculiar ability of these drugs to remedy both manic and depressive phases of bipolar disorder, and the presence of an intermediate inositol concentration, at which there is virtually no effect is consistent with lithium’s lack of mood effects on controls who do not suffer from affective disorders. Acknowledgements Professor R.H. Belmaker, Psychiatric Hospital, Ben Gurion University of the Negev, Beersheba, Israel, is cordially thanked for his continuous support of the studies on which this review is based.

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Moore, G.J., Bebchuk, J.M., Parrish, J.K., Faulk, M.W., Arfken, C.L., Strahl-Bevacqua, J., Manji, H.K., 1999a. Temporal dissociation between lithium-induced changes in frontal lobe myo-inositol and clinical response in manic-depressive illness. Am. J. Psychiatry 156, 1902–1908. Moore, C.M., Breeze, J.L., Kukes, T.J., Rose, S.L., Dager, S.R., Cohen, B.M., Renshaw, P.F., 1999b. Effects of myo-inositol ingestion on human brain myo-inositol levels: a proton magnetic resonance spectroscopic imaging study. Biol. Psychiatry 45, 1197–1202. Novak, J.E., Turner, R.S., Agranoff, B.W., Fisher, S.K., 1999. Differentiated human NT2-N neurons possess a high intracellular content of myo-inositol. J. Neurochem. 72, 1431–1440. O’Donnell, T., Rotzinger, S., Nakashima, T.T., Hanstock, C.C., Ulrich, M., Silverstone, P.H., 2000. Chronic lithium and sodium valproate both decrease the concentration of myo-inositol and increase the concentration of inositol monophosphates in rat brain. Brain Res. 880, 84– 91. Pacheco, M.A., Jope, R.S., 1999. Modulation of carbachol-stimulated AP-1 DNA binding activity by therapeutic agents for bipolar disorder in human neuroblastoma SH-SY5Y cells. Mol. Brain Res. 72, 138 –146. Patishi, Y., Lubrich, B., Berger, M., Kofman, O., van Calker, D., Belmaker, R.H., 1996. Differential uptake of myo-inositol in vivo into rat brain areas. Eur. Neuropsychopharmacol. 6, 73 –75. Paredes, A., McManus, M., Kwon, H.M., Strange, K., 1992. Osmoregulation of Naþ inositol cotransporter activity and mRNA levels in brain glial cells. Am. J. Physiol. 263, C1282–C1288. Pettegrew, J.W., Panchalingam, K., McClure, R.J., Gershon, S., Muenz, L.R., Levine, J., 2001. Effects of chronic lithium administration on rat brain phosphatidylinositol cycle constituents, membrane phospholipids and amino acids. Bipolar Disord. 3, 189–201. Ponte´n, J., Macintyre, E.H., 1968. Long term culture of normal and neoplastic human glia. Acta Pathol. Microbiol. Scand. 74, 465–486. Post, R.M., Weiss, S.R., Chuang, D.M., 1992. Mechanisms of action of anticonvulsants in affective disorders: comparisons with lithium. J. Clin. Psychopharmacol. 12, 23S–35S. Ritchie, T., Cole, R., Kim, H.S., De Vellis, J., Noble, E.P., 1987. Inositol phospholipid hydrolysis in cultured astrocytes and oligodendrocytes. Life Sci. 41, 31–39. Sherman, W.R., Leavitt, A.L., Honchar, M.P., Hallcher, L.M., Phillips, B.E., 1981. Evidence that lithium alters phosphoinositide metabolism: chronic administration elevates primarily D -myo-inositol-1-phosphate in cerebral cortex of the rat. J. Neurochem. 36, 1947–1951. Sherman, W.R., Munsell, L.Y., Gish, B.G., Honchar, M.P., 1985. Effects of systemically administered lithium on phosphoinositide metabolism in rat brain, kidney and testis. J. Neurochem. 44, 798 –807. Schou, M., 1968. Lithium in psychiatric therapy and prophylaxis. J. Psychiatr. Res. 6, 67–95. Schou, M., 2001. Lithium treatment at 52. J. Affect. Disorder 67, 21 –32. Spector, R., Lorenzo, A.V., 1975. The origin of myo-inositol in brain, cerebrospinal fluid and choroid plexus. J. Neurochem. 25, 353 –354. Tricklebank, M.D., Singh, L., Jackson, A., Oles, R.J., 1991. Evidence that a proconvulsant action of lithium is mediated by inhibition of myo-inositol phosphatase in mouse brain. Brain Res. 558, 145 –148. Uldry, M., Ibberson, M., Horisberger, J.D., Chatton, J.Y., Riederer, B.M., Thorens, B., 2001. Identification of a mammalian H(þ)-myo-inositol symporter expressed predominantly in the brain. EMBO J. 20, 4467–4477. Vaden, D.L., Ding, D., Peterson, B., Greenberg, M.L., 2001. Lithium and valproate decrease inositol mass and increase expression of the yeast INO1 and INO2 genes for inositol biosynthesis. J. Biol. Chem. 276, 15466–15471. Vadnal, R., Parthasarathy, R., 1995. Myo-inositol monophosphatase: diverse effects of lithium, carbamazepine, and valproate. Neuropsychopharmacology 12, 277– 285. Weiss, S., Schmidt, B.H., Sebben, M., Kemp, D.E., Bockaert, J., Sladeczek, F., 1988. Neurotransmitter-induced inositol phosphate formation in neurons in primary culture. J. Neurochem. 50, 1425–1433. Wiesinger, H., 1991. Myo-inositol transport in mouse astroglia-rich primary cultures. J. Neurochem. 56, 1698–1704. Williams, R.S.B., Cheng, L., Mudge, A.W., Harwood, A.J., 2002. A common mechanism of action for three mood-stabilizing drugs. Nature 417, 292–295. Wolfson, M., Hertz, E., Belmaker, R.H., Hertz, L., 1998. Chronic treatment with lithium and pretreatment with excess inositol reduce inositol pool sizes by different mechanisms. Brain Res. 787, 34– 40.

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Glial loss in mood disorders and schizophrenia Joseph L. Price Department of Anatomy and Neurobiology, School of Medicine, Washington University, St Louis, MO 63130, USA E-mail: [email protected]

Contents 1. 2. 3.

4.

Depression circuit Cell count data indicating glial loss Glial type—astrocytes or oligodendrocytes? 3.1. Astrocytes 3.2. Oligodendrocytes Concluding remarks

Evidence that there is a low density of glia in the frontal cortex and amygdala in mood disorders and schizophrenia has accumulated from several sources. The evidence is now relatively strong, although it is still based on relatively few cases. At present there is better evidence that oligodendrocytes account for the glial deficit than astrocytes, which could substantially affect the interpretation of the effect. An astrocytic deficit would suggest that the environment within which neurons function might be compromised. Changes in astrocyte roles such as buffering of extracellular Kþ, uptake of glutamate at synapses, and supplying metabolic intermediates to neurons would alter neuronal excitability significantly. If the deficit is due to oligodendrocytes, it is more likely that there is an abnormality in myelinated axons. This could reflect either a difference in myelinated axons, or a more dynamic effect on myelinating oligodendrocytes. 1. Depression circuit Until recently, ideas about the neural basis of major depressive disorder (MDD) and other mood disorders were primarily focused on transmitters such as nor-epinephrine and serotonin. There is now considerable evidence from functional neuroimaging and other techniques, however, that individuals suffering from mood disorders have Advances in Molecular and Cell Biology, Vol. 31, pages 1049–1057 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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functional changes in several specific brain structures. These most definitively include the amygdala, the pre- and subgenual part of the anterior cingulate cortex, and the orbital cortex, but depression related changes have also been seen in the dorsolateral prefrontal cortex, medial thalamus, and hippocampus (Drevets, 2000; Manji et al., 2001; Davidson et al., 2002). Neuroanatomical experiments in monkeys and other experimental animals have also shown that these and other related areas (e.g., the ventral striatum and pallidum) are linked by axonal connections, such that they form a ¨ ngu¨r and Price, 2000). For example, the amygdala has distinct circuit (Fig. 1; O reciprocal connections with parts of the orbital and medial prefrontal cortex, and also projects to the mediodorsal thalamic nucleus, which itself has reciprocal connections with the prefrontal cortex. Both the prefrontal cortex and the amygdala project to the ventromedial striatum, which projects through the ventral pallidum to the mediodorsal thalamic nucleus. This circuit is involved in several functions, including modulation of visceral function in relation to emotional stimuli, appreciation and reaction to reward, and promotion of appropriate decisions, and it appears that it is centrally involved in mood disorders. It is in the structures of this circuit that decreases in glial cell number, density, and/or glia-to-neuron ratio have been found. Many of the studies of glial deficits in mood disorders have also examined schizophrenic cases. This data will be mentioned along with the data on mood disorders. It suggests that there is possibly a similar effect on glia in schizophrenia, although the evidence is more mixed.

Fig. 1. Diagram of connections among forebrain structures that have been implicated in mood disorders. The pointed arrows indicate excitatory (glutamatergic) connections, while the rounded arrows indicate inhibitory (GABAergic) connections. The structures outlined in a heavy border have been shown with functional imaging methods to have activity differences in subjects with MDD and/or BD.

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2. Cell count data indicating glial loss In addition to the functional changes in the subgenual cortex, analysis of this cortex from structural MRI scans has shown that it is also reduced in volume in the brains of individuals who suffer from MDD or bipolar disorder (BD) (Drevets et al., 1997). This observation suggested that there is a cellular change in mood disorders, which could possibly be identified in tissue sections. Tissue from the subgenual cortex was therefore obtained from brain banks (the Harvard Brain Tissue Resource and the Stanley Foundation), sectioned, stained with the Nissl method, and analyzed with stereological ¨ ngu¨r et al., 1998). Somewhat surprisingly, no difference was found in the methods (O number or somatic size of neurons between mood disorder and control brains. A difference was found in glial cells, however, which were significantly reduced in both MDD and BD cases (Fig. 2). This reduction was particularly noted in cases with a family history of mood disorders. A similar difference was not seen in tissue from schizophrenic cases, or in the somatic sensory cortex, suggesting that there is some specificity to the effect. Simultaneously, Rajkowska et al. (1999) identified a similar decrease in glial density in the orbital cortex and the dorsolateral prefrontal cortex. They analyzed cellular changes on a laminar basis, and found the most substantial effects in the deeper layers of the cortex. In the caudolateral part of the orbital cortex, which had the largest change, there was a

Fig. 2. Summary data illustrating lower glial densities in the subgenual cortex in MDD and BD brains, calculated ¨ ngu¨r et al. (1998). * p , 0:05: from data published by O

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lower glial density in MDD cases compared to controls. This decrease was significant in layers IIIc– VI, as well as in the cortex as a whole, but not in the superficial layers. A lesser difference was found in the dorsolateral prefrontal cortex, which was most prominent in layer Va. A variety of differences were also found in glial and neuronal size groups, which suggested that there might be cell shrinkage or other changes in specific cell groups. In a later paper, Rajkowska et al. (2001) reported a significant reduction in glial density in layer IIIc of area 9 (in the dorsomedial prefrontal cortex) from BD cases, as compared to controls. There was also a lower density of neurons in layer III, and of large, pyramidal neurons in layers III and V (Fig. 3). A third study by Cotter et al. (2001a,b) examined the supracallosal part of the anterior ¨ ngu¨r et al. (1998) cingulate cortex, from many of the same cases that were analyzed by O (from the Stanley Foundation brain bank). Using similar stereological counting methods, they found that cases with MDD had a significantly lower glial density in layer VI of the cortex, along with a reduction in neuronal size in the same layer. A similar, but marginally significant reduction was also seen in schizophrenic cases (Fig. 4). In addition to these analyses of cortical areas, Bowley et al. (2002) have recently demonstrated that the amygdala has a markedly lower glial density and glia-to-neuron

Fig. 3. The difference between the density of glia in control over MDD brains, in the caudolateral orbital cortex, calculated from data published by Rajkowska et al. (1999). There was a significantly lower density in layers IIIc, IV, Va, Vb, and VI in the MDD cases, and across all layers together. * indicates p , 0:05:

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Fig. 4. The difference between the density of glia in control over MDD brains, in the supracallosal anterior cingulate cortex, calculated from data published by Cotter et al. (2001a). There was a significantly lower density in layers VI in the MDD cases. * p , 0:05:

ratio in MDD cases than in controls. As in the subgenual cortex, no change was found in neurons. The reduction was mainly accounted for by counts in the left hemisphere. This is possibly in keeping with the results of imaging studies, many of which have indicated that the left hemisphere is more affected in mood disorders than the right (e.g., Drevets et al., 1992, 1997). Average glia measures were not reduced in BD cases, but a small number of BD cases who had not been treated with mood stabilizer drugs had significantly lower glial density. Similar but smaller changes were found in the entorhinal cortex.

3. Glial type—astrocytes or oligodendrocytes? 3.1. Astrocytes In all of the experiments described above, glia were distinguished from neurons by morphological characteristics visible in the Nissl-stained sections (glia have smaller nuclei, lack a nucleolus, and do not have visible cytoplasmic staining). Different types of glia were not distinguished, however, so it is not known whether the glial decrease was due to astrocytes, oligodendrocytes, or microglia. Most discussions have focused on the role of astrocytes in regulating the environment of neurons. The functions of these widely distributed glia include support of the metabolic requirements of neurons and management

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of synaptic glutamate (see chapter by Schousboe and Waagepetersen, this volume), and buffering of extracellular Kþ (see chapter by Walz, this volume). The evidence that astrocytes account for the glial deficit in mood disorders is not strong, however. ¨ ngu¨r et al. (1998) on the subgenual cortex, an attempt was As part of the study by O made to demonstrate an effect on astrocytes by analyzing sections stained for glial fibrillary acidic protein (GFAP). This analysis did not show a difference between control and mood disorder cases, however (unpublished observations). A high degree of variability was found in the staining for GFAP, apparently due to differences in fixation or other parameters and it was considered that this could explain the inability to find a quantitative difference in astrocytes. It is also possible, however, that the negative results were due to lack of involvement of astrocytes in the lower glial density. Subsequent to the analysis of glia in the amygdala, my lab attempted to demonstrate a decrease in astrocytes in the amygdala of mood disorder cases, using antibodies against another astrocyte-specific protein, S-100b (Hamidi et al., 2002). Adjacent sections to those used in the first study were immunohistochemically stained, and S-100b-immunoreactive cells counted. The results indicated that there was no difference between MDD, BD, and control brains in the density of S-100b-immunoreactive astrocytes. Other studies have also produced mixed indications of astrocyte deficits in mood disorders. Johnston-Wilson et al. (2000) used proteomic methods to characterize protein differences in the frontal cortex between mood disorder, schizophrenic and control brains, using the same cases from the Stanley Foundation. They identified four forms of GFAP that are decreased in mood disorder cases, and in schizophrenia. It is not clear from this data, however, whether the decrease is due to fewer astrocytes, or to a decreased expression of GFAP. Recently, Rajkowska et al. (2002) reported that in schizophrenic cases there is a specific decrease in the fraction of tissue occupied by GFAPimmunostained astrocyte processes in layer V of the dorsolateral prefrontal cortex (area 9). This was not accompanied by a comparable loss of GFAP-positive cells (the density of cells was actually increased), suggesting that the difference is due to decreased expression Webster et al. (2001) analyzed immunohistochemical staining for GFAP, also in the cases from the Stanley Foundation. They did not find any significant differences in cortical astrocytes between MDD, BD, or schizophrenic cases and controls, although they did find that fewer of the MDD and schizophrenic cases showed astrocytes around blood vessels. Several other studies have not found a difference in GFAP between mood disorder or schizophrenic cases and controls (see Cotter et al., 2001b for review).

3.2. Oligodendrocytes In comparison to astrocytes, there is a small but increasingly convincing body of data that oligodendrocytes are specifically decreased in mood disorders and schizophrenia. Honer et al. (1999) measured levels of several proteins related to synaptic function, plasticity and myelination in the rostral frontal cortex of control brains and of brains from patients having suffered from schizophrenia or depression. The depressed patients, and many of the schizophrenic patients had died from suicide. The authors found a significant loss of myelin basic protein, without a difference in synaptophysin or GAP-43,

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in depressives and schizophrenics who died of suicide. Because myelin basic protein is a specific marker for oligodendrocytes and for myelin, this suggests a deficit in oligodendrocyte number or function. Hakak et al. (2001) reported similar findings, using genome-wide expression analysis of the dorsolateral prefrontal cortex in controls and schizophrenics. They found changes in genes involved in a number of biological processes, but the most notable were differential expression of genes related to myelination. The same group subsequently reported cellular and stereological evidence for oligodendrocyte abnormalities in frontal cortical area 9 in schizophrenia (Hof et al., 2002a,b). They used an oligodendrocyte specific antibody against the myelin-related enzyme CNPase to identify a substantial, and significant decrease of oligodendrocytes in layer III and the subcortical white matter. Uranova et al. (2001) used electron microscopy to examine pathological changes in schizophrenic and BD cases. They found evidence of degeneration of oligodendrocytes and myelin in the rostral frontal cortex (area 10) and caudate nucleus of both diagnostic groups. Further quantitative analysis in the schizophrenic cases indicated significant nuclear and cytoplasmic changes in oligodendrocytes. Most of the changes were observed in satellite oligodendrocytes adjacent to neurons, but degenerative changes were also observed in myelinated axons, especially in schizophrenics. As part of the analysis of the amygdala in mood disorders in my laboratory, the density of oligodendrocytes and astrocytes was determined from Nissl stained sections of the same brains in which decreased overall glial density was demonstrated previously (Hamidi et al., 2002). Oligodendrocytes were distinguished from astrocytes by their smaller, more condensed and darker nuclei. These counts indicated there was a significant decrease in oligodendrocytes, but not of astrocytes. Further analysis of microglia stained with an antibody against HLA indicated that there was no change in microglia density. The functions of oligodendrocytes have not been investigated as thoroughly as those of astrocytes, but in addition to myelination they are involved in reaction to injury and possibly axonal guidance. In the cortex, many of the oligodendrocytes are satellite or perineuronal cells, which may not have a role in myelination (Peters et al., 1976; D’Amelio et al., 1990). There is very little specific information on the satellite oligodendrocytes, but they may be expected to be involved in maintenance of the extracellular environment for surrounding neurons. In spite of these uncertainties, the primary function of oligodendrocytes is myelination. It is notable, therefore, that many of the cortical deficits in glia have depended on laminar analysis, with the greatest effects in layer III, and in layers V and VI. The intracortical plexuses of myelinated fibers that are known as ‘Bands of Baillarger’ are usually concentrated in layers III and V. The size of these plexuses varies between different cortical areas (the Stria of Gennari in the primary visual cortex is the extreme example). Therefore, if the oligodendrocytes related to these plexuses were affected, it would be expected that different areas would show a greater or lesser deficit. Further, in some studies the greatest effect was in layer VI, which has a relatively large component of myelinated fibers running between the gray and white matter. More investigation of white as well as gray matter is needed to settle the question of whether oligodendrocytes account for all of the glial deficit in mood disorders and schizophrenia, and whether this is related to myelination or other functions.

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4. Concluding remarks The evidence that there is a low density of glia in selective areas or layers of the frontal cortex and amygdala in mood disorders and schizophrenia is now relatively strong. While there are many questions about which areas or layers are primarily involved, several studies have now reported similar findings. A problem with the studies to date is that they are often based on relatively few cases. This should resolve as more studies are done, building on the findings of the previous experiments. At present there is better evidence that oligodendrocytes account for the glial deficit than astrocytes. If this is confirmed, it substantially changes the interpretation of the low glial densities. Low astrocyte densities or astrocyte to neuron ratios would suggest that the many roles of astrocytes in controlling the environment within which neurons function might be compromised. Changes in astrocyte roles such as buffering of extracellular Kþ, uptake of glutamate at synapses, and supplying metabolic intermediates to neurons would be expected to alter neuronal excitability significantly. If the deficit is due to oligodendrocytes, however, a more likely interpretation is that there is an abnormality in myelinated axons. This could either reflect a long-standing difference in axonal number or ramification, or possibly a more dynamic effect on the number or activity of myelinating oligodendrocytes. Although myelin sheaths around axons are usually presumed to be static, there appears toz be relatively little solid evidence that would rule out some degree of plasticity. Finally, other non-myelinating functions of oligodendrocytes could also be affected. Acknowledgements This work was supported by grant DC00093 from the US National Institutes of Health.

References ¨ ngu¨r, D., Price, J.L., 2002. Low glial numbers in the amygdala in mood disorders. Bowley, M.P., Drevets, W.C., O Biol. Psychiatry 56, 404 –412. Cotter, D.R., Mackay, D., Landau, S., Kerwin, R., Everall, I., 2001a. Reduced glial cell density and neuronal size in the anterior cingulate cortex in major depressive disorder. Arch. Gen. Psychiatry 58, 545 –553. Cotter, D.R., Pariante, C.M., Everall, I.P., 2001b. Glial cell abnormalities in major psychiatric disorders: the evidence and implications. Brain Res. Bull. 55, 585–595. D’Amelio, F., Eng, L.F., Gibbs, M.A., 1990. Glutamine synthetase immunoreactivity present in oligodendroglia of various regions of the central nervous system. Glia 3, 335–341. Davidson, R.J., Lewis, D.A., Alloy, L.B., Amaral, D.G., Bush, G., Cohen, J.D., Drevets, W.C., Farah, M.J., Kagan, J., McClelland, J.L., Nolen-Hoeksema, S., Peterson, B.S., 2002. Neural and behavioral substrates of mood and mood regulation. Biol. Psychiatry 52, 478–502. Drevets, W.C., 2000. Neuroimaging studies of mood disorders. Biol. Psychiatry 48, 813–829. Drevets, W.C., Price, J.L., Simpson, J.R., Todd, R., Reich, T., Vannier, M., Raichle, M., 1997. Subgenual prefrontal cortex abnormalities in mood disorders. Nature 386, 824 –827. Drevets, W.C., Videen, T.O., Preskorn, S.H., Price, J.L., Carmichael, S.T., Raichle, M.E., 1992. A functional anatomical study of unipolar depression. J. Neurosci. 12, 3628–3641.

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Hakak, Y., Walker, J.R., Li, C., Wong, W.H., Davis, K.L., Buxbaum, J.D., Haroutunian, V., Fienberg, A.A., 2001. Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl Acad. Sci. USA 98, 4746–4751. Hamidi, M., Drevets, W.C., Price, J.L., 2002. Glial reduction in amygdala in major depressive disorder is due to oligodendrocytes. Soc. Neurosci. Abstracts (Program No. 487.21). Hof, P.R., Haroutunian, V., Byne, W., Friedrich, V.L., Buitron, C., Perl, D.R., Davis, K.L., 2002a. Loss and abnormal spatial distribution of oligodendrocytes in schizophrenia: stereologic analysis of area 9. Soc. Neurosci Abstracts (Program No. 704.14). Hof, P.R., Haroutunian, V., Copland, C., Davis, K.L., Buxbaum, J.D., 2002b. Molecular and cellular evidence for an oligodendrocyte abnormality in schizophrenia. Neurochem. Res. 27, 1193–1200. Honer, W.G., Falkai, P., Chen, C., Arango, V., Mann, J.J., Dwork, A.J., 1999. Synaptic and plasticity-associated proteins in anterior frontal cortex in severe mental illness. Neuroscience 91, 1247–1255. Johnston-Wilson, N.L., Sims, C.D., Hofmann, J.P., Anderson, L., Shore, A.D., Torrey, E.F., Yolken, R.H., 2000. Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. The Stanley Neuropathology Consortium. Mol. Psychiatry 5, 142 –149. Manji, H.K., Drevets, W.C., Charney, D.S., 2001. The cellular neurobiology of depression. Nat. Med. 7, 541–547. ¨ ngu¨r, D., Drevets, W.D., Price, J.L., 1998. Glial reduction in the subgenual prefrontal cortex in mood disorders. O Proc. Natl Acad. Sci. USA 95, 13290–13295. ¨ ngu¨r, D., Price, J.L., 2000. The organization of networks within the orbital and medial prefrontal cortex of rats, O monkeys and humans. Cerebral Cortex 10, 206–219. Peters, A., Palay, S.L., Webster, H. de F., 1976. The Fine Structure of the Nervous System. W.B. Saunders, Philadelphia. Rajkowska, G., Halaris, A., Selemon, L.D., 2001. Reductions in neuronal and glial density characterize the dorsolateral prefrontal cortex in bipolar disorder. Biol. Psychiatry 49, 741– 752. Rajkowska, G., Miguel-Hidalgo, J.J., Makkos, Z., Meltzer, H., Overholser, J., Stockmeier, C., 2002. Layerspecific reductions in GFAP-reactive astroglia in the dorsolateral prefrontal cortex in schizophrenia. Schizophr. Res. 57, 127 –138. Rajkowska, G., Miguel-Hidalgo, J.J., Wei, J., Dilley, G., Pittman, S.D., Meltzer, H.Y., Overholser, J.C., Roth, B.L., Stockmeier, C.A., 1999. Morphometric evidence for neuronal and glial prefrontal cell pathology in major depression. Biol. Psychiatry 45, 1085–1098. Uranova, N., Orlovskaya, D., Vikhreva, O., Zimina, I., Kolomeets, N., Vostrikov, V., Rachmanova, V., 2001. Electron microscopy of oligodendroglia in severe mental illness. Brain Res. Bull. 55, 597–610. Webster, M.J., Knable, M.B., Johnston-Wilson, N., Nagata, K., Inagaki, M., Yolken, R.H., 2001. Immunohistochemical localization of phosphorylated glial fibrillary acidic protein in the prefrontal cortex and hippocampus from patients with schizophrenia, bipolar disorder, and depression. Brain Behav. Immun. 15, 388– 400.

Glutamate excitotoxicity in the immunopathogenesis of multiple sclerosis P. Werner, E. Brand-Schieber and C.S. Raine Contents 1. 2.

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Introduction Multiple sclerosis—an overview 2.1. Introduction 2.2. Multiple sclerosis—disease progression and classification 2.3. Classification of lesions in multiple sclerosis 2.4. Animal models of MS Glutamate receptors in white matter 3.1. Introduction 3.2. AMPA/kainate receptors 3.3. NMDA receptors 3.4. Metabotropic receptors Sources of excess glutamate in MS lesions 4.1. Introduction 4.2. Glutamate production by immune cells 4.3. Relevance to MS 4.4. Release by damaged axons Glutamate homeostasis and MS 5.1. Introduction 5.2. Glutamate metabolizing enzymes 5.3. Glutamate metabolizing enzymes in normal and inflamed white matter Glutamate transporters and MS 6.1. Introduction 6.2. Glutamate transporters in control and MS white matter Concluding remarks

1. Introduction The exact mechanisms that lead to demyelination, axonal damage and death of oligodendrocytes in multiple sclerosis (MS) are still unknown. Among the mechanisms implicated are contact with cytotoxic immune cells, antibodies (Genain et al., 1999), and soluble mediators, especially proinflammatory cytokines (Selmaj et al., 1991). Therefore, Advances in Molecular and Cell Biology, Vol. 31, pages 1059–1083 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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unlike research on most other neurodegenerative diseases, which is commonly characterized by a dearth of proven mechanisms of tissue damage, research on MS suffers from an abundance of cytotoxic mechanisms known to operate in the disease. This embarrassment of riches is a major reason why glutamate excitotoxicity, a concept introduced by Olney over 30 years ago (Olney, 1969), is a late entry into the list of pathologic processes operative in MS. This is in sharp contrast to gray matter conditions such as stroke or Alzheimer’s disease, where glutamate excitotoxicity is a major working hypothesis (Choi, 1988), initially introduced by Olney over 30 years ago (Olney, 1969). In glutamate excitotoxicity, agonist binding to ionotropic glutamate receptors leads to influx of sodium and calcium ions, a cell membrane depolarizing mechanism. Overstimulation, ion influxes and membrane depolarization is then conducive to activation of destructive processes such as interruption of electrolyte and fluid balance, phospholipase and protease activation and formation of free radicals, and activation of cell death pathways (for reviews see: Lipton and Rosenberg, 1994; Meldrum and Garthwaite, 1990; Choi, 1988; Koller et al., 1997). Our review will discuss recent findings that expand the mechanisms of glutamate excitotoxicity from gray matter diseases to MS, a white matter disease. For glutamate excitotoxicity in white matter to be a valid mechanism of damage, the presence of glutamate receptors and one or more of these elements are required: Reduced or absent uptake by glutamate transporters, altered glutamate metabolizing enzymes and a source for extracellular glutamate. The following sections will firstly introduce the major features of MS and then discuss the evidence for the presence of the required elements in white matter and their involvement in white matter pathologies, especially MS. 2. Multiple sclerosis—an overview 2.1. Introduction MS is a debilitating neurological disease with a prevalence of approximately 1 in 1000 in the US and Europe, an onset in early adulthood and a disease course commonly ranging between 3 and 4 decades (Whitaker and Mitchell, 1997). The pathologic substrate in MS is the white matter of the CNS. Pathologic hallmarks of MS are a loss of myelin and oligodendrocytes, the myelin-producing cells of the CNS, reactive astrocytes and axonal damage occurring against a background of inflammation. There are many hypotheses with regard to the underlying mechanisms and initial events that lead to MS. One widely held hypothesis, outlined in Fig. 1, is that viral or microbial infection during childhood (possibly non-symptomatic) occurs in a subject genetically predisposed to the development of the disease. This infection leads to leukocyte activation and cytokine production, which, in turn, up-regulates adhesion molecules on blood vessels. Since activated lymphocytes migrate across endothelium, the permeability of the blood – brain barrier (BBB) for white blood cells and/or infectious agents is increased, and both may enter the normally sequestered CNS tissue and cause damage. T cell sensitization to myelin antigens, thought to be a watershed event for the development of MS, may occur due to exposure of T cells to myelin breakdown products

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Fig. 1. Proposed scheme of MS pathogenesis and progression.

(bystander sensitization), or to molecules displaying homology with myelin sequences (molecular mimicry). Thus, a population of circulating (long-term memory) T cells sensitized to myelin is generated. The above sequence of events occurs without any overt symptoms. After a period of latency (years), the subject is then thought to experience an inflammatory event (e.g., upper respiratory infection), during which lymphocytes are systemically activated and proinflammatory cytokines (e.g., IFNg, TNFa) become elevated. By cytokine-mediated up-regulation of adhesion molecules on endothelial cells throughout the body, including the BBB, the CNS blood vessels become permissive to transmigration by activated lymphocytes. Among the transmigrating cells is the progeny of the myelin-antigen sensitized T cells generated earlier during the initial (childhood) non-clinical sequence of events. The latter cells recognize antigen on myelin, proliferate, and recruit other lymphocytes and monocytes into the tissue—the patient experiences acute clinical signs related to an immune attack on CNS white matter, such as visual impairments, weakness of one or more extremities and paresthesia. Eventually, immunoregulatory mechanisms are triggered, the lesion acquires a regulatory phenotype and the damage and inflammation recede after which some remyelination and astrogliosis may occur. Most patients then experience a period of clinical stability (remission), usually for months to years, until the next adverse event (e.g., respiratory infection, emotional crisis), which precipitates a similar cycle of inflammatory events, a clinical relapse, and the cycle is repeated.

