Progress in Brain Research Volume 15 Biology of Neuroglia

P R O G R E S S I N B R A I N RESEARCH VOLUME 1 5 BIOLOGY OF NEUROGLLA

PROGRESS IN BRAIN RESEARCH

ADVISORY BOARD W. Bargmann H. T. Chang

E. De Robertis J. C. Eccles

J. D. French

H. HydCn J. Ariens Kappers S. A. Sarkisov

J. P. Schadt F. 0. Schmitt

Kiel Shanghai Buenos Aires Canberra Los Angeles

Goteborg Amsterdam Moscow Amsterdam B rookline (Mass.)

T. Tokizane

Tokyo

H . Wael sch

New York

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 15

BIOLOGY O F NEUROGLIA EDITED B Y

E. D. P. DE ROBERTIS Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires (Argentina) AND

R. C A R R E A Instituto Torcuato di Tella. Centre of Neurological Investigations, Buenos Aires (Argentina)

ELSEVIER P U B L I S H I N G C O M P A N Y AMSTERDAM

LONDON

1965

1 NEW

YORK

ELSEVIER P U B L I S H I N G C O M P A N Y

335 J A N VAN GALENSTRAAT, P.O. BOX 211, A M S T E R D A M

A M E R I C A N E L S E V I E R P U B L I S H I N G COMPANY, INC. 52 V A N D E R B I L T AVENUE, N E W Y O R K , N.Y. 10017

ELSEVIER PUBLISHING COMPANY LIMITED R I P P L E S I D E C O M M E R C I A L ESTATE R I P P L E ROAD, B A R K I N G , ESSEX

This volume contains the lectures delivered and the discussion that followed during a symposium on BIOLOGY O F NEUROGLIA

which was held as apart of the 10th Latin-AmericanCongress of Neurosurgery at the Academia Nacional de Medicina in Buenos Aires from 17-18 October, 1963. This Symposium was sponsored by: The Instituto Torcuato di Tella, Buenos Aires (Argentina); The International Brain Research Organization;The Centro de Cooperacidn Cientifca para America Latina de la UNESCO; and partially supported by grants from: The National Institutes of Health, U.S.Dept. of Health, Education and Welfare ( N B 0473541);The Consejo Nacional de Investigacione Cientifcas y Tecnicas, Argentina (No. 1423)

L I B R A R Y O F C O N G R E S S C A T A L O G C A R D N U M B E R 64-18525

W I T H 1 8 1 I L L U S T R A T I O N S A N D 2 3 TABLES

ALL RIGHTS RESERVED T H I S BOOK O R A N Y P A R T T H E R E O F M A Y N O T B E R E P R O D U C E D I N A N Y F O R I N C L U D I N G P H O T O S T A T I C O R M I C R O F I L M FORM, WITHOUT WRITTEN PERMISSION F R O M T H E PUBLISHERS

PRINTED IN THE NETHERLANDS

List of Contributors

L. BAKAY, The Division of Neurosurgery, State University of Jew Tork at Buffa Medical School, Buffalo General Hospital, Buffalo, N. Y. (U.S.A.).

0

M. BRADBURY, Medical Research Council, University College London, London. H. COLLEWIJN, Netherlands Central Institute for Brain Research, Amsterdam. H. DAVSON, Medical Research Council, University College London, London. Institute of General Anatomy and Embryology, Univei sity of E. D. P. DE ROBERTIS, Buenos Aires, Buenos Aires. B. D. DRUJAN, Department of Neurobiology, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas. R. FATEHCHAND, Department of Neurobiology, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas. R. L. FRIEDE, Mental Health Research Institute and Department of Pathology, The University of Michigan, Ann Arbor, Mich. (U.S.A.). R. GALAMBOS, Department of Psychology, Yale University, New Haven, Conn. (U. S.A.). C. A. GARCIAARGIZ,Department of Biochemistry, Faculty of Pharmacology and Biochemistry, University of Buenos Aires, Buenos Aires.

W. HAYMAKER, National Aeronautics and Space Administration, Ames Research Center, Moffett Field, Calif. (U.S.A.). I. KLATZO,Section of Neuropathology, Surgical Neurology Branch, National Institute of Neurological Diseases and Blindness, Bethesda 14, Md. (U.S.A.). A. LASANSKY, Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires.

E. LEVIN,Department of Biochemistry, Faculty of Pharmacology and Biochemistry, University of Buenos Aires, Buenos Aires. J. MIQUEL,National Aeronautics and Space Administration, Ames Research Center, Moffett Field, Calif. (U.S.A.). K. NEGISHI,Department of Neurobiology, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas. G. J. NOGUEIRA, Department of Biochemistry, Faculty of Pharmacology and Biochemistry, University of Buenos Aires, Buenos Aires.

VI

LIST OF CONTRIBUTORS

H. M. PAPPIUS,The Donner Laboratory of Experimental Neurochemistry, Montreal Neurological Institute and the Department of Neurology and Neurosurgery, McGill University, Montreal.

M. POLAK,Fundacion Roux-Ocefa, Laboratory of Histological and Histopathological Investigations, Buenos Aires.

J. P. SCHADB,Netherlands Central Institute for Brain Research, Amsterdam. DE TESTA,Department of Neurobiology, Instituto Venezolano de InvestigaA. SELV~N ciones Cientificas (IVIC), Caracas.

D. E. SMITH,Section of Neuropathology, Surgical Neurology Branch, National Institute of Neurological Diseases and Blindness, Bethesda 14, Md. (U.S.A.). Department of Neurobiology, Instituto Venezolano de Investigaciones G. SVAETICHIN, Cientificas (IVIC), Caracas. I. TASAKI,Laboratory of Neurobiology, National Institutes of Mental Health, National Institutes of Health, Bethesda, Md. (U.S.A.).

F. WALD,Department of Biophysics and Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires. H. WI~NIEWSKI, Section of Neuropathology, Surgical Neurology Branch, National Institute of Neurological Diseases and Blindness, Bethesda 14, Md. (U.S.A.). J. A. ZADUNAISKY, Department of Biophysics and Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires.

VII

Other volumes in this series:

Volume 1: Brain Mechanisms Specific and Unspecific Mechanisms of Sensory Motor Integration Edited by G. Moruzzi, A. Fessard and H. H. Jasper

Volume 2: Nerve, Brain and Memory Models Edited by Norbert Wiener? and J. P. SchadC Volume 3 : The Rhinencephalon and Related Structures Edited by W . Bargmann and J. P. Schade Volume 4: Growth and Mafurafionof the Brain Edited by D. P. Purpura and J. P. Schade

Volume 5 : Lectures on the Diencephalon Edited by W. Bargmann and J. P. Schade

Volume 6: Topics in Basic Neurology Edited by W. Bargmann and J. P. Schade Volume 7: Slow Electrical Processes in the Brain by N . A. Aladjalova Volume 8 : Biogenic Afnines Edited by Harold E. Himwich and Williamina A. Himwich

Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich

Volume 10: The Strucfrrre and Function of the Epiphysis Cerebri Edited by J. Ariens Kappers and J. P. SchadC

Volume 1 1 : Organization of the Spinal Cord Edited by J. C. Eccles and J. P. SchadC Volume 12 : Physiology of Spinal Neurons Edited by J. C. Eccles and J. P. SchadC

Volume 13: Mechanisms of Neural Regeneration Edited by M . Singer and J. P. SchadC

VIlI

Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P. Schad6 Volume 16 : Horizons in Neuropsychopharmacology Edited by Williamina A. Hmwich and J. P. SchadC Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener? and J. P. Schade Volume 18: Sleep Mechanisms Edited by K. Akert, Ch. Bally and J. P. SchadC Volume 19: Experimental Epilepsy by A. Kreindler Volume 20: Pharmacology and Physiology of the Reticular Formation Edited by A, V. Valdman Volume 21 : Correlative Neurosciences Edited by T. Tokizane and J. P. SchadC Volume 22: Brain Reflexes Edited by E. A. Asratyan Volume 23 : Sensory Mechanisms Edited by Y . Zotterman Volume 24: Carbon Monoxide Poisoning Edited by H. Bour and I. McA. Ledingham Volume 25 : The cerebellum Edited by C . A. Fox and R. S. Snider Volume 26 : Developmenfa1 Neurology