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2.2. Multiple sclerosis—disease progression and classification Multiple sclerosis can manifest in several different disease courses (Raine, 1997a,b). The most common form is relapsing– remitting MS. In this manifestation of MS, the patient suffers an initial immune attack usually presenting as sudden neurological impairment. The attack then subsides, and neurological function is partially or often completely regained. Months or years after this initial episode, a second bout of sudden loss of neurological function announces the first relapse, which is again followed by partial or full functional recovery, the remission. Patients with relapsing-remitting MS can often live an essentially normal, unassisted life for decades (Weinshenker, 1997). However, which each subsequent relapse, lasting demyelination and axonal damage, both of which can be visualized by MRI (Barkhof and van Walderveen, 1999), eventually take their toll, and the patient begins to experience neurological deficits even during remissions. Primary progressive MS is characterized by an unrelenting immunologic attack on CNS white matter, with accompanying, downhill progression and often rapid, loss of neurologic function with ensuing disability and death (Thompson et al., 1997). Patients with primary progressive MS typically respond poorly to attempts to moderate or modulate the immune response (Thompson et al., 1997). In contrast to primary progressive MS, secondary progressive MS starts after years or decades of relapsing-remitting MS (Weinshenker, 1997), and usually initiates from an existing level of neurological impairment. However, like primary progressive MS, secondary progressive MS is characterized by an uninterrupted loss of neurological function—the disease is no longer remitting. One explanation for the change from the relapsing –remitting to the secondary progressive form of MS is a change in the type of the aberrant immune response, leading to a loss of the ability to quench the inflammatory activity in CNS (Galboiz et al., 2001). Another explanation is that the disease was never truly dormant in these patients, and that lesion activity continued even in the absence of overt neurological signs (Molyneux et al., 1998). Eventually, the capacity of the CNS to compensate for lost and damaged circuitry is exhausted, and the ongoing lesion activity is now mirrored by a steady loss of neurological function (Bitsch et al., 2000). Immunologic and serial MRI studies suggest that both mechanisms may be operational, possibly side-by-side, adding to the already complex picture of this puzzling disease. 2.3. Classification of lesions in multiple sclerosis MS lesions vary greatly in their degree of inflammatory activity, demyelination and the intensity of damage and repair efforts. Attempts to create a formal decision algorithm that distinguishes different types of lesions have been made (Lassmann et al., 1998). Even within the same patient, multiple lesions can be encountered in various stages of activity, a fact frequently overlooked in studies that classify MS lesions based on limited numbers of samples per case or needle biopsy samples. For example, the CNS of patients with chronic active MS often contains lesions displaying myelin repair, hyperplasia of oligodendrocytes and lesions with oligodendrocyte depletion, sometimes even adjacent to one another. General consensus is that the majority of MS lesions can be classified on the basis of inflammation, demyelination and age (Prineas and McDonald, 1997; Raine, 1997a,b), with

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additional criteria such as axonal pathology, microglia/macrophages, and oligodendrocyte and astroglial response. Categorization based upon oligodendroglial pathology, particularly from biopsies from less typical cases (Lucchinetti et al., 2000), may be difficult to reconcile without survey of many lesions from each case. Such a system might have limited use for routine diagnosis since they require special technologies. In summary, lesion classification based upon inflammation, age and gliotic activity remains the method of choice for most, particularly, since it frequently correlates with clinical and MRI data. With these criteria, the following lesion types can be distinguished. 2.3.1. Acute (early) MS lesions The acute MS lesion is a distinct type generally accepted as the initial manifestation of the MS plaque (Raine, 1997c). It is most frequently encountered in rapid-onset, fulminant, short-term, fatal cases of MS with an onset to death duration of weeks to months— originally described by Marburg (1906). Typically, the lesion has an indistinct margin, is heavily infiltrated by lymphocytes distributed perivascularly and throughout the parenchyma, is highly edematous, contains an abundance of hypertrophic astrocytes, and activated microglia/macrophages filled with recent, myelin-positive debris—an example is shown in Fig. 2. Oligodendrocytes are scattered throughout the lesion and around the margin. Interestingly, acute MS lesions display variable amounts of remyelination (Raine and Wu, 1993; Prineas et al., 1993b), a phenomenon regarded as abortive repair since as the lesion ages over the following months, the response wanes. Interestingly, these lesions are also associated with an abundance of axonal damage, manifested by the presence of axonal spheroids, enlarged dystrophic axons and beaded axonal profiles. These structures, recently shown to display selective binding of antibodies to abnormally dephosphorylated neurofilaments (Trapp et al., 1998), a marker of damaged axons, decrease in number as the lesion resolves but appear again in large numbers with each relapse. Recent data, discussed below, suggest that glutamate excitotoxicity may contribute to these manifestations of axonal damage. 2.3.2. Chronic active MS lesions These lesions are most often found in relapsing– remitting MS and are typically areas of long-standing demyelination and fibrous astrogliosis clearly demarcated from normal appearing white matter (NAWM), along the margins of which recent activity is evident (Prineas and Connell, 1978; Raine et al., 1981). Lesion activity presents as a broad zone of lymphocytic inflammation with prominent perivascular cuffs, ongoing demyelination, microglia/macrophages with recent myelin debris, astroglial hypertrophy, some axonal degeneration and an increase in the number of oligodendrocytes. The ongoing activity extends some distance into the established lesion. Where the lesion abuts NAWM, oligodendroglial hyperplasia is evident and some remyelination is not uncommon. The center of the lesion is completely demyelinated and axonal depletion (40 –50%) is common. The lesion is also rich in highly branched GFAP þ astrocytes, is usually devoid of oligodendrocytes, contains few infiltrating cells, and displays scattered lipid-laden macrophages and what axons are present display little pathology.

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Fig. 2. Demyelination in an active MS lesion. Left panel: A low power overview showing a partial view of an active MS lesion and its surrounding white matter. Note the border between the demyelinated, unstained (white) area to the right and the non-demyelinated white matter to the left of the panel. The red dashed line shows the approximate margin of the lesion. Note the varying degress of demyelination, which is most pronounced towards the lesion center (right) and tapering off towards the margin. One mm epoxy-embedded section, stained with Toluidine Blue. £ 75. Middle panel: Detail view of the center of an active lesion. Myelin debris is readily visible in phagocytic cells (macrophages). An example for hypertrophic astrocytes, commonly found in such lesions, is shown in the center (red A). Faintly staining, possibly surviving oligodendrocytes are present (bottom arrow) as are densely labeled oligodendrocytes (top arrow), the latter possibly in the process of remyelination. One mm epoxy-embedded section, stained with Toluidine Blue. £ 750. Right panel: Electron micrograph from an area of active demyelination. Note several macrophages that contain myelin fragments and lipid droplets, indicating ongoing demyelination. Two oligodendrocytes are shown (asterisks). The oligodendrocyte in the upper right corner of the picture is flanked left and above by an astrocyte. Also note several demyelinated (naked) axons and densely stained fibrous astrocytic processes. Thin epoxy-embedded, carbon-coated, section, stained with uranyl and lead salts. £ 5600.

2.3.3. Chronic silent MS lesions Commonly regarded as the end-stage lesion in MS in which the disease process is burnt-out, the chronic silent MS lesion is the type most encountered at autopsy (Raine, 1997a,b). The lesion edge is sharp and devoid of ongoing activity and the lesion itself is completely demyelinated, fibrous astrogliotic, non-inflammatory and devoid of oligodendrocytes. Remyelination is not prominent. Axonal pathology is not in evidence although variable degrees of axonal loss (30 – 60%) are seen. Vessels frequently display fibrotic changes and a few lipid-laden macrophages persist. 2.3.4. Remyelinated lesions These tend to be single-hit lesions (one inflammatory episode which then resolves), often seen in association with cases showing active lesions, particularly acute MS (Prineas et al., 1993a). They are distinguishable by pale myelin staining and an edge that merges gradually into NAWM. They may contain oligodendrocytes in increased numbers but reactive microglia are rare. Remyelinated lesions may be satellitic to larger areas of lesion activity where they are termed ‘shadow plaques’ (Prineas et al., 1993a). They also exist as

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broad rings or caps of myelin repair surrounding or applied as penumbra to active lesions (Prineas and McDonald, 1997). 2.3.5. Secondary progressive MS lesions This category of MS lesion has been recently defined in cases which originally presented as relapsing-remitting MS before developing a severe progressive course without remission prior to death. They have margins which show low-grade active demyelination in the absence of overt inflammation and activated macrophages or macrophages containing myelin breakdown products. Myelin degeneration is unusual, and occurs in association with clusters of reactive microglial cells aligned along the affected nerve fiber. Hypertrophic astrocytes are not prominent, oligodendrocytes are present in increased numbers, and interestingly, axonal changes are inconspicuous. Significantly, a disease mechanism different from other forms of MS lesion, is suggested. 2.3.6. Other lesion types In addition to the above, a number of variant lesion types can be distinguished which, although noteworthy, are rare and not deserving of separate categorization. These include the well-demarcated hemorrhagic destructive lesions occasionally encountered in acute forms of MS, like Devic’s disease (neuromyelitis optica) and transverse myelitis. These subpial and deep white matter lesions display intense inflammation and several features atypical of MS, such as polymorphonuclear leukocytes, vascular damage, red cell extravasation and tissue necrosis. Such lesions may become cystic with time at which point they comprise fibrous astrogliotic scar tissue and trabeculae. Cystic fibrous astrogliotic MS lesions are also encountered in cases of chronic relapsing MS where they are believed to be related to an antecedent, severe inflammatory phase (Raine, 1997a,b). Another type of lesion, reappraised recently (Peterson et al., 2001), is the cortical (gray matter) lesion of chronic MS that may take several forms depending upon its location within the cerebral cortex. These lesions are apparently relatively common and appear to be non-inflammatory and purely demyelinative. Underlying mechanisms remain to be elucidated. Finally, in some cases of chronic progressive MS, one may find gliotic ‘active’ lesions rich in lipid-laden macrophages and reactive microglia, but little or no evidence of lymphocytic infiltration. By virtue of the abundance of neutral lipid and paucity of myelin abbau (debris) within the macrophages, one assumes that these are long-standing lesions which have lost their lymphocytic inflammatory component but have retained a rich population of activated macrophages. The factors responsible for the chronic retention of these cells within the lesion are yet to be clarified. 2.4. Animal models of MS Two animal models of MS that are frequently employed for mechanistic studies; the inflammatory demyelination induced by Theiler’s virus (Miller et al., 1995), and experimental autoimmune encephalomyelitis (EAE). Since all experimental work on glutamate excitotoxicity in relation to MS has been done in EAE, an introduction into EAE

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is helpful. EAE has fueled much of the current understanding of the immunology of autoimmune demyelination, and most current treatments are derived from initial studies in EAE. EAE is a CD4 þ , Th1-type modulated immune response to myelin antigens. EAE can be induced in most animal species, and is characterized by an initial, acute paralysis, accompanied by white matter involvement and destruction of myelin in proximity of infiltrating lymphocytes and macrophages (Raine et al., 1984). Although EAE is not a complete model of MS, it allows for many insights into the mechanisms of tissue damage which result from the immune response against myelin antigens. The various kinds of EAE differ by the myelin antigen employed to elicit the initial immune response and the mammalian species used. Furthermore the autoimmune reaction can be induced directly in the animal, the so-called active EAE, or by the injection of antigen-primed T cells from syngeneic donor animals into donor animals, the so-called passive or adoptive transfer EAE (Mokhtarian et al., 1984). 3. Glutamate receptors in white matter 3.1. Introduction Several reviews on the various glutamate receptors in the CNS have been published (Choi, 1988; Sprengel and Seeburg, 1993). Therefore, the following paragraphs will only give a short introduction into their basic features, and attempt a summary of their distribution in white matter, the target tissue of MS. Two main types of glutamate receptors exist, the ionotropic and the metabotropic receptors, respectively. Ionotropic glutamate receptors are made up of subunits that are distinct for each receptor subtype. Ionotropic glutamate receptors are ligand-gated ion-channels composed of homo- and heteromers of specific subunits, which form the receptor– channel complex. Three types of ionotropic glutamate receptors are known, each of which is named according to their binding preference for their respective prototypic glutamatergic agonist, namely alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate (KA) and N-methyl-D -aspartate (NMDA) (see Choi, 1988, for review). The subunit composition of the homo- and heteromer determines the ligand binding and ion-conducting property of each receptor. AMPA and KA receptors are typically made up of 4 subunits, likely in the form of a dimer of dimers (Ayalon and Stern-Bach, 2001). AMPA receptors are composed of the subunits GluR1– GluR4, KA receptors are formed by subunits GluR5, 6, 7, KA1 and KA2 and NMDA receptors by the subunits NR1 and NR2. Metabotropic glutamate receptors are divided into three major groups (mGluR I– III), depending on their pharmacological characteristics and their effector mechanism, and have been detected in white matter (Bruno et al., 2001). These receptors differ from ionotropic glutamate receptors in several regards: They do not permit influx of mono- and divalent ions, rather, they are coupled top G-proteins, triggering second messenger cascades that influence kinases and phosphatases. Also, unlike ionotropic glutamate receptors, they are not made up of multiple subunits. Over the last ten years, evidence has accumulated that glutamate receptors are expressed on almost every non-neuronal constituent of white matter, namely astrocytes, microglia and oligodendrocytes, while their expression in axons remains unclear.

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Astrocytes, a key component of the MS lesion, express both ionotropic and metabotropic glutamate receptors (Gallo and Russell, 1995). It is of interest that a recent report (Besong et al., 2002) demonstrated that, activation of group III metabotropic receptors in rat astrocytes inhibited production of the cytokine RANTES in vitro. This suggests that glutamate receptors may be involved in modulating the role of astrocytes in CNS-inflammation. Microglia, the major macrophages in the MS lesion, express both ionotropic and metabotropic glutamate receptors (Tahraoui et al., 2001; Noda et al., 2000). Rodent microglia are similar to astrocytes in their comparable insensitivity to glutamate excitotoxicity, although electrophysiologic studies indicate that the receptors expressed are also functional (Noda et al., 2000). Oligodendrocytes express both metabotropic (Kelland and Toms, 2001) and ionotropic glutamate receptors (Matute et al., 2001, Verkhratsky and Steinhauser, 2000). Currently, only the role of ionotropic receptors in excitotoxic damage to oligodendrocytes is known in some detail. While NMDA and AMPA/KA type glutamate receptors have been reported on oligodendrocytes, both presence and function of NMDA receptors on these cells are in question. Although patch clamp evidence has suggested the presence of NMDA-type glutamate receptor currents in astrocytes and oligodendrocytes in rat spinal cords during development, they appear to undergo down-regulation as the animal matures (Ziak et al., 1998). On the other hand, both expression and functionality of AMPA and KA-type receptors on oligodendrocytes have been well documented (Matute et al., 2001). Oligodendrocytes, like many neurons, are highly vulnerable to AMPA/ kainate receptor-mediated excitotoxicity (Yoshioka et al., 1995; McDonald et al., 1998), and are at risk whenever extracellular glutamate is elevated. Below, we detail the current evidence for the presence of glutamate receptors in white matter and their involvement in white matter excitotoxicity in MS.

3.2. AMPA/kainate receptors Depending on their specific subunit composition, AMPA and KA receptors can possess diverse ion channel characteristics (Ozawa et al., 1998). Diversity in channel attributes can lead to a variety of cellular responses upon receptor activation. To date, most attempts to modulate glutamate excitotoxicity in white matter inflammation used currently available, but rather unspecific, AMPA/KA antagonists (Smith et al., 2000; Pitt et al., 2000). Some of our recent work has thus focused on the dissection of the cellular target for AMPA/KA receptor-mediated excitotoxicity, as well as the precise subunit composition of each receptor. This was performed in light of the desire to eventually develop more selective therapeutic strategies specific for the receptor subtypes involved in destruction of target tissues. The first such study was recently done in mice, which are among the most commonly used animals for the experimental study of CNS diseases in general (Rakic, 2000) and white matter diseases in particular (Werner et al., 1998). The expression sites of AMPA/KA receptor subunits in the cellular constituents of mouse spinal cord were studied using multiple-label immunohistochemistry and laser-confocal microscopy (Brand-Schieber and Werner, 2003).

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White matter was consistently immunoreactive for the AMPA receptor subunit GluR2/3 and the KA receptor subunits GluR6/7 and KA2 (Fig. 3). Interestingly, white matter astrocytes displayed both the AMPA receptor subunit GluR2/3 and the KA subunit GluR6/7, while gray matter astrocytes displayed mostly GluR6/7. The KA receptor subunit KA2, was, exclusively and abundantly, only present on oligodendrocytes and myelin, which were identified by CNPase expression (Fig. 3). KA2 expression was not apparent in the peripheral nervous system (Fig. 3), pointing at KA receptors as a unique mediator for excitotoxicity to CNS, but not PNS, white matter. These findings strongly support the notion that oligodendrocytes are a primary target of glutamate excitotoxicity in white matter. This is of interest in the context of MS, as the loss of oligodendrocytes is a central event in lasting demyelination. A significant depletion of these myelin-producing cells will reduce the capacity of the tissue to repair the damage caused by the immune reaction, i.e., the ability to remyelinate. The migration into or differentiation of oligodendrocyte precursors in the lesion area even in active lesions has been proposed as an important repair mechanism (Capello et al., 1997). This has special relevance in light of the presence of dysfunctional, non-myelinating oligodendrocytes in MS lesions (Wolswijk, 2000), which constitute a sizeable number of the resident oligodendrocytes not only in active, but also in chronic-active and quiescent MS lesions. It is unknown whether glutamate excitotoxicity contributes to this peculiar oligodendrocytic phenotype. Adult oligodendrocytes differ in their expression of certain subunits of AMPA receptors from immature precursor cells, which may display slower inactivating AMPA receptors (Yoshioka et al., 1995; Itoh et al., 2002). The presence of slowly inactivating AMPA receptors on immature oligodendrocytes could render these still developing cells especially vulnerable to excitotoxicity. Interestingly, stimulation of AMPA/KA receptors has been reported to cause lasting changes in the activity of key second-messenger pathways in oligodendrocytes, including inositol triphosphate (IP3) (Liu et al., 1997), suggesting that glutamate receptors may play a role in the regulation of oligodendrocyte differentiation. In this context, it is of interest that KA2 is densely expressed perinodal to the nodes of Ranvier (Fig. 3), but axons showed negligible AMPA/KA receptor expression. The proximity of oligodendrocytic KA2 to the nodal axon and the paucity of axonal AMPA/ kainate receptor expression suggest glutamate excitotoxicity to axons to be a secondary phenomenon, possibly mediated by oligodendrocytes. Indeed, unlike the generally accepted vulnerability of oligodendrocytes to glutamate excitotoxicity, its involvement in axonal pathology is controversial. For example, early experiments by Coyle and colleagues (Coyle et al., 1978), established microinjections with the selective glutamate agonist kainate as a selective method to lesion neurons in rodent striatum without overt damage to passing axons. Similar findings were reported by others using AMPA microinjections (McDonald et al., 1998). However, all these studies used general histology more suited to assess neuronal damage, and overt axonal damage, such as Wallerian degeneration, may not have been present at the time intervals used for sampling. Results in mouse indicated that glutamate excitotoxicity is involved in axonal damage in MS and EAE (see below), and the sampling was likely days after the initial insult, allowing for axonal damage to develop (Pitt et al., 2000; Werner et al., 2000, 2001). The results in the EAE model, coupled with our observation that imbalanced glutamate homeostasis

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Fig. 3. AMPA/KA receptor expression in mouse spinal cord white matter. Mouse spinal cord white matter double-labeled with fluorescent probes for either axonal marker SMI31, astrocytic marker GFAP or oligodendrocytic marker CNPase (all in red) and with green fluorescent probes marking either glutamate receptor subunit GluR2/3 (AMPA) GluR6/7 or KA2 (kainate). Top panels. Left, GluR2/3 (green) labeling traverses, without overlap, areas where several axons (red) are visible. GluR2/3 in peripheral nerve root (bottom of image) shows a different labeling pattern than that of spinal cord/CNS. Asterisks are next to GluR2/3 immunopositive Schwann cell-like structures. Right, yellow regions depicting overlap of GluR2/3 (green) and GFAP (red), confirming GluR2/3 expression in astrocytes. Center panels. Left, no axonal (red) co-localization of intense GluR6/7 (green) immunoreactivity. Peripheral nerve root (bottom of image) does not display GluR6/7 immunoreactivity, while CNS white matter is intensely labeled without axonal co-localization. Right, yellow regions depicting overlap of GluR6/7 (green) and GFAP (red), confirming GluR6/7 expression in astrocytes. Bottom panels. KA2 (green) shows widespread expression between and along axons (red). In contrast, KA2 immunoreactivity is absent from the peripheral nerve root (bottom of image) running along the, well labeled, spinal cord white matter. Right, KA2 immunoreactivity (green) shows extensive overlap (yellow) with the network of CNPase-immunopositive myelin and oligodendrocytes (red). Scale bars ¼ 10 mm.

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correlates highly with axonal damage in MS lesions (Werner et al., 2001), argue for a role for glutamate excitotoxicity in axonal damage. However, the mediators of this damage remain elusive.

3.3. NMDA receptors Numerous studies have implicated NMDA-type glutamate receptors in various CNS pathologies. Inhibition of NMDA receptor stimulation was suggested as a treatment for a wide variety of CNS pathologies ranging from neurodegenerative diseases such as Huntington’s, Parkinson’s (Steece-Collier et al., 2000) and HIV dementia (Epstein, 1998) to epilepsy (Sagratella, 1995), brain ischemia (Williams et al., 2000; Jander et al., 2000), chronic pain (Fisher et al., 2000) and drug and alcohol addiction (Bisaga and Popik, 2000). In spite of the purported absence of NMDA-type glutamate receptors on oligodendrocytes, some studies implicate NMDA receptors in white matter inflammatory demyelinating conditions, including multiple sclerosis. NMDA, when injected directly into the brain, will cause a rapidly developing demyelination (Brace et al., 1997; Arvanitogiannis and Shizgal, 1999) that precedes any immune reaction (Arvanitogiannis and Shizgal, 1999). This suggests that NMDA receptor-mediated glutamate excitotoxicity could be an intrinsic CNS demyelinating mechanism, akin to the demyelination induced by acute stimulation of AMPA/KA receptors in optic nerve (Matute, 1998). CNS1102, a non-competitive NMDA receptor antagonist, protected myelin and axons from damage following focal brain ischemia (Schabitz et al., 2000). With regard to white matter inflammation, increased levels of quinolinic acid, an endogenous NMDA agonist derived from tryptophan, were found in CSF of animals with EAE (Flanagan et al., 1995). However, there is no solid evidence to date that supports a specific mechanism by which to explain the protective effect of NMDA antagonists on white matter. Bolton and Paul (1997) show that the non-competitive NMDA receptor antagonist MK-801 (dizocilpine) reduces neurological dysfunction in rat EAE. The authors attribute the effect of the drug to its ability to inhibit neuroendothelial disruption and BBB dysfunction. Indeed, BBB failure is an important factor in the development of EAE and MS (Brosnan and Raine, 1996). However, evidence suggesting that rat as well as human endothelial cells do not express NMDA receptors (Morley et al., 1998) weakens this hypothesis and the suggestion that NMDA antagonists may remedy BBB disruption in humans. Moreover, the presented data does not support full correlation between the level of BBB disruption and the degree of neurological deficits. A low prophylactic dose of MK-801, which did not significantly decrease BBB dysfunction, provided significant decrease in neurological impairment while a high dose, given later in the course of the disease, partially but significantly improved BBB function but did not significantly affect the neurological score. Therefore, it seems that the efficacy of the NMDA antagonist in reducing neurological impairment, in this EAE model, had a component that is independent from the effect on BBB integrity. In support of that, a study by Wallstrom et al. (1996) suggests a direct involvement of NMDA receptor in the development of acute EAE. Memantine, a non-competitive NMDA receptor antagonist (Bormann, 1989; Chen et al., 1992), reduced neurological deficits in rat EAE without measurable effects on CNS inflammation.

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The authors speculate that blockade of NMDA receptors improved neurological function in their model by interfering with the release of unidentified, nerve conduction blocking, ‘effector molecules’ from an unidentified source. Furthermore, they argue that their data are consistent with NMDA receptor induced ‘neurons-axons damage’ as a basis for neurological impairment and suggest memantine as a possible treatment for neuroinflammatory diseases. It is of interest that a recent study identified a sodium channelblocking peptide that is present in the CSF of MS patients suffering an acute attack (Brinkmeier et al., 2000). However, the relationship of this peptide to NMDA receptor activity is elusive and presently unknown. More studies will be needed to understand the true mechanisms behind the apparent protective effect of NMDA antagonists in EAE. 3.4. Metabotropic receptors Although metabotropic receptors are not included in the classic glutamate excitotoxicity theory, they may be potentially important for MS: Activation of group II metabotropic receptors has been implicated in the protection of neurons against excitotoxic injury in animal models (Bruno et al., 2001), possibly by modulating secondary Ca2þ fluxes. Metabotropic receptors are reportedly expressed by human astrocytes and oligodendrocytes (Mennerick et al., 1996). Furthermore, metabotropic GluR’s (group III) have been recently implicated in inhibiting the production of RANTES, a key proinflammatory cytokine, in glial cells (Besong et al., 2002). However, the role of metabotropic glutamate receptors in the immunopathogenesis of white matter inflammation is presently unknown. 4. Sources of excess glutamate in MS lesions 4.1. Introduction The hypothesis that glutamate excitotoxicity is operative in MS was fueled by reports that activated macrophages and microglia, produce and release significant amounts of glutamate. This is supported by findings of increased extracellular glutamate in the CNS as indicated by increased levels of glutamate in cerebrospinal fluid of patients with a range of CNS inflammatory diseases, from MS to acute encephalitis and meningitis (Buryakova and Sytinsky, 1975; Stover et al., 1997). 4.2. Glutamate production by immune cells The first reports on glutamate release by immune cells came from in vitro experiments by Fontana and colleagues (Piani et al., 1991, 1992), who found that activated rat macrophages and microglia release an excitotoxic agent into their culture medium. When stimulated in vitro, these cells produced a dramatic rise in the levels of the excitotoxin, which killed cerebellar granule cells in culture in an NMDA receptor-dependent manner. The excitotoxin was found to be resistant to heat inactivation, and thus unlikely to be proteinaceous in nature. Fontana and colleagues then identified this agent as glutamate,

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which was produced in larger amounts de novo after stimulation. Furthermore, the excretion of glutamate into the medium was later shown to be an active, energyconsuming process (Piani and Fontana, 1994). Work by others (Curi et al., 1997) showed that other activated immune cells, namely neutrophils, also produce glutamate. The source of this freshly generated glutamate was glutamine, which is the most abundant amino acid in blood and other extracellular fluids. Glutamine uptake is significantly increased in activated monocytes (Ardawi et al., 1991). The production of glutamate by activated immune cells is catalyzed by glutaminase (Newsholme and Calder, 1997), an enzyme that deamidates glutamine to glutamate and ammonia (Eq. (1)). Glutamine þ H2 O ! Glutamate þ NH4 OH

ð1Þ

This reaction is exotherm, and its equilibrium lies on the site of formation of glutamate from glutamine. As discussed below, the reversal of this reaction shown in Eq. (1) by the enzyme glutamine synthetase (GS) requires the consumption of ATP. Two isoforms of glutaminase are known in mammals, one prominently expressed in kidney, and one chiefly found in liver (Curthoys and Watford, 1995). The glutaminase found in kidney is identical to the enzyme expressed by immune cells (N. Curthoys, personal communication). Antibodies to the kidney isoform show excellent crossreactivity to immune cell glutaminase (Werner et al., 2001). The regulation and function of this enzyme has been worked out in exquisite detail for kidney, where it plays a key role in maintaining blood pH (Curthoys and Watford, 1995). In contrast, both the regulation and the metabolic function of glutaminase in immune cells are still elusive. Since macrophages release only a small fraction of the carbon skeleton of the glutamate produced from glutamine as CO2 (Newsholme et al., 1987), it is unlikely to serve as fuel for the increased energy demands of activated monocytes. One possibility is that this reaction allows activated immune cells to maintain its nitrogen balance (Murphy and Newsholme, 1998), as activated monocytes constitutively express nitric oxide synthetase and produce NO. However, this is still hypothetical. 4.3. Relevance to MS A recent study in human autopsy specimen found evidence that glutamate excitotoxicity is indeed operational in MS (Werner et al., 2001). In the same study, upregulation of the glutamate-producing enzyme, glutaminase, was found both in active lesions of mouse-EAE (Fig. 4), and, more importantly, in human tissue (Fig. 4). Both in mouse EAE (Fig. 4) and in MS, increased axonal damage was closely associated with areas of high glutaminase expression. The AMPA/KA antagonist NBQX reduced the extent of axonal damage in the lesion, without any overt effect on glutaminase expression, further supporting glutamate excitotoxicity as a mechanism of axonal damage (Fig. 4). Both activated macrophages and microglia in MS and other inflammatory CNS diseases showed increased presence of glutaminase, with active MS lesions displaying the most robust expression, both regarding the intensity and the density of glutaminase-positive immune cells (Fig. 4). MS lesion tissue was compared to white matter from normal subjects and from other neurologic diseases. Both early and chronic-active MS lesions

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Fig. 4. Glutamate production in control and MS white matter and in EAE. Left panels. Human white matter from control and chronic active MS labeled for the glutamate-producing enzyme, glutaminase (blue), and for the macrophage/microglia marker CD68 (brown). Note the intense glutaminase and CD68 reactivity towards the MS lesion center (full field, bottom left), with brown macrophage/microglial labeling extending into adjacent white matter (upper right) ( £ 75). Lower left panel: High power view ( £ 750) of foamy, active macrophages (brown) with robust glutaminase (blue) expression. Lower right panel: Control white matter processed in parallel shows little or absent glutaminase expression (full field; £ 200). In higher power view ( £ 750), CD68-positive, ramified microglia (brown) are found, but with little or absent glutaminase reactivity (blue). Right panels. Shown is a representative section from a vehicle-treated mouse with EAE (upper left figure). Glutamate production in an acutely demyelinating region of an active lesion is evidenced by intense display of glutaminase immunoreactivity (blue), accompanied by dystrophic, SMI32 positive, axons (brown) ( £ 75). In the detail image below, note the dystrophic axons (brown) surrounded by glutamate-producing macrophages (blue) ( £ 750). In a matching animal treated with the AMPA/KA glutamate antagonist, NBQX, equally intense glutaminase expression is present but axonal damage (brown) is more limited when compared with vehicle treated animals ( £ 75). Below, detailed view further illustrates the marked reduction in the axonal damage ( £ 750).

displayed an abundance macrophages and microglia, which expressed high levels of glutaminase. This abundance of glutamate-producing cells was greatest in the lesion center, and tapered off towards the periphery of the lesion. It is noteworthy that this pattern reproduces faithfully the distribution and migration of immune cells characteristic of active lesions (see Fig. 2). Glutaminase immunoreactivity in CD68-positive macrophages in active lesions was occasionally punctate, in agreement with the mitochondrial localization of this enzyme in neurons and kidney cells (Curthoys and Watford, 1995). Unlike MS lesion tissue, white matter from normal subjects and non-inflammatory other neurological diseases displayed mainly resting microglia, with little or no

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immunoreactivity for glutaminase. In summary, these observations give strong support to the notion that glutamate production by macrophages and microglia is a feature of inflammatory activity, consistent with the well-documented up-regulation of glutaminase in stimulated macrophages in vitro, discussed above. 4.4. Release by damaged axons Axons, under certain conditions, can also release larger amounts of glutamate, likely by reversal of glutamate uptake following reversal of ion fluxes and membrane depolarization (Li and Stys, 2001). This mechanism was shown to be operational in spinal cord anoxia and hypoxia, where it contributed to the oligodendrocytic damage and functional loss in these conditions (Li and Stys, 2001). This is in line with the prominent expression of KA2 receptor subunits in the perinodal region in mouse (Brand-Schieber and Werner, 2003). At present, there is no direct evidence linking axonal glutamate release to white matter damage in either EAE or MS. However, given the suspected impairment of axonal energy supply in MS, it is likely that axonal glutamate release contributes to the excitotoxic damage in MS. Given the documented role of (possibly oligodendrocytemediated) glutamate excitotoxicity in axonal damage in MS (Pitt et al., 2000; Werner et al., 2001), axonal glutamate release may be part of a vicious cycle that amplifies existing white matter damage.

5. Glutamate homeostasis and MS 5.1. Introduction Glutamate is predominantly metabolized by GS and glutamate dehydrogenase (GDH). Excess extracellular glutamate is the result of disturbed homeostasis, which may be the result of excessive release of glutamate (see above), decreased uptake (see below) and metabolism (this section), or all of the aforementioned. While excessive release of glutamate is frequently implicated in both acute and chronic CNS conditions, altered glutamate metabolism can also evoke glutamate excitotoxicity: Some patients with olivopontocerebellar atrophy have been shown to display a mutated form of GDH (Plaitakis et al., 1982), which is a key enzyme of glutamate metabolism. An in-depth review of glutamate metabolism and its role in other CNS diseases can be found in the chapter by Schousboe and Waagepepersen (2003), which is part of this volume.