IX

Preface

The Latin-American Congress of Neurosurgery has been regularly held every two years since 1943 and a Symposium on Neurological Research has been added to each Congress in recent years. The Executive Committee of the 10th Latin-American Congress of Neurosurgery, presided over by Professor Ricardo Morea, decided upon our suggestion that a suitable subject for the 1963 Buenos Aires meeting would be the Biology of Neuroglia and recommended that some related aspects of brain edema should be included. There were three justifications for the choice of this subject: an interdisciplinary presentation on the subject had not been held since the 1956 Conference on the Biology of Neuroglia, organized and edited by W. F. Windle; there was a group of local investigators who could contribute their own experience to the Symposium, and finally, this was a subject of obvious interest to the predominantly neurosurgical audience of the Congress. The responsibility of selecting the subjects and the speakers and of conducting the Symposium was given to us as Coordinator and Secretary of the Symposium, respectively. The Executive Committee of the Congress deserves our gratitude for giving us complete freedom to carry out this responsibility. In the planning of the Symposium, we formulated a program including the morphological, biochemical and neurophysiological aspects of the problem. A small group of distinguished investigators were included who could cover different aspects of these three major subdivisions of the subject. This volume presents enlarged versions of the lectures held, some of which are of a review type as encouraged by Progress in Brain Research. This provides the readers with complementary detailed information, therefore, which could not be made available to the audience of the Symposium. An unrestricted opportunity was given to the members of the Symposium and the invited discussants to express their ideas at the end of each of three sessions. A fairly accurate version of the recorded discussion is also included in this volume at the points where it actually took place, i.e. after each of the sessions and at the end of the Symposium. A considerable amount of additional information can be gathered from this material. The Symposium began with some introductory remarks by Antonio de Veciana from the UNESCO’s Centro de Cooperaci6n Cientifica para America Latina and by Guido Di Tella, Vice-president of the Instituto Torcuato di Tella, both representing the institutional interest in a meeting of this nature. Prof. Bernard0 A. Houssay, President of the Consejo Nacional de Investigaciones Cientificas y Tecnicas of Argentina gave the opening address outlining the history of knowledge about neuroglia. This Symposium was made possible through the joint efforts of the institutions represented

X

PREFACE

by these three speakers and by the U.S. Department of Health, Education and Welfare, Public Health Service (NB 04735-OI), the International Brain Research Organization and the Executive Committee of the 10th Latin-American Congress of Neurosurgery. It was agreed upon from the beginning that the Secretary of the Symposium would not participate in the discussion no matter how tempting it would be to do so. This decision was made not only because the research subject was not his own but also because we thought it more important that one of us should concentrate on keeping the meeting running smoothly. The entire material for this volume was, however, in his hands - which is why it became mandatory for him to be one of the Editors. Last but not least we wish to express our gratitude to our secretary, Mrs. Joan Firmat, who assisted in so many secretarial and other duties. E. DE ROBERTIS R. CARREA

XI

Contents

................................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