5.2. Glutamate metabolizing enzymes Glutamate, once taken up into cells (mainly glia), can, in the presence of ammonia and under consumption of ATP, either be converted back to glutamine by GS (Eq. (2)), or deaminated and oxidized to a-ketoglutarate (Eq. (3)) by GDH. Both enzymes are present

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in mitochondria, where the formation of a-ketoglutarate from glutamate or vice versa is a key link between the tricarboxylic acid cycle and amino acid metabolism. Glutamate þ NH4 OH þ ATP ! Glutamine þ ADP þ Pi þ H2 O

ð2Þ

Glutamate þ NADþ þ H2 O ! a-ketoglutarate þ NADH þ NH4 OH

ð3Þ

The equilibrium of Eq. (3) is almost completely on the site of glutamate production, if a-ketoglutarate and NADH are not withdrawn from the equilibrium by further metabolism, i.e., Krebs’ cycle or respiratory chain activity. Spectrophotometric assays that employ NADH production (Eq. (3)) to determine glutamate concentrations commonly use high concentrations of hydrazine-based compounds to remove the a-ketoglutarate from equilibrium to allow the reaction to proceed (Bernt and Bergmeyer, 1963). In summary, glutamate metabolism is of great importance for glutamate homeostasis. Reduced levels of either GS or GDH would likely aggravate any situation in which glutamate concentrations are increased. This was addressed in a recent study, discussed below. 5.3. Glutamate metabolizing enzymes in normal and inflamed white matter In normal human white matter, both enzymes are expressed primarily by oligodendrocytes (Fig. 5). Similarly, GS- and GDH-positivity was seen on oligodendrocytes in white matter from non-inflammatory controls such as ALS. These findings were consistent with the documented presence of GS in oligodendrocytes in rat white matter (Tansey et al., 1991). The high-level expression of these enzymes in oligodendrocytes in normal and non-MS control tissue further suggested that these cells may have an important role in glutamate homeostasis. In white matter of and around active MS lesions, both amount and distribution of GS and GDH differed vastly from that found in control white matter. Immunoreactivity for either enzyme was lost from oligodendrocytes within the lesion and the surrounding area and appeared instead on astrocytes and microglia. Interestingly, this was also true for silent lesions, where the only detectable GSor GDH-immunoreactivity was in occasional astrocytes or microglial cells. However, previous studies have shown that oligodendrocytes are present in and around both active and silent MS lesions (Raine et al., 1981; Lucchinetti et al., 1996). Thus, the absence of GDH- and GS-immunoreactivity from oligodendrocytes around both active and silent lesions suggests a persistent deficiency in glutamate metabolism by oligodendrocytes, even after the inflammation has subsided. In contrast to MS, GDH and GS immunoreactivity in white matter from autopsy cases with other inflammatory neurological diseases, namely subacute sclerosing panencephalitis and tropical spastic paraparesis (TSP), was robust and localized to oligodendrocytes, similar to normal white matter (Fig. 5). It thus appears that the reduction in glutamate metabolizing enzymes is peculiar for MS, although the reasons remain elusive. One possibility is the intense, focal nature of the inflammation in MS compared to other inflammatory diseases of the CNS. The impact of these changes for survival and function of oligodendrocytes is under study. However, given the importance of both enzymes for overall cellular metabolism in general

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Fig. 5. Glutamate metabolism is disturbed in MS. In control human white matter, immunolabeling for GDH (top panel) and GS (bottom panel) clearly delineates oligodendrocytes. In chronic active MS (MS), only astrocytes are immunopositive for GDH and GS. Note the absence of labeling in oligodendrocytes, which are present in lesions (Fig. 2). In contrast, white matter from other neurological diseases, either inflammatory (TSP) or non-inflammatory (ALS), does not show loss of either enzyme. In TSP, oligodendrocytic GDH and GS expression appears enhanced compared with control. All images £ 480.

and glutamate detoxification in particular, their loss is likely to be a significant factor in the lasting damage characteristic for most MS lesions. 6. Glutamate transporters and MS 6.1. Introduction Glutamate homeostasis depends on glutamate uptake. Reduced uptake of glutamate has been suggested to contribute to excitotoxic damage in conditions ranging from amyotrophic lateral sclerosis (ALS) to stroke (Rothstein, 1996; Choi, 1988). In light of these findings, it is of special interest that tumor necrosis factor alpha (TNF-a), a key cytokine of CNS inflammation in general and MS in particular (Raine, 1995), has been shown to down-regulate both the expression and the activity of the high-affinity glutamate transporters EAAT 1 and EAAT 2 in human and murine astrocytes (Fine et al., 1996). Lastly, reactive oxygen species such as zO2 2 and H2O2, which are commonly produced in by activated macrophages, have also been shown to reduce glutamate uptake by astroglia, although this effect was transient (Piani et al., 1993). Thus, the question is whether glutamate transport is altered in MS. This was addressed in a study of three major glial

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glutamate transporters (GLAST, GLT-1 and EAAT-1) in human white matter (Werner et al., 2001), discussed below. 6.2. Glutamate transporters in control and MS white matter In control human white matter, all three glutamate transporters studied, GLAST, GLT1 and EAAT-1, showed robust expression by oligodendrocytes and low-level expression by astrocytes and microglia, representatively shown is GLT-1 in Fig. 6. This result was unexpected, as glutamate transporters in gray matter are mostly localized to astrocytes (Gegelashvili and Schousboe, 1998). In MS tissue, all three transporters were markedly reduced within lesion centers, from which oligodendrocytes had been depleted. However, unlike GLAST and EAAT-1, the reduction of GLT-1 expression extended far beyond early active lesions into the adjacent white matter, and only faint expression by oligodendrocytes was seen (Fig. 6). In chronic active lesions, decreased expression of GLT-1 was restricted to the perimeter of the lesion while unaffected adjacent white mater displayed control level expression (data not shown). Chronic silent MS lesions exhibited low level GLT-1 expression on macrophages within lesions, but in contrast to early active lesions, displayed control level expression throughout adjacent white matter. The transient

Fig. 6. The glial glutamate transporter GLT-1—localization in control and MS white matter. Left column: GLT-1 expression in control human white matter (chevrons). Top panel: Note the ‘string-of-pearls’ appearance of immunopositive cells, which appear oligodendrocytic. Left column, bottom panel: Double-label for GLT-1 (brown) and CNPase, a marker of oligodendrocytes (blue) verifies that the cells prominently expressing GLT-1 are indeed oligodendrocytes (chevrons). £ 750. Middle column: GLT-1 expression in active MS. Top panel: Center of an active lesion. Note the faint, almost absent label for GLT-1; remaining immunoreactivity appear astrocytic in nature (chevrons). Bottom panel: White matter adjacent to the lesion. Note the reduced, but clearly visible presence of GLT-1 on oligodendrocytes(chevrons). £ 750. Right column: GLT-1 expression in chronic silent MS. Top panel: Center of the lesion. Faint, remaining GLT-1 reactivity is observed on astrocytic processes and macrophages (chevron). Note the absence of oligodendrocytic labeling for GLT-1. Bottom panel: White matter adjacent to the lesion. Note the presence of GLT-1 on oligodendrocytes (chevrons). Other GLT-1 positive cells are likely astrocytes and microglia. £ 750. All images are taken from freshly frozen, acetone post-fixed 10 mm sections from autopsy tissue.

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but widespread down-regulation of a major glutamate transporter associated with the inflammatory activity in MS lesions, is strongly suggestive of a diffusible mediator that down-regulates GLT-1, such as TNF-a. However, the exact causes for this downregulation is still unknown and under investigation. The loss of an important component of the glutamate detoxification pathway can contribute to elevated levels of extracellular glutamate, and thus increases the likelihood of excitotoxic damage. The observations in human autopsy tissue suggest that key glial glutamate transporters, and both principal glutamate metabolizing enzymes, GS and GDH, are prominently expressed by oligodendrocytes in human white matter. In MS, GLT-1, one of the main glial glutamate transporters, and both GS and GDH are strongly reduced in and around the lesion area (Werner et al., 2001). It remains to be seen whether results by others will support or negate the apparent role of oligodendrocytes as the main cell type for glutamate uptake and metabolism in human white matter. Taken together, these observations make it highly likely that glutamate homeostasis in white matter is both acutely and chronically impaired in MS tissue, This reduced capability of the tissue to detoxify glutamate increases the risk of excitotoxicity for the remaining oligodendrocytes and axons. 7. Concluding remarks Glutamate excitotoxicity as a pathologic mechanism in MS is still subject to debate. However, the immunopathology of MS fulfills every single criterion for the presence of glutamate excitotoxicity, as depicted in Fig. 7. Attracted by antigen-stimulated T cells,

Fig. 7. A working model for glutamate excitotoxicity in inflammatory demyelination.

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macrophages and microglia become activated and attracted into white matter. These strongly glutaminase-positive cells, both known to extrude large quantities of glutamate, provide a rich source of extracellular glutamate. In parallel, the capacity of the tissue to absorb the additional glutamate is significantly reduced. This reduced capacity is due to decreased presence of at least three glial glutamate transporters (GLAST, GLT-1, EAAT-1) and two key glutamate metabolizing enzymes (GDH and GS) are depleted or essentially absent from the lesion area. The rise in extracellular glutamate triggers glutamate receptors on oligodendrocytes and, possibly, axons, leading to excitotoxic damage. The situation may be worsened by extrusion of glutamate by damaged axons, adding to the excitotoxic load. The beneficial effects of glutamate antagonists in EAE, an animal model of MS, support the notion that glutamate excitotoxicity is a significant mechanism of damage to both non-neuronal and neuronal targets of MS. Pharmacologic manipulation of the pathways that lead to glutamate excitotoxicity may have therapeutic import.

Acknowledgements We thank Mr Stuart Lowery and Dr Kakuri Omari for their help with the production of this chapter. The authors wish to acknowledge their support by NIH grants NS 41056 (to P.W.), and NS 08952 and NS 11920 (to C.S.R.). A part of Dr Brand-Schieber’s work was conducted as a postdoctoral fellow in the experimental neuropathology training program (NS 07098; director: Dr C.S. Raine).

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Role of glia in prion disease David R. Brownp and Judyth Sassoon Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK p Correspondence address: Tel.: þ 44-1225-323133; fax: þ 44-1223-826779. E-mail: [email protected](D.R.B.)

Contents 1. 2. 3. 4. 5. 6.

Introduction Prion protein expression in glia Mechanisms of toxicity: a role for microglia Microglia – astrocyte interactions Neurone – astrocyte interactions Concluding remarks: a unified cell theory

The prion diseases are notorious neurodegenerative diseases. Their transmission has been linked to an abnormal isoform of the prion protein. Deposition of this abnormal protein isoform in the central nervous system is also associated with the onset of pathological changes and disease progression. The second most significant change to the brain other than neurodegeneration is gliosis involving both microglia and astrocytes. The possible association between gliosis and neuronal death has been investigated using cell culture models. These models, using peptide mimics of the abnormal prion protein isoform, indicate a role for both microglia and astrocytes in the neurotoxic action of the peptide. Microglia activated by the prion protein peptide release both cytokines and toxic substances such as superoxide. The cytokines released by the microglia stimulate astroglial proliferation. Alterations in the interactions between astrocytes and neurones expose neurones to the toxicity of glutamate. Thus a model of neuronal death in prion disease has emerged, indicating that both microglia and astrocytes can either induce or exacerbate neuronal death induced by the abnormal prion protein isoform. 1. Introduction Prion diseases are also called transmissible spongiform encephalopathies. This is because they can be experimentally transmitted between individuals in mouse or primate models (Gibbs et al., 1968; Prusiner, 1982). Additionally, patients or animals with one of Advances in Molecular and Cell Biology, Vol. 31, pages 1085–1104 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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these diseases have vacuolation in the brain to a less or greater extent. However, there are many neurological diseases, which have either gliosis, neurodegeneration or spongiform changes. What is particular to prion diseases is the deposition of some amount (Prusiner, 1997, 1998) of an abnormal, protease resistant isoform (PrPSc) of a normal glycoprotein, the prion protein (PrPc), which is expressed preferentially at synapses (Sale`s et al., 1998). Indeed, it was the development of antibodies to PrPSc in the early 1990s that allowed for reliable positive diagnosis of these diseases. Additionally, most prion disease do not occur as a result of known ‘infection’, and so transmission is not really a major requirement for an individual to have one of these diseases. Furthermore, the disease affects other parts of the body besides the brain. PrPSc can be detected outside the central nervous system. Particular organs are affected more in different prion diseases. However, lymphatic tissue and the spleen seem to be important secondary targets (Glatzel et al., 2001). Recent suggestions have also been made that blood can carry the disease (Schmitt et al., 2002), and that a PrPSc isoform can be excreted in the urine (Shaked et al., 2001). For these reasons it might be advisable to abandon the historical and misleading name of transmissible spongiform encephalopathy in preference to prion disease. The vast majority of human prion disease cases are called Creutzfeldt– Jakob disease (CJD) and occur spontaneously with no known cause (Prusiner, 1991). There are also inherited forms of prion disease, which include Gerstman – Stra¨ussler – Scheinker Syndrome (GSS), Fatal Familial Insomnia and inherited CJD (Prusiner, 1991; Ghetti et al., 1989, 1996). The number of cases where transmission of disease has occurred by ‘infection’ is quite limited. The only confirmed cases are those of iatrogenic transmission resulting from transplantation of human tissue, such as dura mater or central nervous system products (Jaegly et al., 1995). The disease Kuru is believed to have been spread by eating of human brains. Although there are quite a number of unequivocal similarities between Bovine Spongiform Encephalopathy (BSE) and the recently described new variant CJD (nvCJD) (Collinge et al., 1996; Bruce et al., 1997), a causal connection between the two diseases has not been demonstrated. Animal diseases include BSE of cattle (Hope et al., 1988), scrapie of sheep (Prusiner, 1982), Chronic Wasting Disease of deer and elk (Guiroy et al., 1991) and transmissible mink encephalopathy (Marsh et al., 1969). PrPc is a glycoprotein tethered to the outside of the cell by a glycosylphosphatidyl inositol (GPI) anchor (Stahl et al., 1990). The protein is a 253 amino acid polypeptide coded by a single exon in the prnp gene (Basler et al., 1986), and it has been shown to bind copper (Hornshaw et al., 1995; Brown et al., 1997b, 1999, 2001). Four copper atoms can bind to an N-terminal, histidine-rich region of the protein that contains between four and six copies of a repeated sequence of an octamer in mammals or a hexamer in other vertebrates (Wopfner et al., 1999). It is possible that a fifth atom of copper can bind elsewhere in the protein. At present there is no known structure for the holoform of the protein. The apoform of recombinant protein has been studied using NMR in a number of species (Riek et al., 1996; Zahn et al., 2000; Lopez Garcia et al., 2000). The apoform has three helical regions in the C-terminal domain and is unstructured at the N-terminus. Other biophysical techniques indicate that binding of copper causes significant changes to this structure (Miura et al., 1996; Wong et al., 2000a). Therefore the preliminary NMR spectra are probably not an accurate indication of the structure of PrPc in vivo.

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Analysis of the activity of both recombinant and native PrPc has shown that it is an antioxidant with an activity like that of a superoxide dismutase (SOD) (Brown et al., 1999, 2000, 2001). Extracts in which PrPc has been immunodepleted show a decrease in SOD activity (Wong et al., 2000b). Mice in which PrPc expression has been knocked out by genetic ablation show decreased levels of SOD activity as well as altered levels of other antioxidants (Brown et al., 1997c, 2002; White et al., 1999), some of which may be compensations for loss of PrPc expression. 2. Prion protein expression in glia PrPc is expressed primarily in neurones. However there are other cell types that also express PrPc. Thus, PrPc is expressed by microglia (Brown et al., 1998a) and astrocytes (Brown, 1999a). The level of expression is considerably lower than that in neurones (Fig. 1), and astrocytes and microglia express predominantly a single form of PrPc (double glycosylated), while cerebellar neurons express three forms (double, single and not glycosylated). However, this does not mean that PrPc expression has no consequences for the phenotype of glial cells. Changes in the phenotype of glial cells from PrP-knockout mice have been studied. In general, the findings suggest that PrPc-expression modifies glial responses to oxidative stress and possibly protects certain cellular activities that could be inhibited by oxidation. Microglia can be activated by substances like concavalin A or endotoxins such as bacterial lipopolysaccharides (LPS). The activation involves either the release of cytokines or respiratory burst activity leading to the release of large amounts of superoxide. Cultured microglial cells can also be activated by the prion protein peptide, PrP106-126 (Brown et al., 1996a). The degree to which microglia cells can be activated (i.e., how much cytokine or superoxide they release) varies according to the level of PrPcexpression (Brown, 1998). Microglia cells that overexpress PrPc are very sensitive to substances that cause activation. However, knockout of PrPc-expression makes microglia relatively insensitive to activating substances. Furthermore, microglia cells from PrPc

Fig. 1. Prion protein expression in different cell types. Western blots of extracts from cultured murine cerebellar neurones (1), microglia (2) and astrocytes (3). The prion protein was detected with a rabbit antiserum specific for mouse prion protein. Astrocytes and microglia express predominantly a single form of PrPc (double glycosylated) while cerebellar granule neurons express three forms (double, single and not glycosylated).

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overexpressing mice proliferate more rapidly when co-cultured with astrocytes (Brown, 1998). Tga35 mice, which overexpress PrPc have excessive numbers of Mac-1 positive microglia cells in their brain (Brown, 2001). These mice go on in later life to develop ataxia and neuronal loss (Westaway et al., 1994). One possible reason for this is the abnormal expression of PrPc in microglia (Brown, 1998). Astrocytes also respond differently when PrPc expression is ablated. In particular they show changes in glutamate uptake (Brown and Mohn, 1999). Purified astrocytes from PrPc-deficient mice show decreased uptake of glutamate through sodium dependent transport mechanisms. The extent of this decrease is dependent on the culture conditions used. Astrocytes grown in serum-free conditions are affected more than those cultured in the presence of serum. This decrease in glutamate uptake is reversed, when an antioxidant is included in the culture medium. Analysis of kinetic parameters indicates that the alteration in uptake is due to decreased density of glutamate uptake sites (Brown and Mohn, 1999). One possible explanation for the decreased uptake of glutamate by PrPcdeficient astrocytes is an increased susceptibility to oxidative stress. GLAST, an astrocytespecific glutamate transporter (see chapter by Schousboe and Waagepetersen) is particularly sensitive to oxidative stress. Oxidation greatly inhibits its ability to transport glutamate. Uptake of glutamate by wild-type astrocytes is inhibited by the prion protein peptide, PrP106-126 (Brown and Mohn, 1999). This reduction is similar to the decrease in uptake observed for PrPc-deficient astrocytes. However, the peptide does not further decrease uptake by PrPc-deficient astrocytes. There is good evidence that PrP106-126 interacts directly with PrPc itself (Brown, 2000a). Thus, PrP106-126 may exert its effects on both astrocytes and microglia by directly interacting with PrPc and inhibiting its protective function as an antioxidant. PrPc is also expressed in oligodendrocytes (Moser et al., 1995), and there is clear evidence that these cells may also be affected adversely in prion diseases. Damage to oligodendrocytes is apparent in vivo in CJD (El Hachimi et al., 1998). Demyelination is one of the changes that occur in prion disease. At present it is not clear how it is caused but it could also be due to toxic substances from microglia activated by PrPSc.

3. Mechanisms of toxicity: a role for microglia The first studies of neuronal death in prion disease used cell culture models (Mu¨ller et al., 1993; Forloni et al., 1993). This approach has remained the preferred model for the majority of investigators. Mu¨ller et al. (1993) showed that PrPSc is toxic to cultured neurons. That study was also the first to suggest that the mechanism of cell death involved calcium entry via NMDA receptors. The difficulty in isolating PrPSc and the general inability to prove that it is pure has lead investigators to use synthetic peptides. Forloni et al. (1993) identified a twenty-one amino acid peptide (PrP106-126) that appeared to represent the neurotoxic core of human PrPSc. Since then, virtually every group studying neuronal death in prion diseases has used this peptide or sequences equivalent to it. Apart from one very poorly controlled study (Kunz et al., 1999; see Brown, 1999b) the neurotoxicity of this peptide has been consistently and thoroughly confirmed. Indeed, the main concern that it may not be not neurotoxic in vivo has also been shown to be invalid by

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the demonstration that the PrP106-126 peptide is toxic to cells in the retina of rats (Ettaiche et al., 2000). This peptide has many features of PrPSc, which include protease resistance, high beta-sheet content and the ability to form fibrils (Selvaggini et al., 1993; De Gioia et al., 1994). PrP-knockout mice are resistant to prion infection (Bu¨eler et al., 1993). As such mice cannot generate PrPSc it is difficult to determine if PrPSc is toxic in the absence of neuronal expression of PrPc in vivo. However, an ingenious transplantation experiment showed that PrPc expression is necessary for the toxicity of PrPSc (Brandner et al., 1996). Transplantation of embryonic tissue from a mouse overexpressing PrPc was made into a PrP-knockout mouse brain. The transplanted tissue was then infected with mouse scrapie. This transplanted tissue generated PrPSc and showed signs of neurodegeneration and gliosis, while the PrP-knockout brain around remained untouched by neurodegeneration. Although PrPSc accumulated in the brains of the PrP-knockout mice, this did not cause neurodegeneration. These results show that neurones must express PrPc in order to be killed by the agent of neurodegeneration in prion disease. Assuming that this agent is solely PrPSc, it can be concluded that PrPSc requires neuronal expression of PrPc to be neurotoxic. Neurotoxicity of PrP106-126 also requires neuronal expression of PrPc, as first shown in 1994 in cultured cells (Brown et al., 1994). This finding has been confirmed by other groups (Jobling et al., 1999), and the neurotoxicity of PrPSc itself in cultured cells is also dependent upon the expression of PrPc (Giese et al., 1998). As this result has been obtained both in vivo and in vitro, and it has been confirmed by a number of groups, it can be assumed that a requirement for neuronal PrPc expression is an essential component of the mechanism by which PrP106-126 and PrPSc are neurotoxic. Oxidative stress has been shown to be a hallmark of prion diseases (Guentchev et al., 2000; Wong et al., 2001), as is also the case in many other neurodegenerative diseases. Numerous groups have shown that PrP106-126 causes oxidative stress and disturbs the expression of antioxidant proteins, and that the toxicity of PrP106-126 can be inhibited by antioxidants (Brown et al., 1996a; Perovic et al., 1997). Cultures infected with PrPSc are more susceptible to the toxicity of reactive oxygen species (Milhavet et al., 2000). There is currently no evidence that antioxidants can inhibit neuronal death in vivo. However, it is clear that oxidative damage is involved in the mechanism of PrP106-126 in the culture system. Therefore it is a logical to consider induction of oxidative damage as a second component of the mechanism of action of the peptide. There is little doubt that PrP106-126 causes cell death via an apoptotic mechanism. Caspase activation (White et al., 2001), mitochondrial depolarization (O’Donovan et al., 2001) and enhanced calcium entry through either NMDA receptors (Mu¨ller et al., 1993) or L-type voltage gated calcium channels (Brown et al., 1997a) contribute to neuronal apoptosis. It is therefore logical to assume that any or all of these changes might be induced by the peptide or PrPSc. However, none of these effects is specific enough to be safely considered a ‘downstream’ event that occurs as a result of the triggering of apoptosis. Therefore, although these actions are likely parts of the mechanism of action for the peptide, they are not specific. The role of microglia in culture models of prion disease has been reviewed before (Brown, 2001). Microglial cells are activated by either PrP106-126 or PrPSc, as first shown by Brown et al. (1996a). This finding has now been confirmed by a number of groups

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(Giese et al., 1998; Combs et al., 1999; Fabrizi et al., 2001). Activated microglia release toxic substances such as superoxide or TNF-a, which might contribute to indirect toxic effects. Fig. 2 shows that PrP106-126 provokes superoxide formation by microglia, and that this effect is reduced in microglia from PrP knockouts and enhanced in cells from PrP overexpressors (Brown et al., 1996a; Brown, 1998). However, although microglia appears to be a component of the mechanism for PrP106-126 toxicity, it was also shown that the presence of microglia per se is NOT essential, but that these cells serve as a source for generation of oxidative stress (Brown et al., 1996a). Microglia also enhance the toxicity of PrPSc (Giese et al., 1998) and microglia are activated before the onset of neurodegeneration in mouse scrapie (Williams et al., 1994; Giese et al., 1998). Since these first reports it has been independently confirmed that microglia can enhance the toxicity of both PrPSc and PrP106-126 (Bate et al., 2001), implying that the effect is reproducible. Thus, it can be concluded that an indirect effect of substances released by microglia can contribute to the neurotoxic mechanism of prion disease. 4. Microglia –astrocyte interactions Astrogliosis is one of the hallmarks of prion disease. This in itself is not indicative of specific changes in the CNS peculiar to prion disease as most neurodegenerative changes are accompanied by proliferation of astrocytes. These changes are usually a result of

Fig. 2. Superoxide generated by microglia stimulated by PrP106-126. Microglia were prepared from the brains of mice. The mice were either wild-type (open circles), PrP-knockout (filled circles) or PrP-overexpressing (filled squares). Superoxide was measured spectrophotometrically with a nitro blue tetrazolium based assay. Results are means ^ SEM for four experiments.

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damage to or death of neurones. However, in prion disease there is strong evidence that gliosis occurs before neurodegeneration (Jendroska et al., 1991; DeArmond et al., 1992; Giese et al., 1998). Furthermore there is evidence that astrocytes are the cell type in which the abnormal form of the prion protein, PrPSc, is first replicated in the central nervous system (Diedrich et al., 1991). Indeed, mice devoid of murine PrP, but expressing hamster PrP transgenes driven by the astrocyte-specific GFAP promoter, are susceptible to prion disease, despite the fact that these animals only express PrP in astrocytes (Raeber et al., 1997). A recent in vitro study also indicates that the presence of large numbers of astrocytes can accelerate the rate at which neurones are killed by peptide PrP106-126 (Brown, 1999a). This finding does not contradict anything that has been described above. The toxicity of PrP106-126 is not enhanced by astrocytes (Brown et al., 1996a), instead the toxicity of glutamate to neurones can be enhanced by the effect of PrP106-126 on PrPc-expressing astrocytes. A cell culture system is by design a simplification of the natural situation in the brain. As described above the basic mechanism of PrP106-126 toxicity to cerebellar neurons involves microglia. However, when increased number of astrocytes are added into this system to mimic the astrogliosis seen in prion disease, increased toxicity is observed in this system, which can be directly attributed to effects of PrP106-126 on astrocytes (see below). This system therefore represents a more accurate model of the situation in the brain, when the most rapid neurodegeneration is occurring. Thus a model of the end stage pathology must also take into account that large numbers of astrocytes are present, and that they are likely to contribute to the degeneration. Gliosis (of both microglia and astrocytes) is often believed to be a response to damaged neurones. However, this does not imply that the gliosis in prion disease is a response to neuronal damage. As indicated above there are indications that gliosis occurs before neurodegeneration in prion disease begins but does coincide with deposition of PrPSc. Therefore there is the possibility that PrPSc can stimulate gliosis. It has been demonstrated previously that PrP106-126 can induce astrocyte proliferation in culture (Forloni et al., 1994). PrP106-126 can also induce microglia to proliferate (Brown et al., 1996b; Fig. 3). The initial studies of astrocyte proliferation were made on mixed glial preparations from rat cortex (Forloni et al., 1994). However, when astrocytes were purified free of contaminating cells it was found that PrP106-126 could not induce their proliferation, as illustrated in Fig. 3 (Brown et al., 1996a, 1998b). It was found that, like the neurotoxic effect seen in cerebellar cell cultures, the induction of astrocyte proliferation was also dependent on the presence of microglia (Brown et al., 1996b). However, microglia on their own are not sufficient. In order for PrP106-126 to induce astrocyte proliferation, the astrocytes must express the PrPc. PrP106-126 cannot induce proliferation of astrocytes in mixed glial cultures from PrPc-deficient mice (Brown et al., 1996b), and addition of wild-type microglia is insufficient for PrP106-126 to induce PrPc-deficient astrocyte proliferation. (Brown et al., 1998c). Thus, changes induced directly on the astrocyte by PrP106-126 are necessary for the astrocytes to respond to the mitogenic factors released by PrP106-126 stimulated microglia (Brown et al., 1998c). The model of astroglial proliferation described above is similar to the model of neurotoxicity, as it requires (i) a direct involvement of microglia; (ii) the indirect effect of microglial released substance; and (iii) a direct effect on the target cell

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Fig. 3. PrP106-126 induces astrocyte proliferation in astrocyte–microglia co-cultures. 80 mM PrP106-126 (A) or 80 mM bAmyloid peptide (B) was applied to astrocytes either as a single dose (circles) or reapplied after 48 h (triangles). The treatments were either carried out in the presence (open circles, open triangles) or the absence (filled triangle) of microglia. The relative cell number was determined using a standard MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) colorimetric assay. The values for untreated cultures assessed in parallel were equated to 0% and the other values expressed as percentage change in cell number. Results are means ^ SEM of four experiments, with three determinations each.

(neurone or astrocytes), which requires PrPc expression. Interleukin-1 (IL-1) and interleukin-6 (IL-6) were identified as the factors present in the medium of microglia and astrocyte co-cultures necessary for the proliferation of astrocytes induced by PrP106126 (Hafiz and Brown, 2000). Previous studies have shown that PrP106-126 induces release of both cytokines from microglia (Peyrin et al., 1999). Treatment with antibodies against IL-1 and IL-6 inhibits the proliferation of astrocytes mediated by PrP106-126treated microglia (Hafiz and Brown, 2000). PrP106-126 treatment enhances the level of expression of cytokine receptor for IL-1 and IL-6. However, the application of these cytokines in combination with PrP106-126 was insufficient to induce astroglia proliferation in microglia-free cultures, implying that these factors do not completely replace the effect of microglia (Hafiz and Brown, 2000). The effects of IL-1 and IL-6 were potentiated with superoxide generated from xanthine oxidase, and the proliferation of the

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astrocytes in co-culture with microglia was inhibited by SOD, suggesting that superoxide or its products are somehow necessary for the proliferative effect of PrP106-126. Therefore the signal coming from microglia and inducing astrocyte proliferation in the presence of PrP106-126 is a multifactorial signal (Hafiz and Brown, 2000). A role of reactive oxygen species in enhancing cell proliferation appears to be a novel observation, as there seems to be no other previously described system that assigns a role in the induction of cell proliferation to reactive oxygen species. This signal does not appear to include oxidative stress per se as the antioxidant N-acetyl cysteine does not inhibit astrocyte proliferation induced by PrP106-126 treated microglia (Hafiz and Brown, 2000). The multifactorial signal coming from microglia, which was just described, is insufficient to induce proliferation of astrocytes to the same extent as the proliferation induced by PrP106-126 in the astrocyte/microglia co-culture system. Astrocytes expressing PrPc are primed by PrP106-126 to proliferate, when the appropriate signal is received from microglia (Brown et al., 1998c). In other words, a direct effect of PrP106126 on astrocytes enhances the proliferation signal from microglia to induce significant proliferation. The dependence of this astrocyte specific effect on PrPc expression suggests that PrP106-126 may directly interact with astrocytes via PrPc, or that PrPc expression changes astrocytes, so that they respond differently when PrP106-126 is in the environment. Understanding of the direct effects of PrP106-126 on astrocytes that primes them to respond more to proliferation signals, is of considerable interest, as identification of pathways involved in these changes may lead to ways to intervene and prevent astrocyte proliferation in prion disease. Inhibitors of the MAP kinase/ERK pathway in astrocytes prevented the proliferation of astrocytes due to a direct effect of PrP106-126 on astrocytes (Brown et al., 2000). The ratio between phosphorylated and non-phosphorylated ERK proteins in astrocytes was increased after 1 day of exposure of pure astrocyte cultures to PrP106-126. Increased phosphorylation of ERKs is, however, not a specific effect of PrP106-126, as increased phosphorylation of ERKs occurs under many conditions including treatment with bA25-35 (Brown et al., 2000), and b-amyloid decreases, rather than increases, astrocyte proliferation (Fig. 3). PrP106-126 treatment of pure astrocytes greatly enhances cyclin E levels (Hafiz and Brown, 2000). This in itself is suggestive that PrP106-126 treated astrocytes are more likely to progress through to the late stages of the G1 phase of the cell cycle, which precedes DNA replication in S phase (see chapter by Nakatsuji and Miller). However, PrP106-126 does not increase astrocytic expression of PCNA, a proliferating cell nuclear antigen, which is a marker of entry into S phase, indicating that PrP106-126 does not induce increased entry into this part of the cycle (Hafiz and Brown, 2000). However, by inducing astrocytes to progress to late G1 phase, PrP106-126 ‘primes’ astrocytes to respond more rapidly to proliferation signals when they occur. PrP106-126 also induces other direct effects on astrocytes than those related to cell cycle. As already mentioned, PrP106-126 inhibits glutamate uptake (Brown and Mohn, 1999), and it increases the susceptibility of astrocytes to oxidative stress (Brown et al., 1998c). There are similarities between the phenotype of astrocytes deficient in PrPc expression and the susceptibility of astrocytes to various insults in the presence of PrP106-126. PrPc-deficient astrocytes also show increased susceptibility to the toxicity of

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oxidative stress (Brown et al., 1998c) and take up less glutamate than wild-type cells (Brown and Mohn, 1999). These similarities as well as similarities between PrP106-126 treated neurones and PrPc-deficient neurones suggest that a major effect of PrP106-126 may be inhibition of PrPc function. This may once again relate to the recent discovery that PrPc is an antioxidant (Brown et al., 1999), and suggest that changes in astrocytes due to PrP106-126 may be a response to oxidative stress. However, PrPc-deficient astrocytes do not proliferate more rapidly than wild-type cells (Brown et al., 1996b), and there is no indication that these cells respond more rapidly to mitogenic factors. Therefore it is more likely that the changes induced in astrocytes by PrP106-126 leading to proliferation are linked to the ‘stress’ induced on the cells by the peptide. Astrocytes treated with PrP106126, which have been prevented from proliferating by cytosine arabinoside, show increased cell death (Brown et al., 1998c). In this case the astrocytes probably die by activation of a p53 mediated pathway, as is known to be the case for cytosine arabinoside induced cell death (Anderson and Tolkovsky, 1999). It is in support of this conclusion that PrP106-126 treatment enhances p53 expression (Hafiz and Brown, 2000). Therefore increased progression through the cell cycle by PrP106-126 is possibly a way by which astrocytes try to escape commitment to cell death, an option neurones treated with the neurotoxic peptide do not have. Evidence for this concept also comes from studies with PC12 cells and myoblasts, as PrP106-126 is not toxic to undifferentiated PC12 cells or myoblasts, which can divide, but is toxic to differentiated PC12 cells and myotubes, which cannot divide (Brown et al., 1997d, 1998c).