List of contributors Preface

v IX

Some new electron microscopical contribations to the biology of neuroglia (Introduction) 1 E.D.P. De Robertis (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . . . Morphological and functional characteristics of the central and peripheral neuroglia (light microscopical observations) M. Polak (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Enzyme histochemistry of neuroglia R. L. Friede (Ann Arbor, Mich.) . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Functional implications of structural findings in retinal glial cells A. Lasansky (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Observations on penetration of serum proteins into the central nervous system I. Klatzo, H. WiSniewski and D. E. Smith (Bethesda, Md.) . . . . . . . . . . . . . . . 73 Astroglial reactions to ionizing radiation : with emphasis on glycogen accumulation J. Miquel and W. Haymaker (Moffett Field, Calif.) . . . . . . . . . . . . . . . . . . 89 First discussion period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 The extracellular space of the brain H. Davson and M. Bradbury (London) . . . . . . . . . . . . . . . . . . . . . . . 124 The distribution of water in brain tissues swollen in vitro and in vivo H. M. Pappius (Montreal) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The movement of electrolytes and albumin in different types of cerebral edema L. Bakay (Buffalo, N.Y.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changes in the size of astrocytes and oligodendrocytes during anoxia, hypothermia and spreading depression H. Collewijn and J. P. Schadk (Amsterdam) . . . . . . . . . . . . . . . . . . . . . Osmotic behaviour and glial changes in isolated frog brains J. A. Zadunaisky, F. Wald and E. D. P. De Robertis (Buenos Aires) . . . . . . . . . Some aspects of amino acid transport in the central nervous system E. Levin, G. J. Nogueira and C. A. Garcia Argiz (Buenos Aires) . . . . . . . . . . . Second discussion?period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excitability of neurons and glial cells I. Tasaki (Bethesda, Md.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nxvous function based on interactions between neuronal and non-neuronal elements G. Svaerichin, K. Negishi, R. Fatehchand, B. D. Drujan and A. Selvin de Testa (Caracas) Introductory discussion on glial function R. Galambos (New Haven, Conn.) . . . . . . . . . . . . . . . . . . . . . . . . . General discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions of the symposium E. D. P. De Robertis (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . .

135 155

184

. 196

. 219 225 234

. 243 267 278 284

Author Index.

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287

Subject Index.

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1

Some New Electron Microscopical Contributions to the Biology of Neuroglia INTRODUCTION

E D U A R D O D. P. DE ROBERTIS

Institute of General Anatomy and Embryology, University of Biienos Aires, Buenos Aires (Argentina)

When I was asked by the Committee of the 10th Latin-American Congress of Neurosurgery to organize an International Symposium of Neurological Research, as has been done with great success at the previous Congress in Mexico, I thought that a discussion on the Biology of Neuroglia might be of interest to all participants in the Congress. In fact, glia cells contribute greatly to the pathology and clinical treatment of neurological diseases and certainly to most of the surgical treatment. Furthermore, one of the main problems that neurosurgery has to tackle as a complication of traumatisms, tumours or toxic lesions, is that of brain oedema and as will be shown at this Symposium, this pathological condition depends to a certain extent on the glia cells and on their relationship to the blood vessels of the brain. The different types of glial cells constitute quantitatively an important part of the central nervous system (CNS), but until recent years their special functions and relationships with the neurons and vascular elements were practically unknown. The classical work of the Spanish school (Cajal, del Rio Hortega) was fundamental in defining the different types of glial elements -astroglia, oligodendroglia, microglia -and their histogenic relationships, but unable to throw definite light on their physiological significance. Only in recent years, with the development of new cytochemical and ultrastructural techniques and the use of physiological methods for the study of movement of ions and water between the different compartments, of microphysiological techniques for recording potentials in the glia membranes, and the biochemical studies on isolated glial cells in different physiological conditions, has new light been thrown on the role of glial cells in myelination, brain permeability and metabolism. In addition, the possible participation of glial cells in more complex electrophysiological mechanisms of the brain has been suggested. In a review on the ‘Submicroscopic Morphology and Function of Glial Cells’ De Robertis and Gerschenfeld (1961) analyzed some of the possible functions of these cells. Interfascicular OIigodendroglia of the white matter is recognized as having a prevaReferences p . I 1