5. Neurone –astrocyte interactions The way in which astrocytes protect neurones from various insults is under continued investigation. In the cerebellum, where glutamate is a major neurotransmitter (Gallo et al., 1982), the astrocyte population provides effective protection again the excitotoxicity of glutamate (McLennan, 1976). Astrocytes clear the glutamate released by neurones via active uptake through glutamate transporting proteins such as the highly efficient astrocyte-specific glutamate transporters GLAST and GLT-1 (Danbolt et al., 1992; Chaudhry et al., 1995; Lehre et al., 1995; Rothstein et al., 1996—see also chapter by Schousboe and Waagepetersen). Without rapid clearance glutamate would have a toxic effect mediated through binding to NMDA receptors (Choi et al., 1988). The presence of astrocytes in neurone-rich cultures has been shown to reduce the toxic potency of glutamate (Rosenberg et al., 1992; Ye and Sontheimer, 1998). When glutamate is not cleared, levels of glutamate can easily rise activating NMDA receptors, causing increased calcium entry, internal oxidative stress, mitochondrial dysfunction and eventually apoptosis (Choi et al., 1988; Bondy and Lee, 1993; Speliotes et al., 1994; Schinder et al., 1996; Ward et al., 2000). Therefore understanding what regulates glutamate uptake by astrocytes is essential. There is now strong evidence that factors from neurones alter the level of expression of the astrocyte glutamate transporters (Levy et al., 1995). This can result in an increased clearance of glutamate via these transporters (Brown, 1999c, 2000b). Thus, factors released by neurones inform astrocytes of the neurones’ presence. This induces an

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alteration of astrocyte protein expression resulting in increased protection of neurones by astrocytes against glutamate toxicity. Vasoactive intestinal peptide (VIP) has been identified as one such factor released by neurones (Brown, 2000b). It has been shown that substances such as VIP not only alter the effectiveness of glutamate transport by astrocytes, but also stimulate the release of protective factors, which may help neurones resist the excitotoxicity of substances such as glutamate and NMDA (Gottschall et al., 1994; Bassan et al., 1999). In dissecting the mechanism by which glutamate toxicity is prevented in the normal brain, there is clearly a need to understand not only how glutamate uptake by astrocytes is altered, but also which additional mechanisms are involved. The altered astrocyte behaviour is induced by the presence of neurons, but in turn neuronal sensitivity to glutamate is regulated by the presence of astrocytes. Thus, neuronal survival depends also on astrocytic factors other than removal of glutamate by active uptake. Previous studies have shown that mouse cerebellar neurones become dependent on astrocytes for protection from glutamate toxicity (Brown, 1999c,d). Cerebellar neurones co-cultured with astrocytes show an increased sensitivity to the toxicity of glutamate as compared to cerebellar neurones not co-cultured. This effect is not dependent on regional origin of the astrocytes used for co-culture (Brown, 1999c). Region specific effects of astrocytes have been suggested to be contact mediated and the changes to glutamate sensitivity was related to diffusible factors, some of which have been defined (Brown, 2000b). In the presence of astrocytes an increased sensitivity to the toxic effects of glutamate is normally not noticeable, because of the survival-promoting effects of astrocytes by clearance of glutamate and release of protective factors. Increased sensitivity to glutamate toxicity only becomes of consequence, if the protectiveness of astrocytes is compromised. Several mechanisms cause a compromised astrocyte function, which lead to glutamate induced neuronal death: (i) physical removal of astrocytes; (ii) addition of substances which inhibit astrocytic glutamate uptake (Brown, 1999d); (iii) addition of substances which activate astrocytes (e.g., TGF-b) (Brown, 1999d); or (iv) inactivation of protective factors such as IL-6 (Brown, 1999d). This increased sensitivity to glutamate is induced in neurones by a factor released by astrocytes. Neurones treated with conditioned medium from astrocytes exposed to medium from neuronal cultures (NAM) showed decreased survival. This decreased survival was a result of glutamate toxicity, but it was not correlated with any increase of glutamate concentration (Brown, 1999d). Thus, some additional factor released by astrocytes into NAM makes neurones more sensitive to glutamate toxicity. Release of this factor from astrocytes is enhanced by the presence of neurones themselves or by feeding the astrocytes conditioned medium from neurons, such as NAM (Brown, 1999c). Clearly, identification of the factor(s) and the mechanism(s) of action involved would be of great importance, because of the ensuing possibilities for regulation of neuronal sensitivity to excitotoxic death. Following this work, changes in NMDA receptor subunit subtype composition have been identified, which parallel increased sensitivity to glutamate toxicity. It has been suggested that the subunit NR1b is associated with increased sensitivity to glutamate toxicity (Durand et al., 1992; Nakanishi et al., 1992), and this subunit is increased in cerebellar granule neurons co-cultured with an excess of astrocytes or fed NAM (Table 1), which increased both the total amount of NR receptors and the relative contribution by the

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Table 1 Summary of effects on NMDA receptor subunits Subunit

Control

Co-culture

AM

NAM

Co-culture þ PrP106-126

Total NR1 NR1a NR1b Total NR2 NR2a NR2b NR2c

100 ^ 8 57 ^ 6 43 ^ 6 100 ^ 7 40 ^ 4 32 ^ 4 28 ^ 4

123 ^ 15 49 ^ 5* 51 ^ 5* 115 ^ 14 38 ^ 5* 16 ^ 5* 46 ^ 5*

347 ^ 12* 56 ^ 4 44 ^ 4 124 ^ 10 38 ^ 5 36 ^ 5 26 ^ 5

182 ^ 14* 47 ^ 4* 53 ^ 4* 108 ^ 12 57 ^ 3* 13 ^ 3* 30 ^ 4

76 ^ 14* 42 ^ 6* 58 ^ 6* 125 ^ 12* 60 ^ 5* 9 ^ 5* 31 ^ 5

Results of experiments in which cerebellar neurones were cultured with or without astrocytes or exposed to PrP106-126. AM indicates neurones treated with astrocyte conditioned medium while NAM indicates neurones treated with medium from astrocytes treated with neuronal conditioned medium. Treatment of cerebellar neurones with PrP106-126 (40 mM, 24 h) without co-culture had no effect on NMDA subunit expression and composition (data not shown). NMDA receptor composition was determined using RT-PCR as previously described (Daniels and Brown, 2001). The levels of PCR products were analysed by densitometric analysis. All values from densitometric analysis were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used for housekeeping by division of the densitometric value for the receptor (NR1 or NR2) by that of GAPDH. The levels of controls are shown as 100%. The increase relative to the control value is shown as a percentage for each of the treatments. For the subunit subtypes the values shown are the percentage that each subtype represents of the total densitometric measurement (e.g., for NR2a, percentage ¼ (densitometric reading for NR2a £ 100)/(densitometric readings for NR2a þ NR2b þ NR2c)). * indicates a significant difference from the control value according to Student’s t-test ðp , 0:05Þ: Values are mean and SEM for four experiments.

NR1b subunit (Daniels and Brown, 2001). Anti-NR1b oligonucleotide inhibited NAMinduced toxicity, implying that this subunit is involved in the increased glutamate toxicity following NAM treatment. As cerebellar neurones are protected by astrocytic clearance of glutamate when co-cultured, the expression of this subunit in co-cultured cerebellar neurones does not result in increased cell death. Despite its role in sensitivity to glutamate, the NR1b isoform has been shown to be necessary for normal regeneration in certain systems such as the retina (Kreutz et al., 1998). During normal granule cell development there is a progression in the expression of the NR2 subunits subtypes from NR2b towards NR2a and eventually to NR2c (Monyer et al., 1994; Watanabe et al., 1994). This change alters electrophysiological responses of cerebellar neurones (Audinat et al., 1994; Farrant et al., 1994). The NR2 subtype profile of mature cerebellar neurones is more closely mimicked by co-cultured cerebellar neurones than by purified cultures of cerebellar granule neurons. The cerebellar neurones treated with NAM showed an increase in type NR2a, which seemed to be associated with increased sensitivity to glutamate toxicity, which could be blocked by oligonucleotide knockdown of NR2a expression (Daniels and Brown, 2001); in contrast NR2c expression appeared to be protective. Therefore increased sensitivity to glutamate in the NAM treated cerebellar neurones might represent an inadequate transformation towards maturity. Oligonucleotide therapy based on these observations might provide an effective way to combat excitotoxic neurone death in a variety of diseases.

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The importance of these observations in relation to prion disease is highlighted in analyses of survival of cerebellar granule cells co-cultured with large numbers of astrocytes and treated with the PrP106-126 peptide. These cerebellar cell cultures contain a mixture of cells, including microglia, astrocytes and neurones. As illustrated in Fig. 4, specific killing of astrocytes with aminoadipate, a gliotoxin, in such cultures reduces the toxicity of PrP106-126 (Brown, 1999a). In order to test whether PrP106-126 can influence the toxicity of glutamate to neurons via its interaction with astrocytes a culture system was devised, consisting of neurones from PrP-knockout mice, which are resistant to the toxicity of PrP106-126 (Brown et al., 1994), co-cultured with wild-type astrocytes, which react to PrP106-126 (Brown et al., 1998c). Under these conditions PrP106-126 was found to be toxic to the co-cultured PrP-knockout neurones (Brown, 1999a). This toxicity could be inhibited by the NMDA antagonist MK801, suggesting that the toxicity was mediated by glutamate. As described above, co-culture with astrocytes makes neurones more sensitive to glutamate. This increased sensitivity to glutamate has no impact on neuronal survival, when the astrocytes present are able to rapidly clear glutamate and secrete neuroprotective factors. However, when astrocyte protection is inhibited by PrP106-126, then the increased neuronal sensitivity to glutamate makes the level of glutamate in the culture medium potentially more toxic. Thus, in this culture system glutamate-mediated toxicity emerges without any substantial increase in glutamate concentration in the culture

Fig. 4. Wild-type cerebellar cells were treated for 24 h with L -a-aminoadipic acid, a toxin for astrocytes. Cerebellar cells were then exposed to 80 mM PrP106-126 for five days. After this time relative survival was assessed using an MTT assay. The values were compared to cells that had been treated with 80 mM PrP106-126 for five days without prior treatment with L -a-aminoadipic acid. Increasing concentrations of L -a-aminoadipic acid reduced the toxicity of PrP106-126 significantly. Results are means ^ SEM of four experiments, with three determinations each.

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(Brown, 1999a). Preliminary data also suggest that PrP106-126 alters NMDA receptor composition in neurones, which would also increase the toxicity of glutamate to these cells (M. Daniels and D. R. Brown, unpublished data). These findings create a scenario that might explain the rapid increase in neuronal death in the latter stages of prion disease after the onset of clinical signs. At this time neuronal death rapidly follows astrocyte proliferation. Although there would be a continuing neuronal loss mediated by microglia, the addition of increased sensitivity to glutamate, caused by rapid astrocyte proliferation, would initiate cell death without a need for increased extracellular glutamate. Nevertheless, levels of glutamate might also increase due to necrotic cell death, which is known to be a principal cause of the rapid cell death observed in stroke models. Therefore treatments to inhibit glutamate toxicity might prove to be effective in blocking the rapid deterioration of patients with CJD.

6. Concluding remarks: a unified cell theory It is clear from the foregoing that microglia and astrocytes can be involved in the deleterious mechanism of action of the toxic prion protein peptide. However, some researchers have claimed that the neurotoxic effects of PrP106-126 or PrPSc do not require the indirect effects of glia. However, neurones do not exist in the brain in the absence of glia. There is solid evidence from a number of laboratories that glia either mediates or exacerbates the toxic effects, making it absurd to ignore the contribution of glia in the neurotoxic mechanism in the brain in vivo. Furthermore, the same authors who claim that the effects of PrP106-126 are exerted directly on neurons were able to inhibit the toxicity of the peptide with antioxidants (Pillot et al., 2000). Even if glia was not involved, such a result suggests an intermediate between PrP106-126 and the neurotoxic action. Therefore these claims should be considered more carefully. Intracellular accumulation of PrP106-126 has been documented (McHattie et al., 1999). Therefore, it is likely that the peptide will alter intracellular pathways simply by its hydrophobic nature. These changes would constitute direct effects. However, the necessity and importance of such effects are not clear and, indeed, toxicity might still occur in their absence. PrP106-126 has been shown to be toxic in vivo (Ettaiche et al., 2000). Therefore there is evidence that this peptide acts as a good model of PrPSc. There is scant further evidence from in vivo models as to the mechanism of action of PrPSc. Brandner et al. (1996) showed that PrPSc is not toxic to PrP-knockout neurones in vivo, supporting the previous finding in vitro (Brown et al., 1994). This lends credibility to the in vitro studies, on which most models of the toxicity at present rest. Research into this mechanism indicates that there are two types of effects involved in the toxicity of PrP106-126 (Fig. 5). The first are direct effects. These include inhibition of neuronal resistance to oxidative stress. A catalogue of such effects has been collected (Brown et al., 2002). These effects do not necessarily induce toxicity on their own, i.e., they are necessary, but not sufficient. Additionally PrP106-126 has effects on other cell types, such as activation of microglia, inhibition of astrocytic glutamate uptake, and induction of astrocyte proliferation by PrP106126-stimulated microglia. Again these effects are necessary but not sufficient for toxicity.

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Fig. 5. Summary figure showing the details of the theoretical toxic mechanism of PrP106-126 to neurones as described in the text. ROS: reactive oxygen species.

The combination of these two types of effects result in the induction of neuronal death, which progresses by caspase- or Bax-mediated effects (D. R. Brown, unpublished data). Additional factors feed into this system, principally as sources of stress. These include copper released into the cellular environment or the production of reactive oxygen or nitrogen species from other sources. To summarize, two components are needed for PrP106-126 toxicity: firstly, the production or presence of substances that cause oxidative stress and secondly, altered neuronal resistance to the toxicity of these stress agents. Hopefully, future research into this field will use these observations to produce combinatorial strategies that would enhance neuronal self-protection and neutralize stressing agents. Such a strategy might prove beneficial in alleviating neuronal death in patients with CJD.

References Anderson, C.N.G., Tolkovsky, A.M., 1999. A role for MAPK/ERK in sympathetic neuron survival: protection against p53-dependent, JNK-independent induction of apoptosis by cytosine arabinoside. J. Neurosci. 19, 664–673. Audinat, E., Lambolez, B., Rossier, J., Crepel, F., 1994. Activity dependent regulation of N-methyl-D -aspartate receptor subunits expression in cerebellar granule cells. Eur. J. Neurosci. 6, 1792–1800. Basler, K., Oesch, B., Scott, M., Westaway, D., Walchli, M., Groth, D.F., McKinley, M.P., Prusiner, S.B., Weissmann, C., 1986. Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46, 417–428.

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Schwann cells in diabetic neuropathy Andrew P. Mizisin Department of Pathology 0612, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, San Diego, CA 92093-0612, USA Correspondence address: Tel.: þ 1-858-534-5331; fax: þ 1-858-534-1886. E-mail: [email protected](A.P.M.)

Contents 1. 2. 3. 4. 5. 6.

Introduction Diabetic neuropathy Nerve fiber pathology The polyol pathway and nerve injury Neuropathic consequences of exaggerated polyol pathway flux Concluding remarks

This chapter will describe diabetic neuropathy, one of the most common and debilitating effects of diabetes, and review nerve fiber pathology in human and experimental diabetic neuropathy. It will discuss different ways that exaggerated polyol pathway flux, oxidative stress; increased advanced glycosylation end product formation; and deficient neurotrophic support compromise Schwann cells and the axons they ensheath.

1. Introduction While conventional medical treatment of diabetes mellitus markedly prolongs life, serious medical complications affect a large proportion of the more than 10 million people believed to have diabetes in the USA. Diabetic neuropathy is one of the most debilitating complications of this disease (Thomas and Tomlinson, 1993; Powell and Mizisin, 1999; Zochodne, 1999). This chapter will describe diabetic neuropathy and review nerve fiber pathology in human and experimental diabetic neuropathy. Then, after considering the polyol pathway and nerve defects related to aldose reductase activity, neuropathic consequences of exaggerated polyol pathway flux in Schwann cells of myelinated nerve fibers will be examined, revealing that exaggerated polyol pathway flux contributes to several hypothesized mechanisms of diabetic neuropathy. Advances in Molecular and Cell Biology, Vol. 31, pages 1105–1116 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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2. Diabetic neuropathy The most common neuropathy is a diffuse symmetric polyneuropathy involving sensory, motor and autonomic nerves that occurs in both type 1 and type 2 forms of diabetes mellitus as well as in animal models of the disease (see Calcutt et al., 1993 for review). Neurological complications include effects on both nerve function and structure. Nerve conduction and blood flow deficits, resistance to ischemic conduction block and altered perception to thermal, tactile and vibration stimuli are apparent in the early metabolic phase of human and experimental diabetic neuropathy. Later, structural injury affecting both unmyelinated and myelinated nerve fibers becomes evident as chronic neuropathy is established. Despite intensive effort over many years, the cause of diabetic polyneuropathy continues to be actively investigated and debated. Much debate centers on four hypotheses: exaggerated polyol pathway flux; hypoxia and oxidative stress; increased formation of advanced glycosylation end products (compounds formed in a nonenzymatical reaction between glucose and amino groups of proteins); and deficient neurotrophic support. Current thought recognizes that these mechanisms are not mutually exclusive and likely interact to produce diabetic nerve injury. 3. Nerve fiber pathology Nerve injury resulting from diabetes mellitus is characterized by marked changes in Schwann cells and the axons they ensheath. The most obvious structural manifestation of diabetic neuropathy is a loss of both large and small nerve fibers, which is a prominent feature of chronic human (Behse et al., 1977; Bischoff, 1980; Ohnishi et al., 1983; Sima et al., 1988) and long-duration experimental (Powell and Myers, 1983, 1984; Mizisin et al., 2003) diabetic neuropathy. While fiber loss is most prominent distally, it is also apparent in spinal roots, particularly in the dorsal roots. Characteristic degenerative changes of unmyelinated fibers include shrinkage of axons, accumulation of enlarged vesicular elements and deterioration of tubular and filamentous elements of the cytoskeleton (Scott et al., 1999). Edematous Schwann cell cytoplasm has also been observed as well as hyperplasia of the surrounding basal lamina (Bischoff, 1980). In myelinated fibers, axoplasmic dissolution is preceded by glycogen accumulation and dystrophic accumulation of vesicular and cytoskeletal elements (Yagihashi and Matsunaga, 1979; Bischoff, 1980). Complete axonal degeneration is characterized by myelin ovoids within the surrounding basal lamina. Residual Schwann cell basal laminae are frequently circular and may contain regenerative clusters of myelinated sprouts (Yagihashi and Matsunaga, 1979; King et al., 1989). Reports of segmental demyelination provide direct structural evidence of Schwann cell involvement in diabetic neuropathy (Thomas and Lascelles, 1965, 1966; Chopra et al., 1969; Lamontagne and Buchthal, 1970; Behse et al., 1977). Early observations of segmental demyelination without prominent axonal degeneration promoted the view that the Schwann cell was the site of the primary lesion of diabetic neuropathy, although subsequently both primary segmental demyelination and demyelination secondary to axonal degeneration were documented in the same nerve biopsy (Said et al., 1983).

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Demyelination has also been observed in long-term experimental diabetes (Powell et al., 1979; Powell and Myers, 1983, 1984; Forcier et al., 1991; Mizisin et al., 1998b, 2003). Myelin defects, such as splitting and ballooning of the sheath, that appear to precede demyelination have been documented in the dorsal and ventral roots in rodent models of experimental diabetes (Tamura and Parry, 1994; Sasaki et al., 1997; Mizisin et al., 1997a,b, 1998a, 2001) and also in nerves of cats with spontaneously occurring diabetes (Mizisin et al., 1998b, 2003). Subtle changes of the Schwann cell and axon that appear to precede the dissolution of the myelin sheath have been observed in human and experimental diabetic neuropathy (Yagihashi and Matsunaga, 1979; Bischoff, 1980; Bestetti et al., 1981; Forcier et al., 1991; Mizisin and Powell, 1993; Kalichman et al., 1998b; Mizisin et al., 1998b, 2003). Nonspecific, reactive changes in Schwann cell cytoplasm include: accumulation of lipid droplets, paracrystalline inclusions (Pi granules of Reich) and glycogen granules; increased numbers of plasmalemmal vesicles; and cytoplasmic expansion and capping. Enlarged mitochondria with effaced cristae and disintegration of abaxonal and adaxonal cytoplasm and organelles have been described as degenerative Schwann cell changes. Thickening and reduplication of the Schwann cell basal lamina of myelinated fibers have also been illustrated (Bischoff, 1980). Axonal atrophy or the diminution of axonal caliber without myelin or axonal degeneration was suggested by an early report of biopsied teased fibers with long internodes and inappropriately small diameters (Thomas and Lascelles, 1966) and has been extensively documented in experimental diabetes (Jakobsen, 1976; Yagihashi et al., 1990b; Mizisin et al., 1997a,b, 1998a, 2001). However, axonal atrophy in human diabetic neuropathy is controversial, because, despite qualitative descriptions (Yagihashi and Matsunaga, 1979) and multiple-parameter quantitative evidence for its existence (Sima et al., 1988, 1993; Britland et al., 1990), it has not been observed by others (Sugimura and Dyck, 1981; Llewelyn et al., 1991; Engelstad et al., 1997). Paranodal abnormalities described in human and experimental diabetic neuropathy include demyelination, paranodal swelling and axo-glial dysjunction. Demyelination restricted to the paranode is purportedly resolved with selective remyelination by surviving Schwann cells or with the formation of an intercalated internode as noted in teased fibers (Thomas and Lascelles, 1966; Behse et al., 1977; Sima et al., 1988, 1993). Paranodal swelling is thought to precede paranodal demyelination and to be associated with axo-glial dysjunction, the loss of the junctional contacts between paranodal Schwann cell loops and the axolemma on either side of the node of Ranvier. However, the existence of paranodal swelling and axo-glial dysjunction is contentious because, although repeatedly documented by some in experimental and human diabetic neuropathy (Sima et al., 1986, 1988, 1990, 1993; Kamijo et al., 1994), others (Thomas et al., 1996) have not detected these abnormalities.

4. The polyol pathway and nerve injury Since the detection of elevated polyol levels in the lenses of galactose-fed rabbits (Van Heyningen, 1959), the polyol pathway has been implicated in the pathogenesis of peripheral neuropathy and the other complications of diabetes mellitus. In the polyol

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pathway, glucose is converted to fructose in two steps: (1) aldose reductase utilizes NADPH to reduce glucose to its corresponding sugar alcohol, sorbitol; and (2) sorbitol dehydrogenase utilizes NADþ to oxidize sorbitol to fructose. In tissues subject to diabetic complications, flux through the polyol pathway during normoglycemia is limited by low glucose concentrations and by the low affinity of aldose reductase for glucose so that this sugar is metabolized by hexokinase and enters glycolytic pathways. During hyperglycemia, elevated glucose levels saturate hexokinase, which increases the amount metabolized by the polyol pathway and results in accumulation of sorbitol and fructose, and generation of NADPþ and NADH. Aldose reductase will also reduce other hexose sugars and exhibits a lower Km for galactose as a substrate than glucose (Hayman and Kinoshita, 1965; Stewart et al., 1967). The affinity of aldose reductase for galactose coupled with the inability of sorbitol dehydrogenase to oxidize dulcitol results in the rapid accumulation of this polyol that was noted by Van Heyningen (1959) and prompted experimental work leading to the osmotic theory of cataract formation in diabetes (Kinoshita, 1965). After recognition of the polyol-forming capacity of nerve, the osmotic theory of cataractogenesis was extended to this tissue in an attempt to understand the pathogenesis of diabetic neuropathy in terms of exaggerated polyol pathway activity (Gabbay et al., 1966; Gabbay and O’Sullivan, 1968). The polyol-forming enzyme, aldose reductase, was subsequently localized to the Schwann cells of myelinated fibers (Ludvigson and Sorenson, 1980; Kern and Engerman, 1982; Chakrabarti et al., 1987), with little or no aldose reductase detected in Schwann cells of unmyelinated fibers (Powell et al., 1991). In Schwann cells of myelinated fibers, immunostaining for aldose reductase is present in perinuclear, internodal, and paranodal cytoplasm (Powell et al., 1991). Aldose reductase immunostaining has also been detected in endothelial cells and pericytes of endoneurial blood vessels (Chakrabarti et al., 1987), and polyol-forming capability has been observed in skeletal muscle (Cameron et al., 1992; Mizisin et al., 1997a,b). Localization of aldose reductase to key cellular constituents of peripheral nerve and to target tissues highlights the potential for hyperglycemia-induced polyol accumulation and/or flux through the pathway to compromise nerve function. Development of agents that inhibit aldose reductase has been instrumental in investigating the nature and extent of the role of the polyol pathway in the pathogenesis of diabetic neuropathy. Over the last three decades, more than 20 different aldose reductase inhibitors have been used in experimental and clinical studies of diabetic neuropathy. Aldose reductase inhibition improves a wide range of defects associated with diabetes-induced nerve injury (reviewed in Tomlinson et al., 1992; Cameron and Cotter, 1997; Zochodne, 1999). Biochemical parameters responsive to aldose reductase inhibition and therefore related to exaggerated polyol pathway activity include sorbitol, fructose, lactate and malondialdehyde levels, Naþ/Kþ-ATPase activity, and substance P, CGRP, NGF and CNTF content. Physiological parameters linked to the polyol pathway include nerve conduction velocity, resistance to ischemic conduction block, and nerve blood flow and oxygen tension. Inhibiting exaggerated polyol pathway activity protects against myelinated fiber injury by preventing myelin defects, paranodal changes, sodium channel displacement, axonal dwindling and neuroaxonal dystrophy. The wide range of early and late nerve defects improved by aldose reductase inhibitors suggests both direct and indirect actions of these agents. Assuming specificity of action, the efficacy of aldose

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reductase inhibitors has established the polyol pathway as an important pathogenic mechanism of diabetic nerve injury.

5. Neuropathic consequences of exaggerated polyol pathway flux Attempts to understand the pathogenesis of diabetic neuropathy have linked hyperglycemia-induced biochemical aberrations to early functional impairments and to the development of structural changes characteristic of chronic neuropathy (reviewed in Tomlinson, 1992; Stevens, 1995; Tomlinson et al., 1997; Zochodne, 1999; Brownlee, 1992, 2001; Cameron et al., 2001). The biochemical alterations include increased polyol pathway flux, formation of reactive oxygen species, generation of advanced glycosylation end products, and deficient neurotrophic support. The sensitivity of some of the endpoints of these biochemical alterations to aldose reductase inhibition highlights the contribution of polyol-pathway-containing cells and tissues to diabetic nerve injury as well as the complex and interrelated etiology of this neuropathy. Here some functional and structural deficits of diabetic neuropathy will be considered in the context of exaggerated polyol pathway flux in Schwann cells of myelinated fibers, vessels and innervated target tissues. Localization of aldose reductase to peripheral nerve prompted efforts to view nerve conduction velocity deficits in experimental models of diabetes as a consequence of polyol-induced osmotic stress of Schwann cells (Gabbay et al., 1966; Gabbay and O’Sullivan, 1968). However, early studies in galactose-fed (Gabbay and Snider, 1972; Myers et al., 1979) and streptozotocin-diabetic rats (Jakobsen, 1978) noted increased hydration of the endoneurial interstitium instead of the Schwann cell. Subsequently, the sensitivity of hyperglycemia-induced nerve myo-inositol depletion to aldose reductase inhibition and correction of conduction velocity deficits by oral administration of myo-inositol led to the notion linking myo-inositol depletion and phosphoinositide dysmetabolism to decreased Naþ/Kþ-ATPase activity. Conduction velocity deficits were suggested to result from accumulation of intra-axonal Naþ because of deficient Naþ/Kþ-ATPase activity (Greene et al., 1988). Several observations are inconsistent with a link between polyol pathway flux and myo-inositol depletion in Schwann cells, and decreased axonal Naþ/Kþ-ATPase activity. Decreased Naþ/Kþ-ATPase activity is only observed, when diabetic rats are fed a high-sucrose diet (Sredy et al., 1991). Decreased nerve conduction in diabetic rats can be corrected by vasodilators that do not alter nerve myo-inositol or Naþ/Kþ-ATPase activity (Cameron et al., 1991). Further, conduction deficits coexist with increased Naþ/Kþ-ATPase activity in galactose-fed animals (Llewelyn et al., 1987; Lambourne et al., 1987; Mizisin and Calcutt, 1991), develop independently of changes in Naþ/Kþ-ATPase activity in db/db mice (Bianchi et al., 1990), and occur without myo-inositol depletion in human diabetic neuropathy (Dyck et al., 1980). Aldose-reductase-inhibitor-sensitive paranodal swelling, axo-glial dysjunction and nodal Naþ channel displacement have been described in human and experimental diabetic neuropathy (Sima et al., 1986, 1988, 1990, 1993; Kamijo et al., 1994; Cherian et al., 1996). They could conceivably be linked to Naþ/Kþ-ATPase in paranodal Schwann cell loops (Powell et al., 1991), if decreased Naþ/Kþ-ATPase activity promoted paranodal swelling, axo-glial dysjunction and subsequent displacement of Naþ channels. Unfortunately,

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the occurrence of these ultrastructural changes in galactose-fed rats, where Naþ/KþATPase activity is increased, and a failure of others to replicate axo-glial dysjunction (Thomas et al., 1996) do not lend strong support to this possibility. As noted above, demyelination is a feature of human diabetic neuropathy and has been documented in experimental models of long-duration diabetes (Powell et al., 1979; Powell and Myers, 1983, 1984; Forcier et al., 1991; Mizisin and Powell, 1993) as well as in spontaneously occurring feline diabetic neuropathy (Mizisin et al., 1998b, 2003). Myelin defects, such as splitting and ballooning of myelin lamellae, and other reactive and degenerative Schwann cell changes that appear to precede demyelination, have also been observed in rodent and feline diabetic neuropathy (Bestetti et al., 1981; Forcier et al., 1991; Mizisin and Powell, 1993; Tamura and Parry, 1994; Mizisin et al., 1997a, 1998b, 2003) and, with respect to Schwann cell changes, in human diabetic neuropathy (Kalichman et al., 1998b). Several experimental studies have quantitatively demonstrated that Schwann cell injury is sensitive to aldose reductase inhibition, establishing a link to exaggerated polyol pathway flux (Mizisin and Powell, 1993; Mizisin et al., 1997a; Yagihashi et al., 1990a). These observations highlight the significance of polyol-pathwayrelated structural injury to Schwann cells in the pathogenesis of diabetic neuropathy, supporting earlier suggestions by Gabbay and coworkers (Gabbay et al., 1966; Gabbay and O’Sullivan, 1968). Axonal atrophy is present in rodent models of experimental diabetes (Calcutt et al., 1993 and references therein) and has been documented by some in human diabetic neuropathy (Yagihashi and Matsunaga, 1979; Sima et al., 1988, 1993; Britland et al., 1990). Axonal dwindling has been suggested to account for conduction velocity deficits in experimental diabetes (Jakobsen, 1976). Although decreased axonal caliber may exacerbate nerve conduction velocity deficits, it not likely to instigate conduction defects that in some experimental models are established before decreases in axonal diameter are evident (Kalichman et al., 1998a). Inhibiting the polyol pathway prevents axonal atrophy, suggesting the involvement of neurotrophic support deriving from aldose-reductasecontaining Schwann cells and target tissues. Diabetes does alter expression of a variety of axonally transported neurotrophic factors derived from Schwann cells, skin and muscle including CNTF, NGF, BNDF, NT-3, and prosaposin (Hellweg and Hartung, 1990; Calcutt et al., 1992, 1999; Curtis et al., 1993; Rodriguez-Pena et al., 1995; Fernyhough et al., 1995, 1998; Ihara et al., 1996; Mizisin et al., 1999a,b). However, an improved expression after inhibition of aldose reductase has yet only been demonstrated for CNTF and NGF (Mizisin et al., 1997b; Ohi et al., 1998). Both CNTF and NGF have been shown to enhance the synthesis of neurofilaments, important determinants of axonal caliber, in responsive neurons (Wong et al., 1990; Gold et al., 1991). However, while exogenous administration of CNTF, NGF, BDNF, NT-3 and a prosaposin-derived peptide restores motor and/or sensory nerve conduction in experimental diabetes, robust effects on axonal caliber are reported only for NT-3 (Mizisin et al., 1998a, 1999a,b) and prosaptide (Calcutt et al., 1999; Mizisin et al., 2001), suggesting that neurotrophic support influences nerve conduction in other ways. Interestingly, exogenous BDNF administration prevents motor nerve conduction velocity deficits and myelin splitting and ballooning in the ventral roots of galactose-fed rats (Mizisin et al., 1997a), while a prosaposin-derived peptide

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ameliorates motor and sensory conduction deficits, restores axonal caliber and Naþ/KþATPase activity in streptozotocin-diabetic rats (Calcutt et al., 1999). Deficits in nerve blood flow and resultant endoneurial hypoxia have been demonstrated in diabetic animals (see Cameron et al., 2001 for review) and patients (Tesfaye et al., 1993), and linked to nerve conduction deficits. Subsequent studies using a variety of vasodilators and antioxidants have implicated the formation of reactive oxygen species in diabetic neurovascular dysfunction via impaired nitric-oxide- or prostanoid-mediated vasodilation and compromised antioxidant protection mechanisms (reviewed in Cameron and Cotter, 1999). The polyol pathway was linked to these vascular defects by the demonstrations that blocking flux in this pathway prevented nerve blood flow deficits, and that nitric oxide synthase inhibitors completely abolished the beneficial actions of aldose reductase inhibition on nerve conduction velocity and blood flow (Yasuda et al., 1989; Stevens et al., 1994; Cameron et al., 1996). In peripheral nerve, polyol pathway flux and consumption of NADPH in endothelial cells has been suggested to reduce the effectiveness of the glutathione redox cycle by decreasing the formation of the reduced form of glutathione, used to scavenge oxygen free radicals and leading to endothelial damage and consequent loss of nitric-oxide-mediated vasodilation (Low and Nickander, 1991; Stevens, 1995; Cameron and Cotter, 1997). In support of this notion, aldose reductase inhibition has been shown to improve decreased nerve reduced glutathione levels in experimental diabetes (Hohman et al., 1997). Formation of advanced glycosylation end products by auto-oxidative glycosylation of glucose or modification of fructose produced by polyol pathway flux is also a potential source of reactive oxygen species in diabetes and can quench nitric oxide independent of actions as a source of free radicals (Brownlee, 1992, 2001). The link between polyol pathway flux, formation of reactive oxygen species and advanced glycosylation end products, and damage to endothelial cells suggests that other cells containing aldose reductase, including Schwann cells of myelinated fibers, face similar risks in diabetes. Indeed, elevated glucose elicits oxidative stress and weakens antioxidant protection mechanisms in cultured Schwann cells (Miinea et al., 2002), while treatment of diabetic rats with agents known to promote vasodilation and prevent formation of advanced glycosylation end products corrects nerve conduction deficits and ameliorates decreases in myelinated fiber size (Yasuda et al., 1989; Yagihashi et al., 1992). Oxidative stress has been suggested as the cause of vacuolation in dorsal root ganglion neurons and myelin splitting and ballooning in spinal roots reported in long-term diabetic rats (Sasaki et al., 1997). Prevention of myelin splitting and ballooning by aldose reductase inhibition and exogenous BDNF administration (Mizisin et al., 1997a,b) is consistent with this notion, as both treatments increase reduced glutathione levels in vivo (Lou et al., 1988; Hohman et al., 1997) and in vitro (Spina et al., 1992; Gabaizadeh et al., 1997).