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E. D. P. DE R O B E R T I S

lent role in the formation, maintenance and disposal of myelin and thus to be the main point of attack of agents that may cause demyelinating diseases. The possible role of perineuronal oligodendroglia has been mainly studied by HydCn and coworkers (Hyden and Pigon, 1960; HydCn and Lange, 1962; Hamberger and Hyden, 1963), who have obtained data indicating a possible metabolic interaction with the neuronal elements. These studies suggest that neurons and perineuronal oligodendroglia cells are linked in an energetic system, which may react as a functional unit. Astroglia is thought to be involved in the transport of water, electrolytes and metabolites within the brain and to be the site of an active homeostatic mechanism that regulates the content of water and prevents swelling of this tissue. The cell appears to be involved also in the physiologic barriers of the brain including the blood-brain barrier (BBB) and possibly the liquor-brain barrier (LBB) with the cerebrospinal fluid. In the above-mentioned review, together with the data on the bioelectric activity of astrocytes, we also discussed astroglia as a possible pool of electrolytes probably containing a high sodium content. The impact that some of these new concepts have on the physiology of the brain, was reflected on the postulation by Galambos (1961) of a continuous physiological interaction between glial cells and neurons in the electrophysiological and more complex brain functions. The recent evidence supplied by Hydkn and Egyhiizi (1963) that the RNA of glial cells may change during learning experiments in rats, adds even more interest to this new field of research and I am only sorry that Prof. HydCn could not accept our invitation to participate in the Symposium. Since some of the recent experimental works from our laboratory referring to glial cells in the retina and to the ependymoglial cells of amphibian brain will be presented by Dr. Lasansky and Dr. Zadunaisky, I would like to mention here only some of the newer contributions that electron microscopy is making to this field of study. ( a ) A formalin perfusion Jixation method f o r the study of brain First of all I would like to mention to you briefly the method for perfusion fixation with formaldehyde that has been recently developed in our laboratory (Gonzalez Aguilar and De Robertis, 1963) and which permits an excellent preservation of the entire CNS for electron microscopy. For the development of this technique a study was made on the water changes of the tissue after fixation and the components of the solutions for washing of the blood and fixation were so adjusted as to leave the water Fig. 1 . Diagram showing some of the concepts discussed by De Robertis and Gerschenfeld (1961) on the relationshipof astroglia with other cellular components of the CNS and its possible function. In the centre an astroglial cell with the clear cytoplasm and processes that make contact with the basal membrane (bas. m.) of a blood capillary (Cap), with the pial membrane and a neuron. This astroglial cell is supposed to be involved in the blood-brain (BBB), the liquor-brain barrier (LBB) with the cerebrospinal fluid (CSF), and the synaptic barrier (SB). The possible movement of fluids within the astroglial cytoplasm is indicated with arrows. The neuron has surface relationship with astroglia, oligodendroglia (oligo), and the synaptic endings. A microelectrode (elect) recording extraneuronally is supposed to be implanted in a glial process (end = capillary endothelium). This diagram emphasizes the small extracellular space. (For further description see De Robertis and Gerschenfeld, 1961.)

ELECTRON MICROSCOPICAL CONTRIBUTIONS

Fig. 1. For legend see p. 2.

3

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E. D. P. D E R O BER TI S

content of the brain unchanged. The effect of the perfusion with the washing solution was followed by EEG recordings. Excellent preservation of the glial cells and nerve elements in gray and white matter was observed (Figs. 2 and 3). This method has permitted new observations on the disposition of the glial membranes, which will be discussed below. The advantage of this method resides in the use of a cheap, innocuous fixative and a very simple modus operandi. The>otal preservation obtained permits a systematic study of any neuro-anatomical region and the carrying out of histophysiological or neuropathological experiments in the CNS.