6. Concluding remarks Reported nerve fiber pathology resulting from diabetes mellitus ranges from subtle Schwann cell changes and axonal atrophy to complete fiber degeneration. Sensitivity of myelinated fiber injury to aldose reductase inhibition suggests a link to exaggerated polyol

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pathway flux in Schwann cells, vessels and target tissue. Current thought emphasizes the role of oxidative stress, enhanced by polyol pathway activity and advanced glycation end product formation in the vascular dysfunction of the early metabolic phase of human and experimental diabetic neuropathy. The impact of oxidative stress in Schwann cells and target tissues on the establishment of chronic neuropathy has been less extensively studied. However, the sensitivity of markers of oxidative stress and antioxidant defense mechanisms to aldose reductase inhibition highlights the potential contribution of oxidative stress to the Schwann cell injury characteristic of human and experimental diabetic neuropathy. A continuing challenge will be to understand the different ways that exaggerated polyol pathway flux, oxidative stress; increased advanced glycosylation end product formation; and deficient neurotrophic support interact in diabetes mellitus to compromise Schwann cells and the axons they ensheath.

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Powell, H.C., Garrett, R.S., Kador, P.F., Mizisin, A.P., 1991. Fine-structural localization of aldose reductase and ouabain-sensitive, Kþ-dependent p-nitro-phenylphosphatase in rat peripheral nerve. Acta Neuropathol. (Berl.) 81, 529– 539. Powell, H.C., Mizisin, A.P., 1999. Diabetes: effects on the nervous system. In: Adelman, G., Smith, B.H. (Eds.), Elsevier’s Encyclopedia of Neuroscience. Elsevier Science, Amsterdam, pp. 550 –555. Powell, H.C., Myers, R.R., 1983. Schwann cell changes and demyelination in chronic galactose neuropathy. Muscle Nerve 6, 218–227. Powell, H.C., Myers, R.R., 1984. Axonopathy and microangiopathy in chronic alloxan diabetes. Acta Neuropathol. (Berl.) 65, 128–137. Powell, H.C., Ward, H.W., Garrett, R.S., Orloff, M.J., Lampert, P.W., 1979. Glycogen accumulation in the nerves and kidney of chronically diabetic rats. A quantitative electron microscopic study. J. Neuropathol. Exp. Neurol. 38, 114 –127. Rodriguez-Pena, A., Botana, M., Gonzalez, M., Requejo, F., 1995. Expression of neurotrophins and their receptors in sciatic nerve of experimentally diabetic rats. Neurosci. Lett. 200, 37–40. Said, G., Slama, G., Selva, J., 1983. Progressive centripedal degeneration of axons in small fiber type diabetic polyneuropathy. A clinical and pathological study. Brain 106, 791– 807. Sasaki, H., Schmelzer, J.D., Zollman, P.J., Low, P.A., 1997. Neuropathology and blood flow of nerve, spinal roots and dorsal root ganglia in longstanding diabetic rats. Acta Neuropathol. (Berl.) 93, 118 –128. Scott, J.N., Clark, A.W., Zochodne, D.W., 1999. Neurofilament and tubulin gene expression in progressive experimental diabetes. Brain 122, 2109–2117. Sima, A.A.F., Lattimer, S.A., Yagihashi, S., Greene, D.A., 1986. Axo-glial dysjunction: a novel structural lesion that accounts for poorly reversible slowing of nerve conduction in the spontaneously diabetic bio-breeding rat. J. Clin. Invest. 77, 474 –484. Sima, A.A.F., Nathaniel, V., Bril, V., McEwen, T.A., Greene, D.A., 1988. Histopathological heterogeneity of neuropathy in insulin-dependent and non-insulin-dependent diabetes, and demonstration of axo-glial dysjunction in human diabetic neuropathy. J. Clin. Invest. 81, 349– 364. Sima, A.A.F., Prashar, A., Nathaniel, V., Bril, V., Werb, M.R., Greene, D.A., 1990. Overt diabetic neuropathy: repair of axo-glial dysjunction and axonal atrophy by aldose reductase inhibition and its correlation to improvement in nerve conduction velocity. Diabet. Med. 10, 115 –121. Sima, A.A.F., Prashar, A., Nathaniel, V., Bril, V., Werb, M.R., Greene, D.A., 1993. Overt diabetic neuropathy: repair of axo-glial dysjunction and axonal atrophy by aldose reductase inhibition and its correlation to improvement in nerve conduction velocity. Diabet. Med. 10, 115 –121. Spina, M.B., Squinto, S.P., Miller, J., Lindsay, R.M., Hyman, C., 1992. Brain-derived neurotrophic factor protects dopamine neurons against 6-hydroxdopamine and N-methyl-4-phenylpyridium ion toxicity: involvement of the glutathione system. J. Neurochem. 59, 99–106. Sredy, J., Flam, B.R., Sawicki, D.R., 1991. Adenosine triphosphatase activity in sciatic nerve tissue of streptozotocin-induced diabetic rats with and without high dietary sucrose: effects of aldose reductase inhibition. Proc. Soc. Exp. Med. 197, 135 –142. Stevens, M.J., 1995. Nitric oxide as a potential bridge between the metabolic and vascular hypotheses of diabetic neuropathy. Diab. Med. 12, 292– 295. Stevens, M.J., Dananberg, J., Feldman, E.L., Lattimer, S.A., Kamijo, M., Thomas, T.P., Shindo, H., Sima, A.A.F., Greene, D.A., 1994. The linked roles of nitric oxide, aldose reductase and (Naþ,Kþ)ATPase in the slowing of nerve conduction in the streptozotocin-diabetic rat. J. Clin. Invest. 94, 853–859. Stewart, M.A., Sherman, W.R., Kurien, M.M., Moonsammy, G.I., Wisgerhof, M., 1967. Polyol accumulations in nervous tissue of rats with experimental diabetes and galactosemia. J. Neurochem. 14, 1057–1066. Sugimura, K., Dyck, P.J., 1981. Sural nerve myelin thickness and axis cylinder caliber in human diabetes. Neurology 31, 1087–1091. Tamura, E., Parry, G.J., 1994. Severe radicular pathology in rats with longstanding diabetes. J. Neurol. Sci. 127, 29–35. Tesfaye, S., Harris, N., Jakubowski, J.J., Mody, C., Wilson, R.M., Rennie, I.G., Ward, J.D., 1993. Impaired blood flow and arterio-venous shunting in human diabetic neuropathy: a novel technique of nerve photography and fluorescein angiography. Diabetologia 36, 1266–1274.

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Thomas, P.K., Beamish, N.G., Small, J.R., King, R.H., Tesfaye, S., Ward, J.D., Tsigos, C., Young, R.J., Boulton, A.J., 1996. Paranodal structure in diabetic sensory polyneuropathy. Acta Neuropathol. (Berl.) 92, 614– 620. Thomas, P.K., Lascelles, R.G., 1965. Schwann-cell abnormalities in diabetic neuropathy. Lancet 62, 1355–1357. Thomas, P.K., Lascelles, R.G., 1966. The pathology of diabetic neuropathy. Q. J. Med. 35, 489 –509. Thomas, P.K., Tomlinson, D.R., 1993. Diabetic and hypoglycemic neuropathy. In: Dyck, P.J., Thomas, P.K., Griffen, J.W., Low, P.A., Podulso, J.F., (Eds.), Peripheral Neuropathy. W.B. Saunders, Philadelphia, pp. 1219–1250. Tomlinson, D.R., 1992. The pharmacology of diabetic neuropathy. Diabetes Metab. Rev. 8, 67–84. Tomlinson, D.R., Fernyhough, P., Diemel, L.T., 1997. Role of neurotrophins in diabetic neuropathy and treatment with nerve growth factors. Diabetes 46(Suppl. 2), S43– S49. Tomlinson, D.R., Willars, G.B., Carrington, A.L., 1992. Aldose reductase inhibitors and diabetic complications. Pharmacol. Ther. 54, 151–194. Van Heyningen, R., 1959. Formation of polyols by the lens of the rat with ‘sugar’ cataract. Nature 184, 194– 195. Wong, V., Arriaga, R., Lindsay, R.M., 1990. Effects of ciliary neuronotrophic factor (CNTF) on ventral spinal cord neurons in culture. Soc. Neurosci. Abstr. 16, 384. Yagihashi, S., Kamijo, M., Baba, M., Yagihashi, N., Nagai, K., 1992. Effect of aminoguanidine on functional and structural abnormalities in peripheral nerve of STZ-induced diabetic rats. Diabetes 41, 47– 52. Yagihashi, S., Kamijo, M., Ido, Y., Mirrlees, D.J., 1990. Effects of long term aldose reductase inhibition on experimental diabetic neuropathy. Ultrastructural and morphometric studies of sural nerve in streptozotocininduced diabetic rats. Diabetes 39, 690–696. Yagihashi, S., Kamijo, M., Watanabe, K., 1990. Reduced myelinated fiber size correlates with loss of axonal neurofilaments in peripheral nerve of chronically streptozotocin-diabetic rats. Am. J. Pathol. 136, 1365–1374. Yagihashi, S., Matsunaga, M., 1979. Ultrastructural pathology of peripheral nerves in patients with diabetic neuropathy. Tohoku J. Exp. Med. 129, 357 –366. Yasuda, H., Sonobe, M., Yamashita, M., Terada, M., Hatanaka, I., Huitan, Z., Shigeta, Y., 1989. Effect of prostaglandin E1 analogue TFC 612 on diabetic neuropathy in streptozotocin-induced diabetic rats: comparison with aldose reductase inhibitor ONO (2235). Diabetes 38, 832 –838. Zochodne, D.W., 1999. Diabetic neuropathies: features and mechanisms. Brain Pathol. 9, 369–391.

Mu¨ller cells in retinopathies A. Bringmann, M. Francke and A. Reichenbachp Department of Neurophysiology, Paul Flechsig Institute for Brain Research, and Department of Ophthalmology, Eye Clinic, University of Leipzig Medical Faculty, Leipzig, Germany p Correspondence address: Paul Flechsig Institute for Brain Research, Department of Neurophysiology, Leipzig University, Jahnallee 59, D-04109 Leipzig, Germany Tel.: þ49-341-9725731; fax: þ49-341-9725739 E-mail: [email protected]

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction: Mu¨ller cell functions Reactive Mu¨ller cells: definition and roles Mu¨ller cell-derived cytokines: support of neuronal survival The glutamate – glutamine cycle of Mu¨ller cells: challenged in retinal diseases Energy metabolism: Mu¨ller cell– neuron symbiosis, hypoxia and neovascularization Mu¨ller cells and the blood – retina barrier: a vicious circle? Retinal detachment and proliferative retinopathies: special cases of the Janus-faced Mu¨ller cell gliosis An outlook: turning the Janus face towards its good side Concluding remarks

Mu¨ller (radial glial) cells are the principal macroglia of the retina in all vertebrates. They are performing a wealth of glia –neuron interactions, crucial for visual signal processing and even for survival of retinal neurons. Many of the functions of Mu¨ller cells directly or indirectly depend on the abundant expression of inwardly rectifying Kþ (Kir) channels in their membranes. In various cases of retinal injury or disease, the expression of functional Kir channels by Mu¨ller cells is down-regulated. This seems to constitute a key event, resulting in functional deficiency of retinal glia, and aggravating the pathomechanisms of retinal degeneration.

1. Introduction: Mu¨ller cell functions The mammalian sensory retina usually contains three distinct types of glial cells, microglial cells (the blood-borne resident macrophages of the CNS), and two types of Advances in Molecular and Cell Biology, Vol. 31, pages 1117–1132 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1

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Fig. 1. Mu¨ller cells play a wealth of functional roles in supporting neuronal survival and information processing in the retina. (A) Three types of glial cells are present in the sensory retina. Astrocytes occur in the nerve fiber and ganglion cell layers, where they ensheath the blood vessels, whereas microglial cells are present in the nerve fiber/ganglion cell and inner plexiform layers in the healthy retina. Neuronal populations, rods and cones are indicated in the right part of the figure. (B) Mu¨ller cells in a slice of the guinea-pig retina, selectively stained by Mitotracker Orangew. The somata are located in the inner nuclear layer (INL), and two main trunks extend from each soma to the two retinal surfaces. The ends of the vitread main trunks (top) are thickened to form so-called endfeet. The sclerad trunks (bottom) build honeycomb-like leaflets in the outer nuclear layer (ONL), ensheathing the somata of photoreceptor cells. In both synaptic layers of the retina (inner: IPL, and outer plexiform layer: OPL), side branches of the Mu¨ller cell processes contact and ensheath synaptic structures. GCL, ganglion cell layer. Scale bar, 20 mm. (C) Immunocytochemical localization of the Kir4.1 channels mediating Kþ ion homeostasis (confer D, right side). In Mu¨ller cells of the mouse, the Kir4.1 channel protein is predominantly located at sites, where Kþ ions flow out of the cells into sinks (i.e., at the inner limiting membrane adjacent to the vitreous body [arrowheads ] and around blood vessels [arrow ]). Scale bar, 20 mm. (D) Schematic drawing of three different Mu¨ller cell functions. Energy metabolism: Enhanced neuronal activity or pathological anaerobic conditions lead to activation of the glycogen phosphorylase (GP) of Mu¨ller cells, which results in glycogenolysis and, subsequently, glycolysis. Via activation of pyruvate kinase (PK) and lactate dehydrogenase (LDH), Mu¨ller

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macroglial cells (Fig. 1A). Astrocytes are found in the innermost retinal layers (nerve fiber/ganglion cell layers) of most mammals including man. The Mu¨ller (radial glial) cells are the principal glia of all vertebrate retinae. In retinae devoid of blood vessels (most non-mammalian vertebrates, rabbits, guinea pigs, and the fovea centralis of primates) they are the only macroglial cells. Mu¨ller cell processes span all retinal layers (Fig. 1B) and contact virtually all retinal neurons. Mu¨ller cells perform a wealth of functions that are carried out collectively by astrocytes, oligodendrocytes, and/or ependymal cells in other regions of the CNS (Newman and Reichenbach, 1996). They stabilize the retinal architecture, provide an orientation scaffold for neuronal cells, and support neuronal survival and information processing. The importance of Mu¨ller cells is illustrated by the observation that selective Mu¨ller cell destruction causes retinal dysplasia, photoreceptor apoptosis and, finally, retinal degeneration (Dubois-Dauphin et al., 2000). Figure 1D summarizes some of the mechanisms by which Mu¨ller cells contribute to the maintenance and functioning of neurons in the healthy, mature retina (see Newman and Reichenbach, 1996). Under pathological conditions, an overload and/or a breakdown of these mechanisms may occur, and contribute to retinal degeneration. Many of these mechanisms rely upon a high Kþ ion conductance, which indeed is a key feature of normal mature (but not of immature and reactive) Mu¨ller cells (Fig. 2A – C). Specifically, the socalled inwardly rectifying Kþ (Kir) channels mediate the Kþ siphoning currents important for retinal Kþ ion homeostasis (Fig. 1D, right part). For this purpose, Kir channels are expressed at high density in membrane compartments that face ‘extracellular Kþ sinks’ such as the vitreous body, the retinal blood vessels, and the subretinal space (Fig. 1C). The Kir channels are also responsible for the very negative resting membrane potential of normal mature Mu¨ller cells (Fig. 2C), which in turn provides the driving force for the uptake carriers (Fig. 2D) involved in neurotransmitter recycling (Fig. 1D, middle part) and in the synthesis of the free radical scavenger, glutathione. The Kþ channels of Mu¨ller cells seem also to play a role in the triggering and/or maintenance of Mu¨ller cell proliferation (Bringmann et al., 2000), and in the regulation of the glio-neuronal ‘symbiosis’ in energy metabolism (Fig. 1D, left part), where glycogenolysis is stimulated by elevated extracellular Kþ concentrations (Reichenbach et al., 1993); we will show in the following sections that in many retinal diseases, changes in the expression and/or opening probability of Mu¨ller cell Kir channels occur, and crucially contribute to the pathophysiological mechanisms of these diseases.

cells release pyruvate and lactate, which may serve to fuel the Krebs cycle (tricarboxylic acid [TCA] cycle) of the neurons. Neurotransmitter recycling: Mu¨ller cells (in addition to retinal neurons) take up neuronally released glutamate (glu) and GABA and convert these transmitters, under consumption of ATP and ammonia, into glutamine by the glutamine synthetase (GS) reaction. GABA is converted into glutamate via the GABA transaminase (GT) reaction (see chapter by Schousboe and Waagepetersen). Glutamine (gln) is released into the extracellular space, and is used by neurons for the resynthesis of transmitter molecules. Kþ ion homeostasis: Active neurons release Kþ ions into the extracellular space, which are redistributed by Mu¨ller cells via different mechanisms, especially by ‘spatial buffering currents’ through the Mu¨ller cell bodies into the vitreous body, into the blood vessels, and into the subretinal space.

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Fig. 2. Ion channels, uptake carriers, and ligand receptors are important tools of Mu¨ller cells in their interactions with retinal neurons. (A) Mu¨ller cells express different types of Kþ channels in their plasma membranes. The middle trace displays whole-cell Kþ currents of a human Mu¨ller cell, which were evoked by stepping the membrane potential to depolarizing (upward current deflections) and to hyperpolarizing potentials (downward current deflections) from a holding potential of 280 mV. Depolarizing voltage steps evoke—in addition to other Kþ current types—currents, which are mediated by BK channels (calcium-activated Kþ channels of big conductance). Hyperpolarizing voltage steps evoke currents, which are mediated by Kir channels. (B) The expression level of Kir currents is dependent on the differentiation state of rabbit Mu¨ller cells. The high Kþ conductance of Mu¨ller cell membranes in healthy adult retinae is provided by Kir channel-mediated currents, which are increasingly expressed during postnatal development. After 48 h experimental retinal detachment or during proliferative vitreoretinopathy (PVR), the expression of functional Kir channels is significantly reduced (modified after Bringmann et al., 1999 and Francke et al., 2001, 2002). (C) Histogram of Kir channel-mediated current densities vs. resting membrane potentials of human Mu¨ller cells isolated from the retinae of healthy donors (top) and of patients suffering from PVR (bottom). Every point represents an individual cell. The control cells display high current densities (.2 pA/pF) and negative resting membrane potentials (mean, 280 mV: vertical line), whereas the cells from PVR retinae have low current densities (,2 pA/pF) and a wide range of depolarized membrane potentials (mean, 250 mV). (D) The uptake of GABA and glutamate by Mu¨ller cells is voltage-dependent. Current–voltage relationships of GABA (100 mM)- and L -glutamate (1 mM)-evoked currents in Mu¨ller cells from the guinea pig and from the toad, respectively. The more hyperpolarized the Mu¨ller cell membranes, the larger are the uptake currents. Inset: Extracellular L -glutamate (1 mM) evokes an inwardly directed current at a membrane potential of 280 mV. (E) Extracellular application of ATP specifically increases the outward Kþ currents in a rabbit Mu¨ller cell, which are mediated by BK channels, via a P2 receptor-mediated increase of the intracellular free calcium concentration (Francke et al., 2002). (F) The ATP responsiveness of

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2. Reactive Mu¨ller cells: definition and roles Normal Mu¨ller cells are a hallmark of the healthy retina (Fig. 1B); and conversely, reactive Mu¨ller cells (Fig. 3) can be used as indicators of an injured retina. Reactive Mu¨ller cells are characterized by hypertrophy, proliferation, and up-regulation of glial fibrillary acidic protein (GFAP) (Fig. 3B and C); a down-regulation of Kir channels (Fig. 2B and C) and an up-regulation of functional ATP receptors (Fig. 2E and F) seem also to constitute ‘unspecific’ responses of Mu¨ller cells to a variety of different pathogenic stimuli. Mu¨ller cell reactivity (also called ‘gliosis’) may additionally involve specific responses to specific pathogenic agents. As an example, the expression of glutamine synthetase, the key enzyme in neurotransmitter recycling (Fig. 1D, middle part) and ammonia detoxification, may be either down-regulated (after a loss of the majority of glutamate-releasing neurons, such as in cases of photoreceptor degeneration due to light damage or retinal detachment), or upregulated (when the enzyme activity is challenged by elevated levels of ammonia and/or glutamate). When considering the beneficial vs. detrimental effects of Mu¨ller cell gliosis, it should be kept in mind that this is only one component of a complex retinal response to pathogenic stimuli, which may include microglial activation, breakdown of the blood – retina barrier, and immigration of macrophages and lymphocytes into the retina. Generally, Mu¨ller cell activation is thought to represent a cellular attempt to limit the extent of retinal damage, and to promote tissue repair processes. However, reactive Mu¨ller cells may also promote neuronal cell death. Mu¨ller cell gliosis must thus be considered as Janus-faced, depending on the delicate balance between the type, degree, and duration of the pathogenic events on the one hand, and on the effects and side-effects of the Mu¨ller cell responses on the other hand. A main cause of retinal injuries is a deficiency of oxygen and/or glucose supply, due to an impairment of the intraretinal blood circulation (e.g., in diabetes mellitus, hypertension, or glaucoma) or to a detachment of the neural retina from the underlying retinal pigment epithelium-choroid, which normally supplies nutrition to the photoreceptor cells (Stone et al., 1999). However, a variety of different pathogenic mechanisms may cause neuronal cell death in retinopathies. To a varying degree, these events evoke the intraretinal release of proinflammatory and cytotoxic cytokines (e.g., interleukin-1, and tumor necrosis factor, TNF), and an increase of the extracellular Kþ concentration, which causes over-excitation of neurons. Additionally, there are two main mediators of neuronal cytotoxicity within the retinal tissue, (i) glutamate excitotoxicity, underlying death of the neurons within the inner nuclear and ganglion cell layers, which express ionotropic glutamate (AMPA/kainate and NMDA) receptors, mediating excessive cytotoxic calcium influxes during prolonged activation, and (ii) formation of free radicals. Free oxygen radicals cause neuronal necrosis or apoptosis, via massive influx of calcium ions (e.g., through ionotropic glutamate

rabbit Mu¨ller cells depends on their developmental and functional state. The percentage of radial glial (immature)/Mu¨ller cells (mature) responding to extracellular ATP with an increase of the intracellular free Ca2þ concentration, is high in the neonatal retina as well as after experimental induction of PVR in the adult retina, but low in the healthy mature retina.

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Fig. 3. Mu¨ller cells hypertrophy and migrate in rabbit models of experimental retinal detachment, dispaseinduced retinopathy, and proliferative vitreoretinopathy (PVR). (A–D) (Immuno-) Histochemistry of the rabbit retina before (A) and after an intravitreal injection of a dispase-containing solution (B –D); cell nuclei are stained

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receptors) and via inhibition of Naþ/Kþ-ATPase activity, with subsequent inhibition of active Kþ uptake, Naþ extrusion and glutamate uptake. Enzymatically generated NO may exert beneficial effects against bacterial infections and may counteract ischemia by dilating the retinal blood vessels. Low NO concentrations have even been shown to protect neurons from glutamate excitotoxicity, by closing NMDA receptor-gated ion channels (Kashii et al., 1996). However, elevated NO production induces the formation of free nitrogen radicals (peroxynitrite and/or NOz2), which cause protein nitration and dysfunction, particularly, targeting mitochondrial and antioxidant proteins. The inactivation of the mitochondrial respiratory chain results in energy depletion and neuronal cell death. Glutamate, in addition to its direct excitotoxic actions, stimulates the synthesis and release of NO (see chapter by Garcia and Baltrons). Mu¨ller cells may positively or negatively modulate all these mechanisms. Upon pathological stimulation, they release beneficial (but also proinflammatory) cytokines (see chapter by Nakagawa and Schwartz), as well as antioxidant substances. They buffer elevated extracellular Kþ levels, and protect neuronal cells from glutamate and NO toxicity, particularly by uptake and subsequent detoxification of glutamate via glutamine synthesis. However, they also produce free radicals, e.g., by the cytokineinducible NO synthase, iNOS, which is specifically expressed in Mu¨ller cells during ischemic diseases. The hypertrophy and proliferation of reactive Mu¨ller cells result in the formation of glial scars. Glial scars are desirable, as they close small retinal holes or wounds, prevent detachment, and support reattachment of the retina (a negative pressure is established in

with Hoechst dyew (blue), GFAP immunoreactivity is displayed in green, and isolectin B4 label of microglial cells and macrophages is given in red; (A) and (B) represent sections double-labeled for GFAP and lectin, whereas (C) is single-stained for GFAP and (D) for lectin. (A) In the healthy rabbit retina, microglia is confined to the inner retinal layers (ganglion cell and inner plexiform layers; tandem arrowheads), and all Mu¨ller cells are devoid of GFAP immunolabel. (B) After dispase injection, some Mu¨ller cells soon begin to express visible GFAP immunoreactivty (green), and some microglial cells migrate into the inner nuclear and outer plexiform layers (arrow). At places where this occurs, macrophages (double arrowheads) and other blood-derived cells (single arrowhead) occur in the adjacent vitreous body. (C) Later, Mu¨ller cells become hypertrophic, and extend coarse side branches into the retinal tissue (arrowheads) and into the subretinal space where glial scars (‘fibrosis’) develop (arrow). (D) At the same time, activated microglia occupies all retinal layers and even the subretinal space (arrows). (E–G) Rabbit retina 6 weeks after experimental detachment; vimentin immunocytochemistry and counterstaining of cell nuclei by hematoxylin. (E) Survey of a detached retina from a point close to the attached retina (right) to the center of the detached ‘bubble’ (left). Higher magnifications of two different regions are shown in (F) and (G) (localizations indicated by the arrowheads in (E)). The asterisk labels a gliotic scar at the edge of the detached retina, which represents the beginning of a PVR. (F) Even adjacent to the attached portion of the retina, the number of the photorecpetor cells in the outer nuclear layer is reduced, and their long inner and outer segments disappeared. The hypertrophic Mu¨ller cells grow irregular side branches (arrows). (G) In the center of the detached area (i.e., where the largest distance was induced between retina and choroid), only a few photoreceptor cells survived, and the numerical density of retinal neurons is greatly reduced. Nevertheless, the density of Mu¨ller cells is the same as in the healthy retina (or is even slightly enhanced). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRL, photoreceptor segment layer. (H) Transmission electron microphotograph of the inner surface of retina detached for 4 weeks. There is a hole in the vitread basal lamina (arrowheads) through which a Mu¨ller cell (MC) migrates into the vitreous body (vit); ax, axons of retinal ganglion cells.

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the subretinal space if it is closed against the vitreous cavity). However, glial scars are also one reason for the failure of regeneration in the CNS (see chapter by Kalman). In detached retinae for example, Mu¨ller cell processes grow into the subretinal space where they fill the gaps left by the dying photoreceptor cells (Lewis and Fisher, 2000) and form a fibrotic layer (Fig. 3C), which completely inhibits the regeneration of photoreceptor outer segments. 3. Mu¨ller cell-derived cytokines: support of neuronal survival It has been shown that several different neurotrophic factors (brain-derived and ciliary neurotrophic factors, and basic fibroblast growth factor, bFGF) protect photoreceptors, bipolar and ganglion cells from cell death (Faktorovich et al., 1990; LaVail et al., 1992). The protection mechanisms likely involve Mu¨ller cell activation. Neurotrophin-3, for example, mediates its protective effect on photoreceptor cells by stimulation of TrkC receptors expressed by Mu¨ller cells, which mediate a pro-survival signal for photoreceptor cells by increasing the bFGF release from Mu¨ller cells (Harada et al., 2000). Preconditioning of the retina with mild stress protects retinal neurons from degeneration, probably due to stress-induced up-regulation of the release of bFGF and of other neurotrophic factors by Mu¨ller cells (Wen et al., 1995). It must however be kept in mind that the effects of cytokines are not always predictable. For example, nerve growth factor (NGF) administration has been reported to be either beneficial (Hammes et al., 1995) or detrimental (Harada et al., 2000) in different forms of retinal degeneration. 4. The glutamate– glutamine cycle of Mu¨ller cells: challenged in retinal diseases In the healthy retina, neuronally released glutamate (the main excitatory transmitter in the retina) is predominantly taken up by GLAST (L -glutamate – L -aspartate) transporters expressed in Mu¨ller cell membranes (Fig. 1D, middle part). This process is crucial for the rapid termination of the light-evoked activity of retinal ganglion cells (Matsui et al., 1999), and it maintains the extracellular glutamate below neurotoxic levels. After experimental inhibition of the Mu¨ller cell-mediated glutamate uptake, even low concentrations of extracellular glutamate become neurotoxic (Izumi et al., 1999; Kashii et al., 1996). The glutamate uptake is voltage-dependent (Fig. 2D), as depolarization decreases the uptake (Barbour et al., 1988, Reichelt et al., 1997). Retinal ischemia causes a glutamate imbalance because (i) glutamate is progressively released from the depolarized neurons, and (ii) glial glutamate uptake is reduced due to high extracellular Kþ, which depolarizes the Mu¨ller cells (Napper et al., 1999), as well as to an inhibition of the Naþ/Kþ-ATPase activity by free radicals (which reduces the Naþ ion gradient, and, thus, the driving force for the uptake). Mu¨ller cell membrane depolarization is further accelerated by the downregulation of the Kir channels (Bringmann et al., 2000). Under these conditions, there occurs a significant glutamate uptake by retinal neurons, which normally do not accumulate glutamate because it is rapidly cleared by Mu¨ller cells (Barnett et al., 2001). Experimental knockout of GLAST leads to an increased retinal sensitivity to ischemia, and

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to ganglion cell death (Harada et al., 1998; Vorwerk et al., 2000). An impaired glutamate uptake into Mu¨ller cells may underlie neuronal degeneration in various retinal diseases, including glaucoma. However, under certain pathological conditions, an enhanced expression of GLAST by activated Mu¨ller cells was observed (Otori et al., 1994; Reichelt et al., 1997). It has been shown that increased glutamate receptor stimulation activates protein kinase C in Mu¨ller cells (Lopez-Colome et al., 1993), which, in turn, increases the glutamate uptake by phosphorylation and increased expression of the transporter protein (Gonzalez et al., 1999). Once transported into the Mu¨ller cell, glutamate is rapidly degraded by multiple pathways; the main pathway is the ATP-dependent formation of glutamine from glutamate and ammonia by the enzyme, glutamine synthetase, which is exclusively expressed by Mu¨ller cells (Linser and Moscona, 1979) and astrocytes, and which operates in concert with the GLAST transporter (Derouiche and Rauen, 1995). The enzyme is very efficient: glutamate can only be visualized immunocytochemically in Mu¨ller cells, when the glutamine synthetase is inhibited (Pow and Robinson, 1994). Its reaction product, glutamine, is transferred to the neurons, where it serves as precursor of glutamate (‘transmitter recycling’; Fig. 1D, middle part). Experimental inhibition of the glutamine synthetase in Mu¨ller cells resulted in a loss of neuronal glutamate, and the animals became functionally blind within 2 min (Pow and Robinson, 1994). A pathologically decreased activity of the glutamine synthetase (Erickson et al., 1987) may contribute to glutamate excitotoxicity. The problem may be further aggravated in ischemic retinopathies when retinal cells, including Mu¨ller cells, release bFGF which was shown to inhibit the hormonal induction of glutamine synthetase expression in Mu¨ller cells (Kruchkova et al., 2001). Glutamine synthetase activity detoxifies the retina not only from elevated levels of glutamate but also from ammonia, which is released by functionally active retinal neurons. Glutamate and ammonia stimulate glycolysis, via the energy demand of the glutamine synthetase reaction (Poitry et al., 2000) and a possible direct effect of ammonia on energy metabolism. Patients suffering from liver insufficiency display high levels of serum ammonia; this toxic compound must be cleared from the retina by Mu¨ller cells (and astrocytes). Due to the high energy demand of the glutamine synthesis, long-lasting exposure to high ammonia causes energy depletion and, finally, damage to Mu¨ller cells. The failure of Mu¨ller cell functions then impairs neuronal information processing, a process which has been termed hepatic retinopathy (Reichenbach et al., 1995). During this process, the detoxification of glutamate and ammonia by the surviving Mu¨ller cells is further jeopardized by a down-regulation of their Kir channels (Bringmann et al., 1998) and by a failure of their Naþ/Kþ pump due to energy deficiency, two processes which reduce the driving force for glutamate uptake. Another main pathway of glutamate metabolism in Mu¨ller cells is the generation of glutathione, a tripeptide consisting of glutamate, cysteine, and glycine. In its reduced form, glutathione is the main antioxidant in the retina. Glutathione is synthetized exclusively in Mu¨ller cells (Pow and Crook, 1995), and it is rapidly released from Mu¨ller cells and transferred to retinal neurons, particularly to ganglion cells, in cases of hypoxic and hypoglycemic stress (Schu¨tte and Werner, 1998). The inhibition of glutamate uptake under ischemic conditions may result in a glutathione deficiency, which must further

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increase the level of reactive oxygen species. The glutathione content of Mu¨ller cells decreases with age (Paasche et al., 1998), which may contribute to age-related retinal degeneration.

5. Energy metabolism: Mu¨ller cell – neuron symbiosis, hypoxia and neovascularization The energy metabolism of Mu¨ller cells is generally believed to be principally based upon glycolysis, even in the presence of oxygen (Poitry-Yamate et al., 1995). Thus, Mu¨ller cells metabolize glucose to lactate and pyruvate, which may be taken up by the photoreceptor cells to fuel their oxidative metabolism (Poitry-Yamate et al., 1995). Due to their anaerobic energy metabolism and their glycogen deposits (Kuwabara and Cogan, 1961), Mu¨ller cells are resistant to anoxia. Recently, some doubt has been expressed regarding the dominant nature of glycolysis in Mu¨ller cells (see chapter by Chih and Roberts). Mu¨ller cells do possess mitochondria, particularly in most mammalian retinae, which are vascularized (Germer et al., 1998; Paasche et al., 2000), and ischemia does inhibit glutamate uptake (Barnett et al., 2001). In any case, Mu¨ller cells possess oxygen-sensing mechanisms (Eichler et al., 2000), and they are supposed to play a critical role in the development of hypoxia-induced retinal neovascularizations. Neovascular diseases of the retina are a major cause of blindness. Several retinal and choroidal diseases such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, and the wet form of age-related macular degeneration are accompanied by pathological intraocular neovascularization. Hypoxia-induced neovascularization may proceed by multiple mechanisms, including (i) stimulation by vascular endothelial growth factor (VEGF), which is a key regulator of retinal angiogenesis and is released by Mu¨ller cells, especially under hypoxic conditions and during glucose deprivation (Aiello et al., 1995; Eichler et al., 2000), (ii) release of angiogenic cytokines such as transforming growth factor (TGF), bFGF, and TNF from Mu¨ller cells, (iii) release of ATP from Mu¨ller cells (Newman, 2001) and subsequent production of adenosine (via the ectoenzyme 50 nucleotidase: Lutty et al., 2000) activating the adenosine A2a receptors expressed by developing blood vessels, and (iv) an effect of renin, expressed in Mu¨ller endfeet closely apposed to retinal blood vessels (Berka et al., 1995). In chronic diabetes, retinal blood vessel occlusion causes hypoxia, which stimulates abnormal angiogenesis. Mu¨ller cell reactivity is an early feature of experimental diabetes; Mu¨ller cell processes grow into the lumen of occluded vessels where they form a glial scar. An increased density of rat Mu¨ller cells was described after 4 weeks of diabetes, and GFAP expression occurred after twelve weeks (Rungger-Bra¨ndle et al., 2000). The strongly increased expression of advanced glycation end products, AGEs (see chapter by Mizisin) in diabetic retinae may contribute to the induction of VEGF production, via an activation of AGE receptors of Mu¨ller cells (Hirata et al., 1997). During diabetes, Mu¨ller cells express iNOS (Abu El-Asrar et al., 2001). NO is not toxic for Mu¨ller cells themselves, but it may either induce neuronal cell death by free radical generation (Goureau et al., 1999) or support neuronal survival by increasing the retinal blood flow during hypoxia and hypoglycemia (Roth, 1997). NO synthesis by Mu¨ller cells may be

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involved in the pathogenesis of diabetic retinopathy (Roth, 1997). It remains to be proven whether Mu¨ller cell-derived insulin (Das et al., 1987) may rescue retinal neurons from apoptosis (Barber et al., 2001). 6. Mu¨ller cells and the blood – retina barrier: a vicious circle? In addition to the blood –retina barrier of the choroideal circulation, which is maintained by the retinal pigment epithelium (RPE), vascularized retinae of mammals have a second blood –retina barrier which is formed by tight junctions between the endothelial cells of the intraretinal blood vessels. Mu¨ller cells enwrap retinal blood vessels (Fig. 1A) and participate in the establishment of their barrier and in the regulation of the barrier properties, as they produce factors capable of modulating both blood flow and vascular permeability (Behzadian et al., 2001). Glial cell line-derived neurotrophic factor and neurturin, which are secreted from Mu¨ller cells, decrease the permeability of the barrier (Igarashi et al., 2000), while Mu¨ller cell-derived TNF and VEGF open the blood – retina barrier (Aiello et al., 1995; Drescher and Whittum-Hudson, 1996). Thus, reactive Mu¨ller cells may cause a leakage of the blood –retina barrier, and extravasated serum components such as growth factors and plasma proteins (e.g., immunoglobulins), may further trigger Mu¨ller cell gliosis and proliferation. 7. Retinal detachment and proliferative retinopathies: special cases of the Janus-faced Mu¨ller cell gliosis Detachment of the neural retina from the RPE increases the distance between the choriocapillaris and the neural retina, decreasing its oxygen and glucose supply. This was suggested as a cause of photoreceptor cell death and subsequent retinal degeneration (Stone et al., 1999). After experimental retinal detachment, Mu¨ller cells become immediately reactive. Within minutes, Mu¨ller cells show increased protein phosphorylation (e. g., of bFGF receptor-1 and extracellular signal-regulated kinase) and increased production of transcription factors (Geller et al., 2001). Three hours after detachment, neuronal cell bodies are depleted of glutamate, while Mu¨ller cells show an increased glutamine content (Sherry and Townes-Anderson, 2000). Within one day after detachment, Mu¨ller cells begin to proliferate, and display an increased expression of GFAP and vimentin immunoreactivity (Fisher et al., 1991; Fig. 3). This proliferation shows a maximum after 3 –4 days (Fisher et al., 1991). When no reattachment occurs, retinal detachment may develop into a proliferative vitreoretinopathy (PVR) and subretinal fibrosis (Fig. 3C). Proliferating and migrating Mu¨ller cells are one constituent of the epiretinal cellular ‘membranes’ which form during PVR; the contraction of these cellular aggregates leads to further retinal detachment. The detachment-induced Mu¨ller cell reactivity also includes severe alterations of electrophysiological parameters (Bringmann et al., 2000). The plasma membranes of Mu¨ller cells in healthy retinae are highly permeable to Kþ ions, as a consequence of the strong expression of Kþ channels, including (i) the Kir channels, which are open at the resting membrane potential and at hyperpolarized potentials (Brew et al., 1986; Figs. 1C

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and 2A – C), and (ii) calcium-activated Kþ channels of big conductance (BK channels), which open upon membrane depolarization and increases of the intracellular calcium concentration (Puro et al., 1989; Fig. 2A). A prerequisite for effective extracellular potassium clearance by Mu¨ller cells is a very negative membrane potential (normally around 2 80 mV), which is maintained by open Kir channels. Mu¨ller cells from detached retinae show a decrease in their Kir currents, already after 2 days (Francke et al., 2001). Mu¨ller cells from PVR retinae display almost complete absence of Kir currents, which is accompanied by a depolarization of the plasma membrane (Reichelt et al., 1997; Francke et al., 2001). As mentioned above, this must impair the retinal homeostasis of both Kþ and glutamate. On the other hand, the activity of calcium-dependent BK channels is enhanced in Mu¨ller cells from PVR retinae (Bringmann et al., 2000). The activity of these channels is necessary for the proliferation of cultured Mu¨ller cells (Puro et al., 1989). Furthermore, it was recently shown that human Mu¨ller cells from PVR retinae display an increase of ionotropic P2X7 receptor-mediated cation currents (Bringmann et al., 2001), and that rabbit Mu¨ller cells from PVR retinae increase their responses to activation of metabotropic P2Y receptors (Fig. 2E and F) as compared to cells from healthy control retinae (Francke et al., 2002). The increased responsiveness of Mu¨ller cells to extracellular ATP may support their proliferation in proliferative retinopathies. Extracellular ATP enhances the proliferation of cultured Mu¨ller cells in a calcium influx- and BK channel-dependent manner (Moll et al., 2002), and stimulates in a transactivation process (see chapter by Peng) the release of growth factors, such as the heparin-binding epidermal and plateletderived growth factors, from cultured Mu¨ller cells (Milenkovic et al., 2001). As plateletderived growth factor is one of the crucial growth factors involved in the development of PVR (Andrews et al., 1999), the release of ATP and of growth factors by Mu¨ller cells may be a key event in the generation of proliferative retinopathies. 8. An outlook: turning the Janus face towards its good side As Mu¨ller cells survive various pathogenic stimuli, and because they are in intimate contact with the retinal neurons, they may play a central role in novel therapeutic strategies to prevent retinal degeneration. On the one hand, somatic gene transfer to Mu¨ller cells may support their protective role(s) in neuronal cell survival, or depress detrimental Mu¨ller cell actions such as the expression of iNOS. On the other hand, the observation that proliferating Mu¨ller cells may transdifferentiate into retinal precursor cells and even into retinal neurons (Fischer and Reh, 2001), may be used to replace degenerating neurons. In particular, glial scars such as the epiretinal ‘membranes’ in PVR eyes, may constitute a source of dedifferentiated Mu¨ller cells from which a new retina could then be grown in vitro and retransplanted into the eye of the patient. 9. Concluding remarks All available data underline the crucial role(s) of Mu¨ller cells in retinopathies but there are still many open questions, and the need for further investigations in the field is obvious.

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Such future research should be directed towards the elucidation of the inter- and intracellular signaling pathways involved in the development of Mu¨ller cell gliosis (particularly, concerning the regulation of functional Kir channel expression) and also towards the establishment of novel therapeutic strategies targeted at the Mu¨ller glial cells. These strategies may include a pharmacological inhibition of devastating components of the gliotic response, ameliorating gene transfer to the Mu¨ller cells of the injured retina, and/or the use of activated Mu¨ller cells as progenitors of neurons to compensate for degenerative cell loss.

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INDEX

Ab see amyloid b-peptide ABCA1 see ATP-binding cassette transporter protein A-I abetalipoproteinemia 522 acetate metabolism 843– 844 acetylcholine receptors 482– 483 acid extrusion 709–710, 719– 722, 725– 730 acid loading 709, 714, 715– 717 acid shifts 712–713, 728–729 acid – base transporters 709– 710, 727– 728 see also individual transporters acidic FGF see fibroblast growth factor-1 acoustic evoked potentials (AEPs) 623 acquired immunodeficiency syndrome (AIDS) see HIV actin 794 actin binding proteins 149– 150, 157 activity-dependent neurotrophic factor (ADNF) 493 AD see Alzheimer’s disease ‘adaptive’ immunity, human 1002– 1003 ADC (AIDS dementia complex) see HIV-1associated dementia adenine nucleotide carrier (ANC) 985– 986 adenosine 755– 757 adenylyl cyclase second messenger system 449 adherent junctions 299 adhesion molecules anti-psychotic treatments 1008 brain endothelium infiltration 258– 259 gp120-mediated astrocytic dysfunction 936 Schwann cells 335 signal transduction 259– 260 ADNF see activity-dependent neurotrophic factor adrenergic receptors 480–481, 506, 507– 508, 510, 511 adrenocorticotrops 759 adult brain ependymal cells 134– 136 adult cell proliferation 138–139, 141 adult neurogenesis 105 adventitia 219 see also perivascular... AEPs see acoustic evoked potentials AEs see anion exchangers aging 893 AIDS see HIV AIDS dementia complex (ADC) see HIV-1associated dementia alanine synthesis 845– 846

aldol reductase 1108– 1109, 1111 alertness 621–623 Alexander disease alphaB-crystallin 776, 783 astrocytes primary disease 773– 783 clinical subgroups 777 glial fibrillary acidic protein 779–783 leukoencephalopathy 777 pathological features 775– 777 similarity with multiple sclerosis 777 alkaline shifts 712–713 allodynia 955, 959 allopregnenolone 992 alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA) 1066– 1070, 1072– 1073, 1121– 1124 alphaB-crystallin, Alexander disease 776, 783 Alzheimer Type II astrocytosis 984, 987 Alzheimer’s disease (AD) amyloid b-peptide immunization 892 astroglial NOS2 immunoreactivity 579 calcium-binding proteins 652, 653– 654 excitotoxicity 891– 892 glial cell cycle activation 87 glial role 883– 894 glutamate toxicity 891–892 heme oxygenase-1 expression 877– 878 inflammatory hypothesis 886– 887 mitochondria 677– 678 nitric oxide-sensitive guanylyl cyclase 582 peripheral astrocyte processes 159 plaque progression 884– 886 S-100 652 aminoadipate 1097– 1098 4-aminopyridine (4-AP) 651 amitryptiline 1042 ammonia 847, 983– 994 AMPA see alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate amphetamine 454 amphibians see also anurans Necturus 612 amygdala 1050, 1054 amyloid b-peptide (Ab) glutamate toxicity 891–892 immunization 892 microglia activation 887 plaque formation 884– 886 amyloid plaques 884– 885

1146

Index

b-amyloid precursor protein (bAPP) 884 anaplerosis 427– 428, 466 ANC see adenine nucleotide carrier androstenediol 537 androstenedione 537 anesthesia 424– 425, 426 Ang II 281– 287 angioarchitectonic constraints 14, 16 – 18 angiogenesis 18 –22, 302 angiotensin II receptor 506, 511– 512 anion exchangers (AEs) 717– 719, 732– 733 anisomorphic glial reaction 790, 798, 809 ANLSH see astrocyte – neuron lactate shuttle hypothesis ANP (peptide) 281– 287 anti-bipolar drugs 1033–1045 anti-depressants 1020– 1021 anti-inflammatory cytokines 563, 567 anti-oxidant responses 808, 861–862 anti-psychotic therapy 1010, 1012, 1014– 1015 anurans avoidance behaviour 627– 628, 629, 630 glial cell spatial buffering 612, 613 imposed DC shifts 626, 627– 629 optic tectum 613, 616– 621 slow potential shifts 614–624, 630 4-AP see 4-aminopyridine Aplysia punctata 637 apolipoproteins (apo) apoA-I 521, 522 apoD 521 apoE 521, 522, 523– 525, 527– 528, 889–890 apoJ 521 apoptosis 512, 653, 654, 938 bAPP see b-amyloid precursor protein approach behaviour 621, 624 aquaporins (AQP) 752–753, 754, 761, 763 see also water channels arachidonic acid 907, 933 arachnoid 218 arachnoid– dura interface (arachnoid barrier) 226– 227 arginine 780, 782 arginine vasopressin (AVP) astrocytic water content 750– 753 astroglial mitogenesis 191 capillary endothelium 760– 761 choroid plexus 762– 763 CSF regulation 281– 287 hypothalamo-neurohypophyseal system 182, 183 production 748 water channels in non-neuronal CNS cells 747– 764

aromatase (P450 aro) 540, 541 arousal 621– 623 asparaginyl hydroxylase 860 aspartate synthesis 849– 850 astrocyte – astrocyte coupling 168– 169 astrocyte – gp120 interaction mechanisms 928– 930 astrocyte – microglia interactions 1090– 1094 astrocyte – neuron lactate shuttle hypothesis (ANLSH) 391– 403 astrocyte – oligodendrocyte coupling 169– 170 astrocytes see also enteric glial cells; ependymoastroglia; glia; glial reaction activation 801– 802 activity-evoked extracellular acid shifts 728– 729 activity-induced glucose uptake 400–401 afferent nuclei 754– 755 alanine synthesis 845– 846 Alexander disease 773– 783 Alzheimer’s disease 884– 894 anti-bipolar drugs 1033– 1045 apoE synthesis and secretion 524– 525 aromatase and estradiol production 540, 541 ATP release 667– 668 blood– brain barrier 256 brain inflammation 969 calcium waves 199– 200, 661– 678 cell cycle regulation 75 – 89 cell death/neuroprotection signals 174– 175 cell surface increase 6 cell surface molecules 84 – 86 cell volumes 168, 169 cerebrocortical 749– 759 cooperative tissue monitoring 39 – 45 cyclic GMP inactivation 583– 585 cytoarchitectonics 36, 37, 42 cytokine receptors 487– 488 density dependent proliferation inhibition 82 – 86 differentiation 112– 117, 210 distribution in neocortex 44 Donnan-mediated potassium chloride uptake 604, 605 dopaminergic transmission in schizophrenia 1013– 1014 EGF transactivation 509 endothelial cell communication 206, 210 energy metabolism 435–455 energy source distribution 172–173 function 912– 913 functional consequences of receptor activation 489– 493

Index

G-protein-coupled membrane receptors 477– 487, 489, 491 GABA homeostasis 461– 463, 467– 470 gap junctions 45, 166– 170, 441, 599–600, 699– 702, 792 GFAP mRNA expression 188– 189 glial fibrillary acidic protein overexpressor mice 778–779 glutamate 461– 467, 469– 470, 808 glutamine synthetase 151– 156 GnRH neuron interaction 200– 205, 207 gp120-mediated dysfunction mechanisms 930– 938 growth factor receptors 488 high density lipoprotein production 519, 526– 527 HIV infection of 911 HIV virus amplification 928 HIV-1-associated dementia 902– 903, 910– 913 HIV-related glycoprotein gp120-mediated dysfunction 921– 939 hyperammonemic expression alterations 985 hypothalamo-hypophysial system 753– 754 hypothalamo-neurohypophyseal system activation 181– 191 hypoxic response in retina 300 hypoxic– ischemic stress vulnerability 857– 864 IL-1b effects in HAD 909, 910 immunohistochemical labeling 147– 160 injury responses 885 intercellular calcium signaling modeling 699– 702 intracellular receptors 488–489 ion channel-coupled membrane receptors 477– 487, 489, 491 ischemia 837– 850 lactate released in energy production during neural activity 391– 403 loss 1049, 1053– 1054, 1056 mediating thyroid hormone actions 107– 110 metabolism under deep pentobarbital anesthesia 425 metabolite distribution 172– 173 microglial interactions 1090– 1094 morphogenesis 110– 117 morphology/function 952– 953 multiple sclerosis 1063 –1064, 1065, 1067, 1069, 1076– 1077 nerve regeneration inhibition 333 neuron –astrocyte ratio 86 neuronal interactions 104– 117, 551, 672– 675, 702–703, 1094– 1098

1147

neuroprotective transactivation 503– 513 nitric oxide-sensitive guanylyl cyclase regulation 581– 583 oligodendrocytes relationship 54, 55, 64, 169– 170 oxygen consumption 425, 427 Parkinson’s disease 970– 971 peripheral astrocyte processes 147– 160 persistent pain role 951– 962 potassium 170– 172, 595– 605, 923 prion disease 1085, 1087– 1088 proliferation in activated HNS 189– 191 radial glia 130 reactive 605 receptors 475– 493 reorientation in activated HNS 186– 187 retinal stabilizing role 297, 298, 300– 303, 309 retinopathies 1117– 1119 retraction of processes in activated HNS 184– 186 scar formation 330 Schwann cell transplant influence 338– 341 second messenger systems 475– 493 soluble factors 104–106 spatial buffering 599–603 stellation 756, 757– 758 steroid effects 543– 548 strategic position in central nervous system 490– 491 structural organization 167–170 structural plasticity 181– 191 subventricular zone origins 140 supraoptic nucleus 181– 191 swelling 445 synaptic transmission 674– 675 syncytium formation 165– 176 TCA cycle 427– 428 vasculature of retina 301– 302, 303, 309 in vivo neurotrophic factor and cytokine expression 561– 569 volume regulation 720– 721, 748 water channels 752–753 astrocytomas 86, 160 astroglia see astrocytes astroglia-like cells 33 see also ependymo-astroglia astrogliogenesis 111– 112, 801 astrogliosis 774, 952 astrostatine 83 astrotactin, neuronal binding to radial glia 101

1148

Index

ATP astrocytic transactivation receptor selectivity 511 calcium waves 666– 669, 702 hypothalamo-hypophysial system 755– 756 transmitter glutamate effect on astrocytes 672– 673 ATP-binding cassette transporter protein A-I (ABCA1) 522, 527 attention 621– 623 autaptic gap junctions 169 autoimmune systems 321, 347– 359, 1001 automated shimming 416 avoidance behaviour 627– 628, 629, 630 ATP, cytosolic 668– 669 AVP see arginine vasopressin axons astrocyte – neuron interactions 105 atrophy 1110– 1111 glutamate production 1074 regeneration 329– 342, 809– 818 B cells 1014– 1015 bacteria 255, 262– 263 barbiturates 424– 425, 426, 453 basal laminae (BL) anatomy 222, 223– 224 cytokines and growth factors 232 extracellular space 6 magnocellular hypothalamo – neurohypophysial system 240 parenchyma/extraparenchyma boundary 215, 216, 217, 218, 219 role in cell proliferation 233 basic fibroblast growth factor (bFGF) see fibroblast growth factor-2 BBB see blood – brain barrier BCSFB see blood– cerebrospinal fluid barrier BD see bipolar disorder BDNF see brain derived neurotrophic factor behaviour 611– 630 benzodiazepines 453, 479, 489 beta, gamma subunits 507– 508 beta-amyloid precursor protein (bAPP) 884 bFGF (basic fibroblast growth factor) see fibroblast growth factor-2 bicarbonate acid-extruding transporters 719– 722, 725– 730 acid-loading transporters 715– 719 effect on neuronal activity 711–712 bidirectional coupling 702– 703 bipolar disorder (BD) 1033– 1045, 1051, 1054, 1055

BL see basal laminae blood vessel invasion 6, 14, 18 – 22 blood– brain barrier (BBB) anatomy 217, 221 chemical transport 224– 226 cholesterol homeostasis 520, 521– 522 extraparenchymal – neural interaction 245 glial reaction 789, 799, 800, 809, 816 HIV infection process 927 impairment in schizophrenia 1008 infiltration mechanisms 255– 264 inositol 1037 ion and water homeostasis 759– 761 polarized immune response 1005 potassium homeostasis 596– 597 structure 256– 257 tight junctions 256– 257 in vitro model 257 blood– cerebrospinal fluid barrier (BCSFB) 221, 223, 256, 259, 262, 273– 274, 281 blood– retinal barrier (BRB) 303, 1127 bovine spongiform encephalopathy (BSE) 1086 bradykinin receptor 506 brain derived neurotrophic factor (BDNF) 564 branched cell processes 6 BRB see blood – retinal barrier BSE see bovine spongiform encephalopathy ‘burned-out-plaque’ 885 13

C NMR spectroscopy 409– 428, 844, 845– 846 caffeine/ryanodine (CAF/RY)-sensitive calcium stores 645 CAK see cyclin-dependent kinase activating kinase calcineurin 654 calcium arginine vasopressin 750– 751, 754 capacitative entry 647– 651 cyclic GMP in glial cells 585– 586 homeostasis 575, 586 intracellular 173–174, 637–647, 651– 654, 690– 699, 1042– 1044 L-channels 445, 454 mathematical modeling of signaling 689– 703 metabolic stimulation mechanisms 437– 439 regulation of stores in glial cells 635– 655 storage organelles and intracellular calcium release 637– 647 transmitter glutamate effect on astrocytes 672 waves 173– 174, 199, 200, 661– 678, 923, 935

Index

calcium release-activated calcium channel (CRAC) 647, 648, 651 see also store-operated calcium channel calcium-binding proteins 639– 640, 652– 654 calcium-dependent NOS isoforms 576– 578 calexcitin 653 calreticulin (CRT) 639– 640 calsenilin 653 calsequestrin 639 CaM 651–652 cancer 86 – 88, 160, 1036 capacitative calcium entry (CCE) 647– 651 capillaries angioarchitectonics 17 – 18, 21 – 22 angiogenesis 19, 21 epithelial cytoarchitectonics 22, 24 intracerebral network development 20 capillary endothelial cells see endothelial cells Carassius auratus 815– 816 carbamazepine 1033– 1045 carbohydrate epitope CD 15 157 carbon dioxide, interstitial partial pressure 712– 713 carrier-mediated metabolic effects 441– 444, 448 cataractogenesis 1108 catecholamines 755 caudal orbital cortex 1051– 1052 CBF see cerebral blood flow CC chemokine family 563, 567– 568 CCE see capacitative calcium entry CCI see chronic constrictive injury CCR5 chemokine receptor 926, 929 CD4 glycoproteins 926, 929 CDK... see cyclin-dependent kinase... celecoxib therapy 1015 cell-type-specific polarities 6 – 7 cells adhesion molecules 331– 332 communication 224– 229, 245– 247 contact relations 1 – 47, 930 cycle 75 – 89 death 174– 175 development 8, 15, 31 – 32 junctions 791– 792 migration 795– 796 origins 2 proliferation 81 – 86, 233, 246, 795– 798, 953 shapes 1 – 47 structures 1 – 47, 791, 792 surface extensions 6, 9– 10, 33, 36, 37 –39 territories 2 – 3, 45 volume regulation 720– 721, 748

1149

cellularization 165– 166 cerebellum 107– 110 cerebral blood flow (CBF) 418, 419 cerebral ischemia 839– 840 cerebral oxygen consumption 422 cerebrocortical astrocytes 749– 753 cerebrospinal fluid (CSF) see also blood – cerebrospinal fluid barrier apolipoproteins 521 choroid plexus 270, 597 filtration 1015 high density lipoprotein 521 hydrocephalus disorders 269– 287 MNC communication 187 neuroendocrine regulation 281– 287 pH regulation 708– 709, 731– 735 production 270, 272– 274 schizophrenia 1008– 1009 shunts 287 ventricular system 128 CFTR see cystic-fibrosis airway epithelial cells channel-mediated effects 444– 445, 448 chemokines HIV-1-associated dementia 907 receptors 486– 487 release at blood– brain barrier 259 viral entry into CNS 261 in vivo astrocyte expression 562, 563, 567– 568 chicken embryo ganglia 399–400 a-chloralose anesthesia 426 chloride transport 272– 274, 281, 285 cholesterol 519– 529, 991– 992 choroid plexus (CP) anatomy 217, 219– 220, 221, 223 AVP-induced epithelial changes 284– 285 basolateral spaces 284– 285 blood – cerebrospinal fluid barrier 256 brain barrier functions in ion and water homeostasis 761– 763 cerebrospinal fluid 731 ‘dark’ and ‘light’ epithelial cells 280, 284– 285 epithelial cells 269–287, 731– 734 function 270, 271–274 hydrocephalus 275– 282, 283– 284 immunosurveillance of CNS 259 neuroendocrine CSF regulation 281– 287 neuropeptide receptors 282 normal ventricular relationships 271– 274 pathogen entry into CNS 262 potassium homeostasis 597 structure and development 271– 272

1150

Index

choroid plexus –CSF – brain nexus 270– 281 chronic acid loading 709 chronic constrictive injury (CCI) 955 chronic multiple sclerosis lesions 1063– 1064, 1077 chronic pain 951– 962 chronic progressive multiple sclerosis 1065 CICR 642– 644 ciliary neurotrophic factor (CNTF) 305– 307, 566 4-CIN see alpha-cyano-4-hydroxycinnamate Cip/Kip family cyclin-dependent kinase inhibitors 79 – 81, 83 – 84, 87 circumventricular organs (CVOs) 754 CJD see Creutzfeldt – Jakob disease CKIs see cyclin-dependent kinase inhibitors Cl– HCO3 exchangers 717– 719, 734– 735 clonidine 449 closed injuries 790, 797 clustering of calcium release channels 694–699 CNS see central nervous system CNTF see ciliary neurotrophic factor co-transporter mediated effects 445–446 see also individual transporters cocaine 454 communication 65 – 66, 224– 229, 245– 247 compartmentalization 7, 147– 148, 644– 646 composite perivascular glial sheath 39, 41 concentration gradients 170 conductances, acid-loading 714 confocal scanning laser microscope 662 congenital hyperammonemia 990 congophyllic deposits 884 connective tissue see also meninges anatomy 216, 218 basal lamina 223– 224 role in brain function 245– 246 connexins 157– 158, 168– 170, 332, 441, 664– 666, 668– 669 contact inhibition 86 see also density dependent inhibition contact relations 10 continuity equation 690 cooperative tissue monitoring 39 – 45 Cop-1 antigen (glatiramer acetate) 359 copper 1099 cortex see caudal orbital...; subgenual...; supracallosal anterior cingulate... corticosteroids 544 Cox-2 975 CP see choroid plexus CPA-sensitive calcium stores 645

CRAC see calcium release-activated calcium channel Creutzfeldt– Jakob disease (CJD) 87, 1086 Crohn’s disease 321, 324 CRT see calreticulin CSF see cerebrospinal fluid CVOs see circumventricular organs CX3C chemokine family 563, 567, 568 CXC chemokine family 563, 567, 568 alpha-cyano-4-hydroxycinnamate (4-CIN) 400 cyclic GMP 580– 586 cyclin-dependent kinase activating kinase (CAK) 77 cyclin-dependent kinase inhibitors (CKIs) 76 – 77, 79 – 80, 83 – 84, 87, 88 cyclin-dependent kinases (CDKs) 76 – 81 cyclins 76 – 77, 81 – 82, 86, 87 cystic fibrous astrogliotic MS lesions 1065 cystic-fibrosis airway epithelial cells (CFTR) 668– 669 cytoarchitectonics 1 – 47 cell layering 217, 224– 227 descriptors 10 – 12 ependymo-astroglia 33 – 45 microglia 24 – 28 oligodendroglia 28 – 33 parenchyma/extraparenchyma 217, 219 supraoptic nuclei 239 terminal vascular bed endothelia 14 – 24 cytokines see also individual cytokines anti-depressants 1020– 1021 bacterial entry into CNS 262 basal lamina 232 brain 228– 229 calcium wave propagation 665 cell proliferation role 233 chemokine receptors 486– 487 choroid plexus stroma 221, 223 CNS network 1001– 1002 crossing blood brain barrier 227– 228 enteric glia 323, 324 expression in astrocytes in vivo 561, 562, 563, 566–569 extraparenchymal tissue 229– 233, 245, 246 glial reaction regulation 799– 805 HIV-1-associated dementia 906– 910 HIV-1-induced changes in production 934– 935, 936– 937 hypothalamo– hypophysial systems 243– 244 inhibitors 961 major depression 1012 Mu¨ller cell-derived 1124 multiple sclerosis 1061

Index

pain transmission 958– 960 parenchymal cells 228– 229, 234– 235 Parkinson’s disease 975– 976 photoreceptor protection 304– 305 prion disease 1085, 1092– 1093, 1095 psychoses 999– 1022 receptors 487– 488 release at blood– brain barrier 259 schizophrenia 1005– 1015 ‘sickness behaviour’ 1015–1016 cytomegalovirus 1007 cytoprotection 512 cytoskeleton 792– 794 cytotoxic effects 803 DAG see 1,2-diacylglycerol ‘dark’ epithelial cells 280, 284– 285 DBI see diazepam binding inhibitor DC shifts see imposed DC shifts De Young– Keizer model 691, 696 death proteins 913 debris elimination 805–807 dehydration 183– 195 dehydroepiandrosterone (DHEA) 537 dementia 775, 901–913 see also Alzheimer’s disease demyelination 1059 –1061, 1062– 1065, 1069, 1070, 1106– 1107, 1110 dendritic response 614– 621 density dependent inhibition 82 – 86 deoxyglucose (DG) 401, 413, 442, 443 depolarization, glial 601, 613– 614, 615 depression 1049– 1054 see also bipolar disorder; major depression detached retina 300, 308– 309 deterministic modeling 693 development brain 492–493 endothelial cells 15 intracerebral vascularization 14, 18 – 22 nervous system 97 – 118, 128– 133 neuroepithelial organisation 6 Devic’s disease 1065 dexmedetomidine 504, 507 DG see deoxyglucose DHEA see dehydroepiandrosterone diabetes 1126– 1127 neuropathy 1105– 1112 retinopathy 303–304, 309 1,2-diacylglycerol (DAG) 1034, 1035– 1036 diazepam binding inhibitor (DBI) 984, 990 diffuse amyloid b-peptide plaques 884, 885 dizocilpine 1070

1151

Donnan-mediated potassium chloride uptake 604, 605, 749 dopamine heme oxygenase-1 induction 878 receptors 481 schizophrenia 1001, 1013– 1014 transmitter effects on metabolism 450, 452 double synapses 185– 186 drug effects on astrocytic energy metabolism 453– 454 drugs of abuse 454 dura 218 dura– arachnoid interface see arachnoid-dura interface dynamic isotopomer analysis 413, 414, 415– 416 dystrophia 874, 884 EAAT1/EAAT2 464 see also glutamate transport EAE see experimental autoimmune encephalomyelitis early gene expression 804– 805 early multiple sclerosis lesions 1063 EC see ependymal cells ECF see extracellular fluid ECM see extracellular matrix ECS see extracellular space ‘ecstacy’ see 3,4methylenedioxymethamphetamine EGC see enteric glial cells EGF see epidermal growth factor EGFR see epithelial growth factor receptor electrical stimulation 618 electrogenic NBC activity 725, 726– 727 electrophile response element see anti-oxidant responses embryonic development 128– 133 encephalitis 256, 903– 905 endogenous PTBR ligands 990 endoplasmic reticulum (ER) 636– 647, 649, 670– 671 endothelial cells adhesion molecules 258– 259 blood – brain barrier 221, 225, 245, 255– 264 capillary 734– 735, 759– 761 cellular communication 199– 200, 206– 210, 225– 226, 228 developmental cytoarchitectonics 15 GnRH control 199 –200, 206–210 immortalized lines 257– 258 nitric oxide synthase 207– 209 steroid effects 541, 543 terminal vascular bed 14– 24