(b) The problem of the extracellular space in the CNS The second part I would present, refers to the problem of the extracellular space in the brain. Physiologically, the extracellular space of a tissue can be determined by calculating the volume distribution of a substance incapable of penetration through cell membranes (Robinson, 1960). In the case of the brain this operational approach has given figures that vary considerably. Using radioactive C1- spaces of 31.4 to 50% and with radioactive Na+, spaces of 26.6 to 40% were calculated. From the above figures down to 4-5 % with 35S04-or inulin in vivo, many intermediary ones have been obtained. With in vitro techniques the most accepted figures vary between 1 6 1 5 % for inulin or sucrose and 17 for ferrocyanide (see Table I in De Robertis and Gerschenfeld, 1961). From the very early observations with the electron microscope (lit. in De Robertis and Gerschenfeld, 1961), it was demonstrated that in the CNS there are no large extracellular spaces and that the membranes of all the cellular components of the nervous tissue are in intimate contact among themselves and with the basal membrane of capillaries. Only fine spaces in the order of 100 to 250 A were observed in between the adjacent membranes. This morphological extracellular space was calculated by Horstmann and Meves (1959) to be not larger than 5 %. This apparent contradiction with the physiological data became more marked when Gerschenfeld et ul. (1959) found that on incubation of brain slices in vitro, as is done for the determination of the extracellular space, there is a marked swelling of the cell body and processes of astrocytes, but absolutely no change of the extracellular spaces. These studies demonstrated also that, at least in the gray matter of the brain, there is not a true extracellular oedema, but an intracellular swelling of the astrocytes. The implications of these observations in the pathology and treatment of human cases of brain oedema need no further comment. As a consequence of these experimental findings and those which will be reported by Dr. Zadunaisky at this Symposium, the following hypotheses on the function of astroglia can be advanced. (I)Astroglia constitutes a cellular compartment that has a special osmoticbehaviour reacting with a swelling in different conditions in vitro or in vivo when the BBB mechanism is altered. (2) In addition to having important mechanical and homeostatic functions, astroglia constitutes a pool of water and electrolytes interposed between the blood plasma,

ELECTRON MICROSCOPICAL CONTRIBUTIONS

5

Fig. 2. Electron micrograph of the cerebral cortex of a rat showing a pericapillary region. cl = capillary lumen; e = endothelium; bm= basement membrane; As = astrocytic feet. With arrows are indicated two tight junctions at which the astrocytic processes are adherent. See therest of theneuropi1 with small intercellular spaces. Fixation by formaldehyde perfusion followed by osmium and uranyl acetate in all Figs. 2-5. x 68,000.

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E. D. P. DE R O B E R T I S

the neurons and other glial elements. Gerschenfeld et al. (1959) found that under in vivo conditions of water overload as marked as to produce a 40% increase in the total extracellular space of the body, no real brain oedema could be produced. This was explained as being due to the presence of a BBB mechanism preventing the entrance of excess water and ions and/or an actively pumping back of ions and water into capillaries. Either or both mechanisms would prevent the swelling of the brain in the living animal. The narrow gaps observed under the electron microscope led some authors to think that they could not adequately serve as diffusion channels and an extreme view that in the CNS there is not a true extracellular space, was sometimespropounded. The experimentsof Lasansky and Wald (1962) on the retina, in which with the electron microscope ferrocyanidewas observed to diffuse in between the intercellular gaps with great rapidity, indicated that these gaps are truly extracellular in their function and therefore that the neuronal membrane is exposed to an extracellularfluid (see Lasansky, this Symposium). These findings are not necessarily opposed to the concept that the access of solutes to the extracellular space can be regulated by the glial cells by way of the astrocytic feet (Fig. 1). Recently Farquhar and Palade (1963) have drawn attention to the fact that at the junction between certain epithelial cells there may be occluding zones in which the intercellular gap disappears. At these points the membranes of adjacent cells are completely adherent. The presence of these so-called tight junctions could explain the impermeability shown by the furface of some epithelia. Zones of adhesion between the plasma membrane of adjacent astrocytic feet were observed by Gray (1961) and have been observed by us in specimens perfused with formaldehyde in the best conditions for the preservation of the tissue (Figs. 2 and 3). Further studies should be carried out to determine if these occluding zones are constant in all asstrocytic feet and if they are permanent or transient phenomena in the astrocytes. The presence of series of tight junctions around the capillaries would have the effect of occluding the intercellular spaces. If this is the case, the water, electrolytes and other solutes that traverse the endothelium and the basement membrane, should find a barrier and be taken up by the glial cell before entering into the open extracellular spaces. ( c ) Morphological bases of the synaptic barrier Another interestingproblem is that of the relationship that glial elements may have with synaptic transmission. The idea that glial processes might be interposed in between endings and neurons and involved in synaptic activity, which was especially propounded by De Castro (1951), has not been substantiated by electron microscopy. Starting with the work of De Robertis and Bennett (1954, 1955), and Palade and Palay (1954), all subsequent studies have demonstrated that glial processes are not interposed at the synaptic junction and that at this level a direct contact of the two neuronal elements exists. Recently the finer organization of the synaptic junction has been determined in synapses of the CNS (De Robertis, 1962). The synaptic cleft of about 300 A is crossed by a series of fine (50 A) intersynapticfilaments that join the