1152

Index

viral infection 261 endothelial – glial communication 209– 210 endothelial – neuronal signaling 206– 209 endothelin receptors 484, 506 endothelins 484, 756, 757– 759, 760 energy demands 808 energy metabolism 435– 455 energy sources 172– 173, 391– 403 enkephalins 484 eNOS see nitric oxide synthase, NOS3 ENS see enteric nervous system enteric glial cells (EGC) 315– 324, regulation of neuronal function 317– 319 enteric nervous system (ENS) 315–324 envelope proteins 925– 926 ependymal cells (EC) brain barrier functions in ion and water homeostasis 763 choroid plexus epithelium comparison 278 function and morphology 128, 134– 136 hydrocephalus effects 275, 277 NOVOcan protein 137–138 origins 133– 134 subependymal layer 138 tanycytes 136 ventricular zone cells relationship 128, 133– 134 ependymo-astroglia cell processes 37 – 39, 40, 41, 43 cytoarchitectonics 23, 24, 33 –45 development 8 functional differentiation 35 histoarchitectonic differentiation 35– 37 morphogenetic programming 36 – 37 multiple functional potentials 33 – 34 structural components 37 – 39 subcellular differentiation 34 – 35 ependymoglial cells see ependymal cells epidermal growth factor (EGF) 81 – 82, 525 epidermal growth factor (EGF)-related peptides 202– 203 epidermal growth factor receptor inhibitor (ERI) (neurostatin) 83 epilepsy 677 epithelial cells 218, 221, 223, 269– 287, 731– 734 epithelial growth factor receptor (EGFR) 503, 505– 509 see also erbB signaling ‘equilibrated inositol space’ 1042 ER see endoplasmic reticulum erbB signaling 202– 203, 206 ERK see extracellular-signal regulated kinase

ERM see ezrin/radixin/moesin protein family estradiol 537, 540– 541, 548 estrogen receptor 506, 512 estrogens 549 estrone 537 ethacrynic acid 446 evoked potentials 622– 623 exaggerated pain states 955–956 exaggerated polyol pathway flux 1109– 1111 excitability 935, 983– 994 excitable cellular-automaton model 694 excitotoxicity see also glutamate excitotoxicity calcium waves 676, 677 inflammatory response 891– 892 lactate utilization under stress 400 experimental autoimmune (allergic) encephalomyelitis (EAE) 258, 259, 1065– 1066, 1070–1071, 1072– 1073 experimental hyperammonemic syndromes 988– 990 external zone of the median eminence 202– 203, 204– 205, 209– 210 extracellular ATP 666– 669 extracellular fluid (ECF) 708, 727– 728, 735 extracellular matrix (ECM) 81 – 82, 333, 334, 335 basal lamina 223 meningeal – vascular system 221, 245, 246 peripheral astrocyte processes 157 extracellular potassium 597–598, 611– 630 extracellular space (ECS) 6, 597– 598 see also ventricles extracellular-signal regulated kinase (ERK) 504, 507 extraparenchymal tissue basal lamina 223– 224 cell types 215– 216, 218– 223 cytokines 229– 233 functional syncytium with neural cells 245– 246 growth factors 229– 233 magnocellular hypothalamo– neurohypophysial system 235– 240 role in brain function 215– 247 extrinsic to intrinsic signal transduction pathways 58, 59 eye 286, 353– 355, 1108 see also optic nerve; retina ezrin 160 ezrin/radixin/moesin (ERM) protein family 149– 150

Index

facial nerve transection 953 fatal familial insomnia 1086 ferritin synthesis 862– 863 FGF-1 see fibroblast growth factor-1 FGF-2 see fibroblast growth factor-2 fibers, diabetic neuropathy pathology 1106 –1107 fibroblast growth factor-1 (FGF-1) 524– 525, 563, 565 fibroblast growth factor-2 (FGF-2) brain injury 528 photoreceptor protection 305, 307 PNS regeneration 331 in vivo astrocyte expression 563, 565– 566 fibroblasts 218– 219 ‘fibrous gliosis’ see glial reaction filopodia astrocytes 147, 148– 149 cytoarchitectonics 9 –10 ependymo-astroglia 33, 34, 37 – 39 glycogen as energy reservoir 441 fire-and-diffuse model 694 fish arousal response 622 CNS regeneration and glial reaction 815– 816 slow potential shift changes during habituation 629 fluid dynamics 285– 287 fluorescent probes 662 fluoxetine (Prozac) 454 fractones 223, 240 frogs see anurans fucose 1044, 1045 functional syncytia 166, 167– 168, 612 furosemide 445 G-protein-coupled receptors (GPCRs) 477– 487, 489, 491, 503– 513 G1/G2 cell cycle stage 76 – 77, 81 GABA astrocytic homeostasis 461– 463, 467– 470 metabolism 468– 469 neuronal – astrocytic interactions 848–850 receptors 479, 992– 993, 994 release 462, 463, 467– 468 transporters 462 uptake 463, 468 GABA – glutamate – glutamine cycle 466, 469, 843 galactosylceramide (GalCer) 929 gamma-aminobutyric acid see GABA ganglion cells 297– 298 ganglionic eminences 131 –133

1153

gap junctions astrocytes 166– 167, 441, 599– 600, 792 astroglial cell networks 43, 45 calcium waves 173– 174, 664– 666 connexins 168– 170 meninges 218, 219–221, 246 metabolite distribution through CNS 172–173 modeling of intercellular calcium signaling in astrocytes 699– 702 nerve regeneration 335– 336 neuroprotective role 174– 175 ventricular lining 135 gastrointestinal tract enteric glial cells 315–324 enteric nervous system 315– 324 epithelial and vascular barriers 322, 323– 324 immune responses 321, 323– 324 inflammation 319, 321– 324 GAT1– 4 carriers 468 GCs see guanylyl cyclases GDH see glutamate dehydrogenase GDNF see glial-derived neurotrophic factor gene expression brain development 131– 132 glial reaction 804– 805 gp120-mediated astrocytic dysfunction 938 hypoxia-ischemia 860– 863 peripheral-type benzodiazepine receptor 985– 988 genetic determination 12 – 13 Gerstmann – Stra¨ussler – Scheinker syndrome (GSS) 87, 1086 GFAP see glial fibrillary acidic protein GLAST see EAAT1; glutamate transport glatiramer acetate (Cop-1) 359 glia limitans 297, 299– 300, 309 glia activation 955– 958 Alzheimer’s disease role 883 –894 amyloid plaque association 884, 885 calcium store regulation 635–655 cholesterol homeostasis in the brain 519– 529 cyclic GMP targets and actions 585– 586 depolarization 612– 613, 614, 615 diversity 24 ependymo-astroglia 33 –45 facial nerve transection 953 function 952– 954 HIV-1-associated dementia 902, 910– 913 injury to sensory afferents 954– 956 iron deposition 875– 876 length constants 613 loss 1049– 1056 markers 135– 136, 147– 160

1154

metabolism 409– 428 microglia 24 –28 morphology 952– 954 nervous system role 98 –99 neuron– glial interactions in development 97 – 118 neuronal sensitisation 618, 620 nitric oxide-cyclic GMP pathway 575– 587 oligodendroglia 28 – 33 Parkinson’s disease 967– 977 pathology 159– 160 pH regulation 713– 730 PNS/CNS comparison 333 precursors 81 prion disease 1085– 1099 proportions of CNS 24, 25, 30 5a-reductase and 3a-hydroxysteroid dehydrogenase 538– 540 retinopathies 1117 –1119 retraction 184–186 scarring 333, 334 schizophrenia 1013– 1014 Schwann cell transplants into CNS 338 slow potential shifts 611–630 spatial buffering of potassium 612– 615 steady-state pHi 710 structural plasticity 181– 195 syncytium 165– 176, 599, 601, 612 tumors 160 gliagrana 637 glial fibrillary acidic protein (GFAP) Alexander disease role 779–782 astrocyte differentiation 113– 115 astrocyte structural plasticity 188– 189 astrogliosis 774, 952 brain inflammation 969 function 774– 775 glial activation marker 954–955 human GFAP gene 777– 778 mood disorders and schizophrenia 1054 mutations 779– 783 steroid effect on astroglia 543– 545 glial fibrillary acidic protein-immunopositivity glial reaction 789, 792, 793–794, 796, 797, 798, 818 nonmammalian CNS regeneration 815– 816 glial heme oxygenase-1 869– 879 glial limitans ventral to supraoptic nucleus (SON-VGL) 183, 186– 188, 191 glial reaction 787– 818 age factors 812– 815 anisomorphic phase 790, 798, 809 astrocyte recruitment 795– 796 astroglial phases 798

Index

axon regrowth 809– 818 brain injury 562 ciliary neurotrophic factor 566 cytoskeleton 792– 794 demarcation 809 energy demands of astrocytes 808 feedback effects/balancing 802, 803 functions 805– 809 GFAP-immunopositivity 789, 792, 793– 794, 796, 815–816, 818 humoral regulation 800, 803– 804 hypothalamo– hypophyseal axons 810 immature mammalian CNS, glial reaction 812– 815 inflammatory phase 797– 798, 806– 807 intracellular regulation 804– 805 isomorphic phase 790, 798 lower vertebrates 815– 816 neuroprotection 803, 807– 809 noncytoskeletal protein markers 794 pathological conditions 86 – 87 physiological/clinical importance 789 potassium homeostasis 605 protease systems 806 reactive astrocytes structure/cell junctions 791– 792 regulation 799– 805 scarring 798– 799, 802– 803 sequence/phases 796– 799 tissue destruction 800– 801, 807 types 790– 791 glial restricted precursor (GRP) 54 glial-derived neurotrophic factor (GDNF) 316, 317– 318, 320, 973–974 glial-neuronal interactions 200– 205, 235, 241– 243 glial-neuronal-endothelial interactions 199– 210 glial-targeted metabolic inhibitors 961 glio –meningeal scar formation 814 gliogenesis 140, 801 gliomas 86, 87 –88, 160, 1036 gliosis see also glial reaction definition 300 retinal 300 GLT see EAAT1/EAAT2; glutamate transport GLT-1 see glutamate transport glucocorticoids 537, 544, 551 gluconeogenesis 397 glucose brain concentration 418– 420 glutamate synthesis 840– 842 metabolism 392, 393, 394, 424, 425

Index

oxidation compared to lactate oxidation 396– 397 pentobarbital anesthesia effect on brain content 424– 425, 426 phosphorylation 442– 443 potassium-induced metabolic enzyme stimulation 446 transmitters affecting metabolism in mouse astrocytes 450 glucose transport astrocyte syncytium 172–173 glucose transporter-1 393, 394, 543 glucose transporter-3 393, 394, 401 glucose uptake, activity induced 400– 401 GLUT1/GLUT2 see glucose transport glutamate astrocytic homeostasis 461– 467, 469– 470 calcium wave signaling 663– 664, 672– 675 cycle 152, 400–401 cytotoxicity 976 excitotoxicity 848, 1059– 1079, 1095 –1098 homeostasis 1074– 1078 inhibition 1093– 1094 metabolism 464– 467 neurotransmitter networks 115– 117, 410– 411 receptors 477–479, 1066– 1071, 1088– 1089, 1121 –1124 release 462, 463– 464, 913 sodium ion-driven active uptake 448 synthesis 840– 842 toxicity 353– 357, 891– 892 turnover 422– 423 uptake 463, 464, 808, 912– 913, 930– 931, 932, 933 glutamate dehydrogenase (GDH) 1074 –1076 glutamate transport 462, 463 astrocyte syncytium 172–173 bicarbonate-independent acid-loading transporters 716– 717 EAAT1/EAAT2 464 Mu¨ller cells 1124– 1126 multiple sclerosis 1076 –1078 peripheral astrocyte processes 158 prion disease 1088, 1094– 1095 glutamate – glutamine cycle dynamic isotopomer analysis 416 GABA metabolism 469 glial metabolism 421– 428 glutamate metabolism 410– 411, 465– 466 metabolic astrocytic – neuronal interaction 843 Mu¨ller cells 1124– 1126

1155

glutamatergic neuronal – astrocytic interactions 846– 848 glutaminase 1072– 1074 glutamine transmitters affecting metabolism in mouse astrocytes 450 transport 172– 173 turnover 423–427 in vivo labeling 13C NMR detection 416 glutamine synthetase (GS) enteric glia 318, 320 glial metabolism 423–424 multiple sclerosis 1072, 1074– 1076 pathological conditions 159 peripheral astrocyte processes 151– 156, 158, 159 pyruvate carboxylase 427, 428 retinopathies 1118– 1119, 1125 glutathione 1119, 1125–1126 glycerol-3 phosphate shuttle 397 glycine receptors 480 glycogen 416– 421, 439– 441 glycogenolysis adrenergic stimulation 449– 450 benzodiazepines 453 drugs of abuse 454 metabolic stimulation mechanisms 437– 441 potassium-stimulated 445 serotonin 451 glycolysis activation during increased energy demand 395– 396 astrocytes 394, 397– 398, 808 ATP generation in neurons and astrocytes 397– 398 neurons 394, 397 –398 glycophospholipids 929 glycoprotein gp120-mediated astrocytic dysfunction 921– 939 glycosphingolipid microdomains 929 glycosylphosphatidyl inositol (GPI) 1086 GnRH see gonadotrophin releasing hormone goldfish 815– 816 see also fish Go¨mo¨ri astrocytes 808 gonadotrophin releasing hormone (GnRH) see luteinizing hormone releasing hormone (LHRH) gp120 911, 928– 930 gp120-mediated astrocytic dysfunction adhesion molecules 936 apoptosis 938 arachidonic acid 933 cytokines 934– 935, 936– 937

1156

Index

gene expression 938 glutamate uptake 930– 931, 932, 933 HIV-related 921– 939 mechanisms 930– 938 neuronal excitability 935 pH regulation 930 potassium channels 930, 931– 932, 933 GPCRs see G-protein-coupled receptors GPI see glycosylphosphatidyl inositol grafting see transplantation GRASP (heparin-binding glycoprotein) 66 – 67 gray matter cells 61, 62 ‘growth cones’ 809 growth factors astrocyte – neuron interactions 104 basal lamina 232 cell cycle regulation 76, 81 – 82 cell proliferation 233 choroid plexus stroma 221 crossing blood – brain barrier 226– 228 extraparenchymal tissue 229–233, 245–246 hypothalamo– hypophysial systems 243–244 Mu¨ller cells 1124, 1126 oligodendrocyte development 57 – 58 parenchymal cells 234– 235 production 229, 232, 245– 246 receptors 86, 488 role 229, 246 shedding in response to transactivation 503– 504 T3-dependent modulation 107 growth inhibitors 810– 811, 812, 816–817 GRP see glial restricted precursor GS see glutamine synthetase GSS see Gerstmann – Stra¨ussler – Scheinker Syndrome guanylyl cyclases (GCs) 580 gut see gastrointestinal tract H-K pumps 723– 724 HAART see highly active antiretroviral therapy habituation 629– 630 HAD see HIV-1-associated dementia haloperidol 1042 HAM see Human T-cell leukemia type 1associated myelopathy HB-EGF see heparin-binding EGF-like growth factor HDL see high density lipoprotein HE see hepatic encephalopathy HEK cells 675 heme catabolic pathway 870, 871 heme oxygenase-1 872– 873 dystrophic effects in neural tissues 874

expression 877– 878 glial iron deposition 875– 876 induction 875 glial 875 intraglial oxidative stress 876 mitochondrial permeability transition pore 876 neuroprotective role 873– 874 pathological neuronal – glial interaction model 876– 877 upregulation, mitochondrial iron trapping 875– 876 heme oxygenases 870–872 hemodynamics, choroid plexus 271–272 heparin-binding EGF-like growth factor (HB-EGF) 504, 508– 509, 512 heparin-binding glycoprotein 66 – 67 hepatic encephalopathy (HE) 986– 987 HER family of EGF receptors 509, see also erbB signaling heterogeneous oligodendrocyte phenotype/morphology 53 – 68 hexokinase phosphorylation 413 HIF1 see hypoxia inducible factor 1 high density lipoprotein (HDL) 519, 520, 521, 526– 527 high density lipoprotein-like particles 522, 523, 524 high-affinity myo-inositol uptake systems 1039, 1042 highly active antiretroviral therapy (HAART) 902, 922 hindbrain rhombomeres 131 histamine 452, 482 HIV-1 255– 256, 261, 911, 924– 927 HIV-1 encephalitis (HIVE) 903– 905, 923 HIV-1-associated dementia (HAD) 901– 913 arachidonic acid metabolites 907 astrocytic functions 912– 913 astroglial dysfunction 910– 913 chemokines 907 cytokines 906– 907, 908– 910 envelope proteins 925–926 glycoprotein gp120-mediated astrocytic dysfunction 921– 939 IL-1b effects 909– 910 microglia – astrocyte interactions 908– 910 neurotoxicity 913 platelet activating factor 907 proteases 908 reactive oxygen species 908 tumour necrosis factor-alpha 908– 909 viral proteins 911

Index

HIV-2 922 HIVE see HIV-1 encephalitis HNS see hypothalamo-neurohypophyseal system holistic morphogenetic organization 13 homeostasis arginine vasopressin and water channels 747– 764 astrocytic GABA 461– 463, 467– 470 astrocytic glutamate 461– 467, 469– 470 calcium 575, 586, 636– 637, 654 cholesterol in the brain 519–529 choroid plexus fluid balance 281– 287 enteric glia 318, 320 potassium 441– 447, 595– 605 retina 295– 310 hormonal influences 803– 804 see also sex steroids HPA see hypothalamic– pituitary – adrenal axis HRE see hypoxia-response element 3a-HSD see 3a-hydroxysteroid dehydrogenase HSP27 776, 783 5-HT see serotonin HTLV-I see Human T-cell leukemia type I human glial fibrillary acidic protein (hGFAP) gene 777– 779 human immunodeficiency virus see HIV human inflammatory bowel disease 321, 323– 324 Human T-cell leukemia type 1 (HTLV-1) 256, 261– 262 Human T-cell leukemia type 1-associated myelopathy (HAM) 261–262, 1075 humoral regulation 800, 803– 804 hydrocephalus choroid plexus epithelium 269– 287 functional effects 280– 282, 283– 284 neuroendocrine CSF regulation 281– 287 nNOS effects 286 structural effects 275–280, 282 types and causes 274 hydrogen peroxide 654 24S-hydroxycholesterol 521, 529 6-hydroxydopamine (6-OHDA) 972 3a-hydroxysteroid dehydrogenase (3a-HSD) 538– 540 hyperalgesia 955, 959 hyperammonemia 983– 994 hyperexcitability 598 hypoglycemia 308, 417– 420 hypophysial – parvocellular secretory system 242– 243 hypothalamic– pituitary – adrenal (HPA) axis 244, 1017

1157

hypothalamo– hypophysial system anatomy 235– 241 axonal glial reaction 810 cytokines 243– 244 growth factors 243– 244 ion and water homeostasis 753– 759 magnocellular system 235– 240 neuron – glial interactions 235, 237, 241– 243 neuropeptide CSF regulators 282 parvocellular systems 235, 241, 242–243 hypothalamo-neurohypophyseal system (HNS) 181– 195 hypoxia inducible factor 1 (HIF1) 860 hypoxia effects on choroid plexus 280– 281 extracelluar potassium clearance 443 lactate utilization under stress 400 metabolic protection of photoreceptors 307– 308 Mu¨ller cell –neuron symbiosis 1126– 1127 retinal detachment 300, 308– 309 retinal neovascularization 303– 304, 309 retinal pathology 300, 303– 304, 308– 309 retinal vascular development role 302 hypoxia-inducible responses 860– 861 hypoxia– ischemia 857– 864 hypoxia-response element (HRE) 860 IACW see mathematical modeling, intracellular calcium signaling IBP see isoquinolone carboxamide binding protein ICAM see intercellular adhesion molecule ICC see immunocytochemistry ICP see intracranial pressure IDO see indoleamine 2,3-dioxygenase IFAP see intermediate filament associated protein IGF-I see insulin-like growth factors IICR see IP3R-mediated calcium release IL see interleukins ILM see inner limiting membrane imaging studies 662, 677 see also individual imaging studies immature mammalian CNS 812– 815 immune system anti-depressants 1020– 1021 cells blood – brain barrier penetration 255 glutamate production 1071– 1074 CNS injury responses 351– 353 CNS relationship 348– 349 development/mode of action 1002– 1005 glial cells activation 952, 953

1158

‘innate’ 1000, 1002–1003, 1009 major depression 1001, 1016– 1017, 1020– 1021 mediators in persistent pain treatment 956– 958, 961 protective autoimmunity 347–359 psychoses 999– 1022 schizophrenia 1002– 1015 immunocytochemistry (ICC) 188, 954, 956 immunohistochemistry 147– 160, 1037 immunopathogenesis 1059– 1079 imposed DC shifts 626–629 in vivo metabolic studies 409– 428 indoleamine 2,3-dioxygenase (IDO) 1018– 1019 inflammation Alzheimer’s disease role 883– 884, 886 astrocytic cytokine receptors 487– 488 brain 562, 968– 970 cytokine/chemokine release 259 immune cell recruitment 258 viral entry into CNS 261 CNS injury 347, 349– 351 glial reaction inflammatory phase 797– 798, 806– 807 glial role in degenerative disease 886– 887 role in healing 357– 358 information transfer 595, 596 inherited CJD 1086 injury responses astrocytes 86, 87, 492, 885 brain apoE synthesis and secretion 524, 527– 528 central nervous system 329– 330, 332– 334 glial activation 954– 956 glial reaction 787– 818 glial steroidogenesis 540– 541 glutamate toxicity protection 353–357 microglia 347, 885 peripheral nervous system 329–332 polyol pathway and nerve 1107– 1109 reactive gliosis 562 sensory afferents 954– 956 INK4 family CKIs 79 – 80, 88 ‘innate’ immune system 1000, 1002– 1003, 1009 inner limiting membrane (ILM) 299, 300 iNOS see nitric oxide synthase, NOS2 inositol anti-bipolar drug effects 1036– 1042 content/formation 1036– 1037 formation from IP 1035 pools 1038, 1040– 1042

Index

transporters 1037– 1038, 1039 uptake inhibitors 1044, 1045 uptake systems 1037– 1038, 1040– 1042 inositol monophosphatase 1036 inositol-1,4,5-trisphosphate (IP3) 693, 697– 703, 1034, 1035 inositol-1,4,5-trisphosphate receptor (IP3R) 640, 641–642, 645, 690– 691, 694– 699 insulin-like growth factors 546, 563, 565 integrins 81 – 82, 85 intercellular adhesion molecule-1 (ICAM-1) 258– 260, 936 intercellular calcium signaling 699– 702 intercellular calcium wave propagation 664– 666, 673 intercellular clefts 6 intercostal nerve segments, transplantation of 350 interdigitation 36, 41 interfascicular oligodendrocytes 65 interferon gamma 1018, 1019, 1021 interkinetic nuclear migration 129, 130 interleukins (IL) anti-depressants 1021 astrocytic cytokine receptors 487– 488 IL-1, gp120-mediated astrocytic dysfunction 937 IL-1b, HIV-1-associated dementia 909– 910 IL-6 anti-psychotic therapy 1010 central nervous system 1002 major depression 1016–1017 pain 959 schizophrenia 1000, 1009 major depression 1016– 1017 microglia in HIVE 906 pain 958– 960 schizophrenia 1000, 1009, 1010– 1011 intermediate filament associated protein (IFAP) 794 interstitial fluid 707– 735 interstitial partial pressure of CO2 (PCO2) 712– 713 intracellular calcium 173– 174, 637– 647, 690– 699, 1042– 1044 intracellular pH 707–710, 711 intracellular proteins 970 intracellular receptors 488– 489 intracellular regulation 804– 805 intracerebral vascularization 6, 14, 18– 22 intracranial pressure (ICP) 281– 283, 287 see also hydrocephalus; ventricular system

Index

intraglial oxidative stress 876 intramitochondrial calcium concentration 437– 439 intraventricular pressure see hydrocephalus; intracranial pressure; ventricular system intrinsic signalling pathways 58 invertebrates 3 inwardly rectifying potassium channels 602 iodoacetate 443 ion channel-coupled membrane receptors 477– 487 ion effects on astrocytic energy metabolism 441– 448 ion transport arginine vasopressin 747– 764 brain barrier functions 759– 764 cerebrocortical astrocytes 749– 750 choroid plexus 272–274, 281, 283, 285 gap junctions 166– 167 non-neuronal parenchymal cells 749– 759 ionotropic receptors 478– 479, 483, 1066– 1071 IP3 see inositol-1,4,5-trisphosphate IP3R see inositol-1,4,5-trisphosphate receptor IP3R-mediated calcium release (IICR) 640– 642 IRCW see mathematical modeling, intercellular calcium signaling iron deposition 875–876 ischemia astrocytes, non-MRS studies 844– 845 13 C magnetic resonance spectroscopy studies 845– 846 effects on choroid plexus 280 GABA release 467– 468 GABAergic neuronal – astrocytic interactions 848– 850 glutamatergic neuronal – astrocytic interactions 846– 848 neuronal dysfunction, astrocytes contribution 837– 850 ischemic core 846– 847 isomorphic glial reaction 790, 798 isoquinolone carboxamide binding protein (IBP) 985– 990, 994 isotopomer methods see dynamic isotopomer analysis Janus-faced Mu¨ller cells 1127– 1128 K+ see potassium kainate (KA) 1066– 1070, 1072– 1073, 1121 –1124 kernicterus 874 a-ketoglutarate 415, 416, 426 kidney 270, 285– 286

1159

Kir channels 1117– 1120, 1127– 1129 Kolmer cells 278 Kuru 1086 kynurenine metabolism 1013– 1014 L-channels 445, 454 lactate astrocytic metabolism 391– 403 oxidation compared to glucose oxidation 396– 397 role as energy substrate in brain 402– 403 transport, astrocyte syncytium 172– 173 lactate dehydrogenase (LDH) 395– 396, 399, 402, 1118– 1119 lactation 183– 186, 188 lamellipodia 9 – 10, 33, 34, 37 – 39, 441 laminin 798 lateral ventricle 141 LDH see lactate dehydrogenase LDL see low density lipoprotein learning 439– 440, 449– 450 leech 612, 713, 720, 725, 726 length constants 600, 601, 613 leukocytes 258– 259 leukoencephalopathy 777 LHRH see luteinizing hormone releasing hormone Li– Rinzel model 691, 693, 697 ‘light’ epithelial cells 280, 284– 285 lipopolysaccharide (LPS) 955, 974 lipoproteins 519, 521– 524, 526– 527 see also high density...; low density... lithium astrocyte phosphoinositide system effects 1033– 1045 inositol formation inhibition 1035– 1036 inositol pool size 1040, 1041 transmitter-induced intracellular calcium changes down-regulation 1043 lithium– pilocarpine test 1044, 1045 liver failure 983, 984, 986– 990 low density lipoprotein (LDL) 521 low-affinity uptake system 1039, 1042 lower vertebrates 3, 815– 816 luteinizing hormone releasing hormone (LHRH) 199– 210, 545– 547 macroglia cytoarchitectonics 24 ependymo-astroglia 33 –45 oligodendroglia 28 – 33 retinal stability 295– 310 retinopathies 1117– 1119

1160

Index

macrophages see also Kolmer cells HIV-1 encephalitis 903– 905 injury responses 331, 333 meningeal/perivascular network 218– 219 nerve regeneration role 340 PNS regeneration 350 spinal cord injury 351 Magistretti– Shulman hypothesis see astrocyte – neuron lactate shuttle hypothesis magnetic resonance studies (MRS) 409–428, 844, 845– 846 magnocellular hypothalamo –neurohypophysial system (mHNS) 235– 242 magnocellular neuroendocrine cells (MNCs) 182– 188 major depression clinical significance 1000 cytokines 1012, 1016– 1017 glial loss 1049– 1054 immune activation 1001, 1016– 1017 melancholic versus non-melancholic 1019 norepinephrine 1017 ‘sickness behaviour’ similarity 1015– 1016 suicidality 1018– 1019 malate– aspartate shuttle 397, 422–423 malic enzyme 843 manic– depressive illness see bipolar disorder MAPK/ERK pathway 1093 massed unit activity 625 mathematical modeling 690– 702 mature mammalian CNS, glial reaction 809– 812 MBP see myelin basic protein MCT see monocarboxylate transporter median eminence 199– 210 megalencephaly 775 melancholic depression 1019 see also major depression memantine 1070 memory 439– 440, 449– 450 see also learning meninges anatomy 217, 218– 221 cell types 218 extraparenchymal – neural functional syncytium 245– 246 gap junctions 218, 219– 221, 245– 246 infection 255– 256, 262– 263 neural plasticity role 215– 247 perivasculature 219 pial microvessels 221 projections in brain 218– 221 signaling molecule production 245

mesenchymal tissue surfaces 36 metabolic astrocytic – neuronal interaction 840– 844 acetate metabolism 843– 844 13 C magnetic resonance spectroscopy (MRS) analysis 844 GABA – glutamate – glutamine cycle 843 glutamate synthesis from glucose 840– 842 glutamate – glutamine cycle 843 metabolic fluxes, measurement of absolute 415– 416 metabolism astrocytic energy 435– 455 inhibitors in pain suppression 961 ion effects 441– 448 lactate released from astrocytes 391– 403 neuro-glial 409– 428 steroid hormone 536– 541 stimulation mechanisms 437– 441 metabolite exchange at gap junctions 166– 167, 172– 173 metabotropic glutamate receptors (mGluRs) astrocytic 478 astrocytic transactivation receptor selectivity 510– 511 bidirectional neuron – astrocyte coupling 702– 703 GPCR transactivation 506 multiple sclerosis 1066, 1071 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 972, 973 N-methyl-D -aspartate (NMDA) Mu¨ller cells 1121– 1124 multiple sclerosis 1066– 1067, 1070– 1071 prion disease 1088– 1089, 1094– 1098 receptor ATP release 672 synaptic transmission 675 3,4-methylenedioxymethamphetamine (‘ecstacy’) 454 MGCs see multinucleated giant cells mGluRs see metabotropic glutamate receptors Michael reaction acceptors 862 microglia see also glia Alzheimer’s disease 883– 894 amyloid b-peptide activation 887– 888 beneficial activation 893 brain inflammation 969 capacitative calcium entry 651 cytoarchitectonics 24 –28 cytokines 906– 907 distribution 25

Index

dopaminergic transmission in schizophrenia 1013 –1014 feedback effects on glial reaction 803 functions 26 – 27 glutamate buffering 356 glutamate release 891 HIV-1 encephalitis 904– 905 HIV-1-associated dementia 902– 903, 906– 910 injury responses 351, 492, 493, 885 mobility 26 morphology/function 952– 953 multiple sclerosis 1067, 1077 nerve regeneration 333 neurotoxicity 887, 1088– 1090 origins 298 persistent pain role 951– 962 prion disease 1085, 1087– 1088 proliferation 27 protective autoimmunity 347– 359 retina 298 retinopathies 1117– 1119 spacing 25 – 26 steroid effects 548 structural plasticity 192–195 T-cell interaction 355– 358 tumour necrosis factor-alpha 906 microglia – astrocyte interactions 908– 910, 1090 –1094 microscopy, confocal scanning 662 migration of neurons 99 – 103 mitochondria benzodiazepine receptors see peripheral-type benzodiazepine receptors calcium storage 646– 647 calcium wave signaling 671– 672, 677– 678 alpha-cyano-4-hydroxycinnamate effect 400 function, hyperammonemic PTBR changes 991 intracellular calcium signaling modeling 691– 692 iron trapping, heme oxygenase-1 upregulation 875– 876 permeability transition pore, heme oxygenase-1 876 receptors, astrocytic 489 mitosis 76, 129– 130, 795 MNCs see magnocellular neuroendocrine cells models see also mathematical modeling intracellular calcium signaling 689– 703 multiple sclerosis 1065 –1066 Parkinson’s disease 972– 973

1161

pathological neuronal – glial interaction 876– 877 vulnerability – stress model 1000 modulation of neuronal activity 173– 174 molecular networks 12 monoaminergic neurons 489 monocarboxylate transporter (MCT) 398, 724 monocytes 258, 261, 1013 mood disorders 1049– 1056 see also bipolar disorder; major depression mood-stabilizing drugs 1034, 1036, 1040– 1045 morphine treatment 958, 962 morphogenesis 12 – 14, 202– 203, 204– 205, 209– 210, 492– 493 morphology glial cells 952– 954 HIV-1 virion 924– 926 oligodendroglia heterogeneity 28 –29 structural dynamics 10 motivation 624– 626, 627– 628 motor neuron degeneration 104– 105 mouse models 450, 778– 779, 973 MPTP see 1-methyl-4-phenyl-1,2,3,6tetrathydropyridine MRS see magnetic resonance studies mucosal gastrointestinal barrier 321 Mu¨ller cells blood – retina barrier 1127 cytoarchitectonics 33, 35, 36, 37 cytokines 1124 development 8 differentiation into astrocytes 130 energy metabolism 1126– 1127 ependymal cells relationship 128, 133– 134 function 298– 299, 1117– 1120 glia limitans structure 299– 300 glutamate –glutamine cycle 1124– 1126 hypoxic response 300 metabolic support for photoreceptors 307– 308 neurogenesis 129– 131 neuron migration 99– 103 neuron – glial interactions 99 – 104 neuronal morphogenesis 103– 104 neuronal precursors 100– 101 Notch pathway 111 NOVOcan protein 137– 138 photoreceptor protection factors 305– 307, 309 potassium siphoning 170– 172 reactive 1121– 1124 retinopathies 1117– 1129 spatial buffering 599 stabilizing role 297

1162

Index

transformation to astrocytes 104 vascular development 302 ventricular zone relationship 129– 130 wrapping photoreceptors 304 multicellular networks 11 multifunctional potential 13 – 14 multinucleated cells see syncytia multinucleated giant cells (MGCs) 902, 903, 923, 927 multiple sclerosis (MS) animal models 1065– 1066 disease classification 1062 disease progression 1059 –1062 glutamate excitotoxicity 1059– 1079 glutamate homeostasis 1074– 1078 glutamate receptors in white matter 1066– 1071 glutamate transporters 1076– 1078 immunopathogenesis 1059–1079 lesion classification 1062– 1065 leucocyte infiltration 255– 256, 258–259 sources of excess glutamate 1071– 1074 multiple sclerosis lesions, acute 1063 muscarinic receptors 483 myelin axonal differentiation role 65 – 66 diabetic neuropathy 1107, 1110 gene transcription 57– 58 multiple sclerosis 1059– 1061, 1062– 1065, 1069, 1070 oligodendrocytes 56, 57 – 59, 65 – 68, 1049, 1055 peptides, immune response 352, 358– 359 process of myelination 56, 60 –61 reconstructing optic nerve 337, 341 regeneration 66 – 68, 1064– 1065 regeneration inhibition 333 related growth inhibitors 350 Schwann cells 549– 550 transcription factors 60 – 61 myelin basic protein (MBP) 57, 60, 61 myo-inositol 1038– 1039, 1042 N-cadherins 792 Na+/K+-ATPase blood– brain barrier 596– 597 capillary endothelium 759– 760 choroid plexus 597, 761, 762 diabetic neuropathy 1109– 1110 metabolic stimulation mechanisms 437–438 potassium uptake in astrocytes 604, 749 Na+, K+, 2Cl– cotransporter capillary endothelium 760– 761 choroid plexus 761– 762

ion effects on metabolism 437, 442, 445– 446, 455 potassium ion clearance from extracellular space 750, 752 Na-driven Cl-bicarbonate exchanger (NDCBE) 730 Na– H exchangers (NHEs) acid-extruding bicarbonate-independent transporters 719–722 capillary endothelial cells 734– 735 choroid epithelial cells 731– 732, 733 gp120-mediated dysfunction 930– 932 Na/Bicarbonate Cotransporters (NBCs) bicarbonate-dependent acid-extruding transporters 725–729 bicarbonate-dependent acid-loading transporters 719 oligodendrocytes 727 pHi and pHECF changes 728 NADH shuttles 394, 397 NADPH-oxidase 975 NAWM see normal appearing white matter NBCs see Na/Bicarbonate Cotransporters NDCBE see Na-driven Cl-bicarbonate exchanger necrosis 512 Necturus 612 Neisseria meningitidis 263 neovascularization 303– 304, 309, 1126– 1127 nerve damage see injury nerve growth factor (NGF) 564 nerve regeneration antibody therapy 359 CNS in glial reaction 809– 818 CNS hostile environment 330, 333, 350 CNS/PNS comparison 329– 330 failure 817– 818 glial effects 333– 334, 338– 341 glial reaction 787– 818 lower vertebrates 815– 816 optic nerve 329– 342 postmyelinating oligodendrocytes 66 – 68 Schwann cell role 329– 342 steroid effects on astrogliosis 547 nestin 793, 796 networks 11 – 12, 165– 176 neural pain mediators 956– 958 neural plasticity 215– 247 neural tube development 129 neuregulins 102, 202 neuritic plaques 884– 886, 887– 888 neuroactive steroids 535– 552 neurodegenerative disorders see also Alzheimer’s disease

Index

Alexander disease 773– 783 brain inflammation 968– 970 glial cell cycle activation 87 glial heme oxygenase-1 induction 878 HIV-associated dementia 901– 913 new variant CJD 1086 Parkinson’s disease 877– 878, 967– 977 therapeutic vaccination 357– 358 neurodevelopmental schizophrenia hypothesis 1007 –1008 neuroendochrine regulation cytokine relationship 235 magnocellular hypothalamo– neurohypophysial system 235– 240 neuron –glial interactions 241– 243 parvocellular hypothalamo– adenohypophysial system 241 steroid effects on astroglia 545– 547 neuroepithelium cytoarchitectonics 4 –5 development transformations 6 oligodendrocyte lineage 55 stem cells 6 neurogenesis see also development astrocyte – neuron interactions 104, 105 radial glia role 129–131 subventricular zone 141 neuroglia see glia neuromyelitis optica 1065 neuron-derived growth factors 112– 115 neuron– glial interactions astrocytes 104–117 astrogliogenesis 111– 112 behaviour 611–612 hypothalamo– hypophysial systems 235, 237, 241– 243 nervous system development 97 – 118 neuron migration 99 – 103 neuronal morphogenesis 103–104, 106 pathological, model 876– 877 prion disease 1094– 1098 radial glia 99 – 104 thyroid hormone actions 107– 110 neuronal – endothelial signaling, GnRH release 206– 210 neurons activity modulation, astrocyte syncytium 173– 174 astrocyte proliferation regulation 86 astrocytic interactions 551, 672– 675, 702– 703, 1094–1098 compartments 147– 148 death, CNS injury 332

1163

excitability 935, 983– 994 gp120 effects 935 hyperammonemia 983– 994 ischemia-induced dysfunction, astrocytes contribution 837– 850 lactose/glucose utilization 392– 400, 401– 402 migration, ventricular zone 131 morphogenesis, neuron– glial interactions 103– 104, 106 myelin role 65– 66 myelination 56 – 58 oligodendrocyte communication 65 – 66 pH effects on activity 711–712 pHECF 711– 713 polarity, astrocyte – neuron interactions 106 population maintenance 104– 105 precursors, radial glia 100– 102, 130 processing, extracellular potassium 598 radial migration 99 – 103 responsivity, imposed DC shifts 626– 627 role 285– 287 neuropathology HAD 923 HIVE 903, 904 neuropathy see diabetic neuropathy neuropeptides 452 neuropil 401 neuropoietic cytokines 563, 566– 567 neuroprotection astrocyte syncytium 174– 175 astrocytic transactivation 503– 513 astroglia 803 brain glycogen during hypoglycemia 417– 418 glial reaction 807– 809 heme oxygenase-1 873– 874 injury therapy approach 349, 351, 358– 359 steroid effects on astroglia 547–548 neurostatin see epidermal growth factor receptor inhibitor neurosteroids 536, 991– 993 neurosupport cells 315–324 neurotoxicity 913, 1088– 1090 neurotransmission astrocyte differentiation 115– 117 astrocytic GABA homeostasis 461– 463, 467– 470 astrocytic glutamate homeostasis 461– 467, 469– 470 IL-6 effects 1002 major psychoses 1000 transmitter effects on astrocytic energy metabolism 436, 448– 451

1164

Index

transmitter-induced calcium down-regulation 1043 neurotrophic factors CNS neuron death 332 enteric glia 317– 318, 320 expression on astrocytes in vivo 561, 562, 563, 564– 566, 568– 569 nerve regeneration 331 Schwann cells 335 in vivo astrocyte expression 563, 564– 565 neutrophils 1072 new variant Creutzfeld –Jakob disease (nvCJD) 1086 Nexus multiprotein complex 665– 666 NF-kappa-B see nuclear factor-kappa-beta NGF see nerve growth factor NHEs see Na – H exchangers nicotinic cholinoreceptors see ionotropic... nifedipine 445 nitric oxide (NO) calcium wave signaling 669– 670 formation in glial cells 576– 579 GnRH release 207– 210 gp120-mediated astrocytic dysfunction 937 signaling, enteric glia 318– 319, 320 nitric oxide synthase (NOS) calcium-dependent isoforms 576– 578 endothelial– neuronal communication 207– 209 NOS1 285– 287, 578– 580 NOS2 induction and regulation 578– 579 NOS3 immunoreactive astrocytes 578 role in choroid plexus 285– 287 nitric oxide-cyclic GMP (NO-cGMP) pathway 575– 587 nitric oxide-sensitive guanylyl cyclase (sGC) 580– 583 nitrous oxide (NO) 1123, 1126 NMDA see N -methyl-D -aspartate nNOS see nitric oxide synthase, NOS1 NO see nitric oxide... NO synthase (NOS) 975 NO-cGMP see nitric oxide-cyclic GMP non-mammalian nervous systems 3, 614– 624, 629, 815– 816 non-melancholic depression 1019 non-neuronal parenchymal cells 749– 759 non-phenomenological modeling 693 non-radial glioblasts 8 noncytoskeletal protein markers 794 nonmyelinating oligodendrocytes otmp gene 61 –63 perineuronal 61

phenotype description 56, 61 precursors 65 ‘nonpermissive’ reactive glia 811, 812, 814 noradrenaline astrocytic receptors 480– 481 developmental role 492 major depression 1017 phosphoinositide system 1034– 1035 transmitter effects on metabolism 437, 449– 451 nordidemnin 1044, 1045 normal appearing white matter (NAWM) 1063, 1064 NOS see nitric oxide synthase Notch pathway 111– 112 novocan gene 67 – 68, 137– 138 NOVOcan protein 67 –68, 137–138 nuclear factor-kappa-beta (NF-kappa-B) 87, 960, 976 nuclear receptors, astrocytic 488 nvCJD see new variant Creutzfeldt – Jakob disease octadecaneuropeptide (ODN) 984, 990 6-OHDA see 6-hydroxydopamine olfactory epithelium 4 – 5 OLGs see oligodendrocytes oligodendrocyte progenitor (OLP) 55, 57 Oligodendrocyte Trans Membrane Protein (OTMP) 56, 62 – 63 oligodendrocyte – astrocyte coupling 169– 170 oligodendrocytes (OLGs) see also glia calcium release mechanism 645– 646, 647 cell distribution 32 – 33 cell processes 29, 30 cell shaping 29, 31 cytoarchitectonics 24, 28 – 33 development 31 –32 extrinsic to intrinsic signal transduction pathways 58, 59 ferritin synthesis 862– 863 function 1055 glial loss and mood disorders/schizophrenia 1049, 1054– 1055, 1056 glutamine synthetase 155– 156 hypoxic– ischemic stresses vulnerability 857– 864 injury response 795 morphology/origin links 63 – 65 multiple sclerosis 1059– 1060, 1062– 1064, 1067, 1068– 1070, 1075– 1078 myelin role 54 myelinating phenotypes 56 –61

Index

myelinogenesis transcription factors 60 – 61 Na/Bicarbonate Cotransporters 727 neuron communication 65 – 66 neuron connections 31 neuron –glial interactions 111 nonmyelinating phenotypes 56, 61 – 63 origins 54, 55 Parkinson’s disease 974 phenotypical/morphological heterogeneity 53 – 68 polymorphism 28 – 29 precursors 55, 57, 65 regeneration 66 – 68, 137, 333 Schwann cell transplant influence 338–341 steroid effects 548–549 subventricular zone origins 140 olivopontocerebellar atrophy 1074 OLM see outer limiting membrane OLP see oligodendrocyte progenitor ontogenesis 107– 110 open injuries 797, 798 opioid receptors 484– 485 opioids 958, 962 optic nerve 329– 342, 352 organelles 637–647 see also mitochondria organum vasculosum lamina terminalis (OVLT) 754 orienting behaviour 621, 622, 624, 625 ornithine transcarbamylase (OTC) 984, 990 osmotic theory of cataractogenesis 1108 OTC see ornithine transcarbamylase OTMP see Oligodendrocyte Trans Membrane Protein otmp gene 61 – 63, 68 outer limiting membrane (OLM) 299– 300 OVLT see organum vasculosum lamina terminalis OX see oxytocin oxidative metabolism glial NMR 421– 422, 425, 427, 445, 446 glucose vs lactate 396– 397 oxidative stress astrocytes vulnerability 858– 860 diabetic neuropathy 1111, 1112 glial heme oxygenase-1 induction 875 intraglial heme oxygenase-1 876 oligodendrocytes vulnerability 858– 860 prion disease 1087– 1090, 1091– 1092, 1099 retinopathies 1121– 1123 2-oxoglutarate see a-ketoglutarate oxytocin (OX) 182, 183, 185, 191

1165

P2Y receptor 506 p27Kip1 79 – 88 p53 87, 88 P450 aro see aromatase PAF see platelet activating factor pain, persistent 951– 962 palisade-like arrangement 790, 796, 809 PAP see peripheral astrocyte processes paranodal abnormalities 1107 paraventricular nucleus (PVN) 182–183, 237– 240, 753, 754 parenchymal cells 218, 234– 235 Parkinson’s disease (PD) animal models 972– 973 astrocytes/microglia alterations 970– 971 deleterious effects 974– 976 protective effects 973– 974 cytokines 975– 976 definition 968 glial cell activation initiation 970 glial cells role 967– 977 glial-derived neurotrophic factor 973–974 heme oxygenase-1 expression 877– 878 oligodendrocytes 974 pathological features 971 paroxetine (Paxol) 454 PARs see protease-activated receptors parvocellular hypothalamo– adenohypophysial system 241, 242– 243 pathology calcium waves 676– 678 diabetic neuropathy 1106– 1107 enteric glial cells 319, 321– 324 glial cell cycle regulation 86 – 87 infiltration of brain endothelium 255– 264 neuronal-glial interaction model, heme oxygenase-1 876–877 Parkinson’s disease 971 retina 300, 303– 304, 308– 309 syncytium formation 166 Paxol see paroxetine PCO2 see interstitial partial pressure of CO2 PD see Parkinson’s disease PDEs see phosphodiesterases PECAM-1 (adhesion molecule) 258– 259, 260 penetrative injuries 86, 87, 566– 567 pentobarbital anesthesia 424–425, 426 pentoxyfylline 961– 962 penumbra 847–848 peptide receptors 281– 287, 452, 485– 486 pericytes 256 perineuronal oligodendrocytes 54, 61, 65 perineuronal satellites 32

1166

Index

peripheral astrocyte processes (PAP) 147– 160 additional labeling 158– 159 ezrin/radixin/moesin protein family 149–150 functional aspects 151–155 glutamine synthetase 151– 156 glycogen 441 pathology 159– 160 preferential labeling 157– 158 structure 148– 151 peripheral nervous system (PNS) enteric glial cells 315– 324 implants, optic nerve regeneration 334– 335 nerve transplants into CNS 349, 350 regeneration 329– 332 steroid hormones 535– 536 trauma, central glial activation 954–956 peripheral-type benzodiazepine receptors (PTBR) 453 congenital hyperammonemia 990 experimental hyperammonemic syndromes 988– 990 gene expression 986– 988 mitochondrial receptors 489 steroidogenesis in hyperammonemia 991– 993 subunits 985, 986 upregulation in hyperammonemic syndromes 983– 994 perisynaptic astrocyte processes 147, 148, 152– 154 perivascular astrocyte processes 152– 154 perivascular oligodendrocytes 65 perivascular tissue anatomy 217, 219 basal lamina 223– 224 cell types 218, 233 CNS plasticity mediation 215– 247 permeability 751, 752, 761 ‘permissive’ reactive glia 811, 814 persistent pain 951– 962 therapeutic targets 960–962 pH regulation capillary endothelial cells 734– 735 cerebrospinal fluid 708– 709, 731– 735 choroid epithelial cells 731– 734 extracellular fluid 708, 711– 713, 727– 728, 735 general cellular activity 710– 711 glial cells 713– 730 gp120-mediated astrocytic dysfunction 930 intracellular 707– 710, 711 neuronal activity 711– 712 non-neuronal brain cells and interstitial fluid 707– 735

phagocytosis 805– 806, 807, 969 phenomenological modeling 693– 694 phorbol ester-induced HB-EGF shedding 505 phosphatases 76 – 78, 86 phosphatidyl 4,5-bisphosphate (PIP2) 1034, 1035– 1036 phosphodiesterases (PDEs) 583– 584 phosphoinositide system 448–449, 475– 493, 1033– 1045 phosphorylation 442, 443 photoreceptors lactate/glucose utilization 398– 399 macroglial stabilization 304–308 metabolic protection 307– 308 protection factors 309 vulnerability 298 pia 218, 221 PIP2 see phosphatidyl 4,5-bisphosphate pituicytes see also posterior pituitary arginine vasopressin-induced calcium elevation 754 endothelin reversion of stellation 758– 759 stellation 756, 757, 758– 759 structural plasticity 186, 188, 189– 191 PK see pyruvate kinase PK11195 985 PKG see protein kinase G plaque progression 884– 885 plasma membrane Ca2+-ATPase (PMCA) 715– 716 plasticity mediation 215–247 platelet activating factor (PAF) 907 plectin 794 PLP see proteolipid protein PMCA see plasma membrane Ca2+-ATPase PNS see peripheral nervous system Po gene expression 550 polarized immune response 1003– 1005, 1013 polyneuropathy see diabetic neuropathy polyol pathway 1107– 1111 portacaval shunting 988– 989 posterior pituitary astrocyte proliferation 189– 191 astrocyte structural plasticity 181, 182, 184, 186, 188 glial retraction 184, 186 microglial structural plasticity 193 postmyelinating oligodendrocytes 66 –68 potassium brain activation and extracellular concentration 436 cerebrocortical astrocytes 749– 750, 751 channels 930, 931–932, 933

Index

glia-derived slow potential shifts in relation to behaviour 611– 630 homeostasis 318, 320, 441– 447, 595– 605 ion siphoning, astrocyte syncytium 170– 172 motivation 624– 626 spatial buffering 923 spreading depression 444 transient storage sites 603– 605 pRb see retinoblastoma tumor suppressor protein prebeta-HDL-like particles 523 precapillary vessels 17, 18 pregnancy, schizophrenia 1007– 1008 pregnenolone 537, 992 prey-catching behaviour 617– 621, 624, 626, 627– 629, 630 primary progressive multiple sclerosis 1062 prion disease glia 1085– 1099 glial cell cycle activation 87 microglia – astrocyte interactions 1090 –1094 neurone– astrocyte interactions 1094– 1098 toxicity mechanism 1088– 1090 unified cell theory 1098– 1099 pro-inflammatory cytokines Parkinson’s disease 975– 976 persistent pain 958– 960, 961 in vivo astrocyte expression 563, 567 Probst-bundles 814– 815 process formation 9 progesterone 537, 538, 539, 550 progressive multifocal leukoencephalopathy 87 proinflammatory agents 887– 890 proliferative vitreoretinopathy (PVR) 1120, 1122 –1123, 1127– 1128 propentofylline 961– 962 prostaglandins 975, 1016, 1021 prostanoid receptors 486 protease receptors 486 protease systems 806 protease-activated receptors (PARs) 486, 511 proteases 908 protein kinase cascades 58 protein kinase G (PKG) 669– 670 protein markers 794 proteoglycans 67 – 68, 811, 885– 886 proteolipid protein (PLP) 57, 60 Prozac see fluoxetine psychomotor retardation 775 psychoses 999– 1022 PTBR see peripheral-type benzodiazepine receptors purine receptors 452, 483–484, 666–667, 678 PVN see paraventricular nucleus

1167

PVR see proliferative vitreoretinopathy pyruvate carboxylation 427, 428, 466, 467 pyruvate kinase (PK) 1118– 1119 quarantine analogy, amyloid plaque 884– 886 radial migration 99 – 103 radial unit hypothesis 131 radioligands 413, 414, 415, 985 reactive glia see glial reaction reactive Mu¨ller cells 1121 –1124 reactive nitrogen species (RNS) 975 reactive oxygen species (ROS) HIV-1-associated dementia 908 Parkinson’s disease 974, 975 prion disease 1087– 1090, 1091–1092, 1099 receptor tyrosine kinases (RTKs) 81 –82, 504 recovery capability 280 5a-reductase (5a-R) 538– 540 Reelin pathway 102– 103 regeneration see axons; nerve regeneration regulation astrocyte cell cycle progression 75 – 89 enteric glia 317– 319 glial reaction 799– 805 relapsing – remitting multiple sclerosis 1062 ‘remote glial reaction’ 791 reproductive hormones 199– 210, 537– 539, 542, 545– 547 retina detachment 308–309, 1127– 1128 glia limitans 297, 299– 300 ischemia 1124– 1125 macroglial stability role 295– 310 neovascularizing diseases 303– 304, 309 neuron – glial interactions in development 112– 113 pathology 300, 303– 304, 308– 309 photoreceptor protection factors 304– 305 potassium ion siphoning 170– 172 stress conditioning 304– 307 structure 296 vascular development 301– 302 vascular stability 303– 304 retinal ganglion cells (RGCs) 330, 337– 338, 354– 355 retinal pigment epithelium (RPE) 296, 298, 299– 300, 1127 retinitis pigmentosa 309 retinoblastoma tumor suppressor protein (pRb) 78 –79, 83, 84, 88 retinopathies hypoxia-induced 303 Mu¨ller cells 1117– 1129

1168

Index

blood– retina barrier 1127 cytokine production 1124 energy metabolism 398–399, 1126– 1127 functions 1117– 1120 glutamate – glutamine cycle 1124– 1126 reactive 1121– 1124 retinopathy of prematurity 309 Reye syndrome 983, 984 RG (radial glia) see Mu¨ller cells RGCs see retinal ganglion cells rheumatoid arthritis 1011 ‘the right place at the right time’ see structural plasticity del Rio-Hortega, P. 53, 54, 63, 65 RNS see reactive nitrogen species ROS see reactive oxygen species Rosenthal fibers 773, 775–778 RPE see retinal pigment epithelium 5a-R see 5a-reductase RTKs see receptor tyrosine kinases RyR (ryanodine receptor) 640, 642– 644, 645 S-100 652– 653 S100b marker 158 sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) 638– 639, 671 scalp lipid signals 416 scarring 798– 799, 802– 803 schizophrenia B cells 1014– 1015 blood– brain barrier 1008 celecoxib therapy 1015 clinical significance 1000 cytokines 1005– 1015 cytomegalovirus 1007 dopaminergic neurotransmission 1001, 1013– 1014 glia cells 1013– 1014 glial loss 1049– 1056 immune system dysbalance 1005 ‘innate’ immune system 1009 interleukins 1009, 1010– 1011 neurodevelopmental hypothesis 1007– 1008 rheumatoid arthritis connection 1011 type 1/type 2 immune response 1010– 1011 viral hypothesis 1006– 1007 Schwann cells anion exchange activity 718 cytoarchitectonics 3 diabetic neuropathy 1105– 1112 enteric glia comparison 316– 317 myelinating reconstructed optic nerve 337, 341 neuron– glial interactions 111

normal signaling relationships 331 optic nerve regeneration 329–341 peripheral nervous system injury 330– 332 steroid effects 548– 551 steroidogenesis 538 tight/gap junctions with nerve axons 335– 336 transplantation 334– 341 sciatic nerve 527– 528 second messenger systems 448– 449, 475– 493, 1033– 1045 secondary degeneration 349, 351– 359 secondary progressive multiple sclerosis 1062, 1065 secondary tissue damage 805– 807 secreted b-amyloid precursor protein (sAPP) 888– 890, 891 segmental demyelination 1106 ‘self’ recognition 352 self-antigens 351–358 sensory afferents 954– 956 sensory neuronal activity 614– 621 SERCAs see sarco(endo)plasmic reticulum Ca2+-ATPases serotonin (5-HT) astrocytic receptors 482 developmental role 492 5-HT2A receptor 506 major depression 1017, 1018 phosphoinositide system 1034– 1035 receptors 482 transmitter effects on metabolism 450, 451– 452 sex steroids 199–210, 537– 539, 542, 545– 547 SFO see subfornical organ sGC see nitric oxide-sensitive guanylyl cyclase sgp130 1009 shadow plaques 1064 sICAM-1 see soluble intercellular adhesion molecule 1 ‘sickness behaviour’ 1015– 1016 signal exchange, gap junctions 166– 167, 173– 175 signaling calcium in glial cells 640, 641 calcium waves 199– 200, 661– 678 astrocyte syncytium 173– 174 cell death/neuroprotection, astrocyte syncytium 174– 175 epithelial adhesion molecules 259–260 glial receptors 953 modeling of calcium 689–703 parenchymal/extraparenchymal 224– 229, 245– 247

Index

transduction pathways 910 sIL-6R see soluble IL-6 receptor sleep – wake cycle 420 slow potential shifts (SPSs) arousal and attention 621– 623 behaviour 614 glia-derived 611– 630 habituation 629– 630 intact animals 614– 621 motivation 624 Smase see sphingomyelinase SMC see smooth muscle cells SMIT see sodium-dependent myo-inositol transporter smooth muscle cells (SMC) 218 SNpc see substantia nigra pars compacta SOC see store-operated calcium channel SOD see superoxide dismutase sodium see Na... sodium-dependent myo-inositol transporter (SMIT) 1037–1038, 1040, 1042 Sokoloff method 413, 414 soluble IL-6 receptor (sIL-6R) 1009 soluble intercellular adhesion molecule 1 (sICAM-1) 1011– 1012 SON see supraoptic nucleus SON-VGL see supraoptic nucleus-ventral glial limitans source density 691 spasticity 775 spatial buffering 170– 172, 599– 603, 612– 614 specific serotonin re-uptake inhibitors (SSRIs) 454 ‘specificity pain theory’ 954 sphingomyelin 520, 526– 527 sphingomyelinase (Smase) 526, 527 spinal cord 55, 65 spinal glial activation 958 spreading depression 444, 446, 447, 596, 598 SPSs see slow potential shifts SSRIs see specific serotonin re-uptake inhibitors stab wounds 86, 87, 566– 567 stability maintenance 295– 310 steady-state pHi 709– 710 stellation 756, 757, 758– 759 stem cells 100– 101, 141 steroids astroglia 543– 548 brain endothelial cells 541, 543 CNS receptors expression 541 functional implications of glial 540–541 hyperammonemic PTBR changes 991– 993 microglia 548 neuroactive 535– 552

1169

non-neuronal cells 536–538, 541– 551 oligodendrocytes 548– 549 5a-reductase/3a-hydroxysteroid dehydrogenase in glia 538– 540 role 535– 536 Schwann cells 548– 551 synthesis and metabolism 536– 541 stochastic modeling 694– 697 store-operated calcium channel (SOC/CRAC) 647, 648, 651 Streptococcus pneumoniae 262–263 stress see also oxidative stress; vulnerability– stress model calcium waves 676 CNS trauma signals 351– 353 fiber depolymerization 650 lactate utilization 400 retinal conditioning 304– 307 structural dynamics 10 structural plasticity 181–195 subacute sclerosing panencephalitis 1075 subcellular networks 11, 12 subependymal layer see subventricular zone subfornical organ (SFO) 754 subgenual cortex 1050, 1051 substance P 485– 486 substantia nigra pars compacta (SNpc) 967, 968, 971, 972 subventricular zone (SVZ) cells adult 138 –139, 141 embryonic development 140 neurogenesis 140– 141 NOVOcan protein 137 suicidality 1018– 1019, 1054– 1055 superoxide dismutase (SOD) 1087– 1090, 1091– 1092 supracallosal anterior cingulate cortex 1052– 1053 supraoptic nucleus (SON) anatomy 237– 240 astrocyte structural plasticity 181– 195 astrocytes in hypothalamo-hypophysial system 753– 754 basal lamina 222, 224 catecholamines 755 dehydration activation 183– 195 lactation activation 184– 186, 188 microglial structural plasticity 192– 195 neuron – glial interactions 241– 243 supraoptic nucleus-ventral glial limitans (SON-VGL) 183, 186– 188, 191 surface specializations see cell surface extensions

1170

SVZ see subventricular zone symbiosis 1126–1127 synapses 200, 400, 545, 674– 675 syncytia astrocytes forming 165– 176 definition 165– 166 extraparenchymal – neuronal tissue 245 functional 612 gap junction role, astrocytes 166– 167, 170– 175 spatial buffering 599, 601 synthesis cell cycle stage 76 systemic stresses 234 T-cells 352– 355, 357 tanycytes brain barrier functions in ion and water homeostasis 763 cytoarchitectonics 34, 35 –36, 37 endothelial cell communication 206, 209– 210 function 136 GnRH neuron interaction 201, 202– 205 neuroendocrine regulation 546– 547 NOVOcan protein 136, 137– 138 tat 911 TBOA see threo-b-benzyloxyaspartate TCA cycle see tricarboxylic acid cycle tectum 613, 614, 615–621 tenascin 191, 811 terminal vascular bed endothelia 14 –24 testosterone 537, 538, 539 thapsigargin 650– 651 Theiler’s virus 1065– 1066 therapeutic vaccination 358– 359 thioacetamide-induced liver failure 988– 989 threo-b-benzyloxyaspartate (TBOA) 464 thrombin 506, 511 thyroid hormone actions 107– 110 tight junctions blood– brain barrier 256– 257 endothelial cells and pial microvessels 221 molecular structure 256– 257 nerve regeneration 335– 336 time-lapse imaging studies 662, 677 time-resolved label incorporation see dynamic isotopomer analysis tissue destruction 800– 801, 807 growth 21 – 22 homeostasis 912 organization 3 – 5 polarity 4 – 5, 6 – 7, 28 related cell structures 45

Index

structure 10 – 12 TNF see tumor necrosis factor toads see anurans toxins 806, 808, 903 see also neurotoxicity tracer methods 412– 413, 414, 415, 985 transactivation 503– 513 transcription factors 60 – 61 transcriptional responses 860– 862 transcytosis 262– 263 transendothelial migration 259, 263 transforming growth factor alpha (TGFa) 202– 203 transforming growth factor beta (TGF-b) 563, 565 transient potassium storage sites 603– 605 translational responses 862– 863 transmissible spongiform encephalopathies see prion disease transmitter effects on astrocytic energy metabolism 436, 448– 451 transmitter-induced calcium response downregulation 1043 transplantation 334– 341, 349, 350, 351 transport functions 930– 938 transverse myelitis 1065 trauma see injury tricarboxylic acid (TCA) cycle 464– 465, 467 3,5,30 -triidothyronine (T3) 107– 110 tropical spastic paraparesis (TSP) see Human T-cell leukemia type I-associated myelopathy tryptophan metabolism 1018 tryptophan– kynurenine pathway 1013– 1014 TS see tuberous sclerosis TSP (tropical spastic paraparesis) see Human T-cell leukemia type I-associated myelopathy tuberous sclerosis (TS) 677 tumor necrosis factor (TNF) 488, 906, 908– 909, 936, 937, 959– 960 tumor suppressor pathways 88 tumors 86, 87 – 88, 160, 1036 type-1-serotonin-link 1018– 1019 type-1/type-2 immune response 1000, 1003– 1005, 1010–1011, 1013– 1014 typographical variables/invariables 45 ubiquitin – proteasome pathway 77, 80 unit activity 624– 625 upregulation 983– 994 uptake systems 1037– 1038, 1040– 1042 urea cycle disorders 983, 984

Index +

vacuolar-type (V-type) H pump 722– 723 valproic acid 1033– 1045 vascular cell adhesion molecule-1 (VCAM-1) 258– 259, 260 vascular endothelial growth factor (VEGF) 302– 304, 563, 566 vascular tissue see also perivasculature barrier, enteric glia 323 basal lamina 222, 223– 224 cell types 218 choroid plexus 271–272 connective tissue relationship 246 extraparenchyma 216, 218 intracerebral vascularization 6, 14, 18 – 22 terminal vascular bed endothelia 14 – 24 vasoactive intestinal peptide (VIP) astrocytic receptors 485 developmental role 493 neurotrauma 492 prion disease 1095 transmitter effects on metabolism 452 vasopressin see arginine vasopressin VCAM-1 see vascular cell adhesion molecule-1 VDAC see voltage-dependent anion channel VEGF see vascular endothelial growth factor venous occlusive disease 309 ventral glial limitans of supraoptic nucleus (SON-VGL) 183, 186– 188, 191 ventricular lumen (VL) 218, 221, 223 ventricular system fractones (basal lamina) 223, 240 hydrocephalus 275– 281, 283, 287 lining cells 127– 142 normal 271– 274 structural effects 275–280 tissue surfaces, ependymo-astroglial cells 35 – 36 ventricular zone (VZ) cells

1171

embryonic development 128– 133 ependymal cell relationship 133– 134 gap junctions 135 NOVOcan protein 137– 138 radial glia relationship 129– 130 vesicular ATP 669 VGCGs see voltage gated calcium channels vimentin 778, 793, 796 VIP see vasoactive intestinal peptide viruses encephalitis, HIV-1-associated dementia 901– 913 entry into CNS 255, 261– 262 HIV infection process 927 proteins, HIV-1-associated dementia 911 schizophrenia hypothesis 1006– 1007 visual evoked potentials 622 voltage gated calcium channels (VGCGs) 641 voltage-dependent anion channel (VDAC) 985, 986 voltage-dependent calcium channels 447 voltage-dependent H+ conductance 714 volume regulation 720– 721, 748 vulnerability-stress model 1000 VZ see ventricular zone Wallerian degeneration 330– 331, 335 water channels 747– 764 see also aquaporins white matter 61, 62 – 63, 1066– 1071, 1075– 1076 ‘worm-like’ stimuli 618, 624, 627 wound healing 787–818 see also injuries; scarring xanthine derivatives 961– 962 xanthine oxidase 1092– 1093