ELECTRON MICROSCOPICAL CONTRIBUTIONS

7

Fig. 3. The same description as Fig. 2 with a very long tight junction between astrocytic feet (arrows). se = synaptic ending; is = intercellular space in the neuropil; mi = mitochondria. x 45,000. Referenccs p . 11

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E. D. P. D E R O B E R T I S

Fig. 4. Synaptic region of the hypothalamus of a rat, showing the synaptic vesicles (sv), the synaptic membranes (sm) and subsynaptic web (ssw). The synaptic complex is surrounded by glial processes (gp), which at certain points (marked with arrows) are adherent to the ending. This may be the basis of the synaptic barrier. mi = mitochondria. x 120,000.

ELECTRON M I C R O S C O P I C A L C O N T R I B U T I O N S

9

Fig. 5. The same as Fig. 4, showing between arrows several tight junctions between glial processes and nerve endings. x 105,000. References p . I 1

10

E. D. P. DE R O B E R T I S

two synaptic membranes and in the postsynaptic cytoplasm there is an irregular system of filaments, the subsynaptic web. Both synaptic membranes are so tightly bound that upon homogenization of the brain the isolated synaptic endings generally carry with them the postsynaptic component. Surrounding this synaptic complex (Fig. 4) glial processes may be frequently observed. Recent observations on brains fixed by formaldehyde perfusion (Gonzalez Aguilar and De Robertis, 1963), have led us to observe the rather frequent presence of tight junctions around the endings. At certain points the plasma membrane of the glial process adheres to the membrane of the ending (Figs. 4 and 5). This sealing, either total or partial, of the extracellular space around the synaptic complexes may physiologically act as a kind of synaptic glial barrier slowing down or preventing diffusion of transmitters released at the junction to penetrate into the intercellular gaps. Physiological evidence of barriers to the diffusion of transmitters has been observed in different synapses in which there is a prolonged or residual action after a presynaptic stimulus (Curtis and Eccles, 1959). In fact, repetitive discharges due to longer excitatory postsynaptic potentials have been observed at certain synapses in response to the firing of a single presynaptic volley. Studies with microelectrodes in which drugs were injected electrophoretically, have also indicated the possible existence of such a synaptic barrier (Curtis and Eccles, 1958a, b). The finding of tight junctions between glial processes and the synaptic complexes thus appears as the most conspicuous morphological basis for a synaptic barrier. By this mechanism glial cells may certainly influence in a subtle manner the physiology of synaptic transmission. I hope that these few examples, in which submicroscopic studies contributed to the Biology of Neuroglia, may serve as introductory remarks to the important papers that will follow and in which the chemical, physiological and pathological aspects of glial cells will be discussed by some of the best experts in the field. I would like to express my sincere gratitude to the eminent foreign scientists who have come from such long distances to take part in this Symposium, thus giving to all of us the light of their wisdom and their stimulus and inspiration to carry on our own humble share in the progress of science. SUMMARY

References are made to a previous review on the submicroscopic morphology and function of glial cells published in the International Review of Neurobiology, Vol. 3, p. 1, 1961, De Robertis, E.D.P., and Gerschenfeld, H.M. Some recent contributions are related to the problem of fixation of the CNS. A new method of formalin perfusion which gives the best preservation of glial and nerve elements at the electron microscope level, is presented. The problem of the extracellular space in the CNS and the discrepancy between physiological methods and electron microscopy is discussed. The bases for an agreement are mentioned.

ELECTRON MICROSCOPICAL CONTRIBUTIONS

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Astroglia constitutes a cellular compartment with a special osmotic behavior that reacts with swelling in several ‘in vivo’ and ‘in vitro’ conditions. The presence of tight junctions between the glial processes surrounding the capillaries, may occlude the intercellular space and thus the astroglia may have a regulating effect on the composition of the extracellular fluid. The so-called synaptic barrier is discussed and findings on the presence of tight junctions between glial processes and nerve endings, which could be the bases for such a barrier, are described. REFERENCES CURTIS, D. R., AND ECCLES, R. M., (1958a); The excitation of Renshaw cells by pharmacological agents applied electrophoretically. J . Physiol. (Lond.), 141,435-445. CURTIS, D. R., AND ECCLES, R. M., (1958b);The effect of diffusional barriers upon the pharmacology of cells within the central nervous system. J. Physiol. (Lond.), 141,44&463. CURTIS, D. R., AND ECCLES, J. C., (1959); The time courses of excitatory and inhibitory synaptic actions. J. Physiol. (Lond.), 145,529-546. DE CASTRO, F., (1951); Aspects anatomiques de la transmission synaptique ganglionnaire chez les mammifckes. Arch. int. Physiol., 59, 479. DE ROBERTIS, E., (1962); Fine structure of synapses in the CNS. Proceedings of the International Congress of Neuropathology, Munich, Vol. 2 (p. 35). DE ROBERTIS, E., AND BENNETT, H. S., (1954); Some features of the submicroscopic morphology of synapses in frog and earthworm. Fed. Proc., 13,35. DE ROBERTIS, E., AND BENNETT, H. S., (1955); Some features of the submicroscopic morphology of synapses in frog and earthworm. J. biophys. biochern. Cytol., 1,47-58. DE ROBERTIS, E., AND GERSCHENFELD, H. M., (1961); Submicroscopic morphology and function of glial cell. Znt. Rev. Neurobiol., 3, 1. FARQUHAR, M. G., AND PALADE, G. E., (1963); Junctional complexes in various epithelia. J. Cell Biol., 17,375-412. GALAMBOS,R., (1961);Aglia-neuronal theory of brain function. Proc. nut. Acad. Sci. (Wash.),47,129. GERSCHENFELD, H. M., WALD,F., ZADUNAISKY, J. A., AND DEROBERTIS, E. D. P., (1959);Function of astroglia in the water ion metabolism of the central nervous system. Neurology, 9,412-425. GONZALEZ AGUILAR, F.,AND DE ROBERTIS, E., (1963); A formalin-perfusion fixation method for histophysiological study of the central nervous system with the electron microscope. Neurology, 13,758-771. GRAY,E. G., (1961); Ultrastructure of synapses of the cerebral cortex and certain specialization of neuroglial membranes. Electron Microscopy in Anatomy. London, E. Arnold, Publishers (p. 54). HAMBERGER, A., AND HYDEN,H., (1963); Inverse enzymatic changes in neurons and glia during increased function and hypoxia. J . Cell Biol., 16,521-525. HORSTMANN, E., AND MEVES,H., (1959); Die Feinstruktur des molekularen Rindengraues und ihre physiologische Bedeutung. Z. ZeNforsch., 49, 569404. HYDBN,H., AND EGYH~ZI, E., (1963); Glial RNA changes during a learning experiment in rats. Proc. nut. Acad. Sci. (Wash.),49,618-624. HYDEN,H., AND LANGE, P. W., (1962);A kinetic study of the neuron-glia relationship. J. Cell Biol., 13,233-237. HYDBN,H., AND PIGON, A., (1960);A cytophysiological study of the functional relationship between oligodendroglial cells and nerve cells of Deiters’ nucleus. J. Neurochem., 6, 57-72. LASANSKY, A., AND WALD,F., (1962); The extracellular space in the toad retina as defined by the distribution of ferrocyanide. A light and electron microscope study. J. Cell Biol., 15,463-479. PALADE, G. E., AND PALAY, S. L., (1954); Electron microscope observations of interneuronal and neuromuscular synapses. Anat. Rec., 118, 335-336. ROBINSON, J. R., (1960);Metabolism of intracellular water. Physiol. Rev., 40,112-149.

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Morphological and Functional Characteristics of the Central and Peripheral Neuroglia (Light Microscopical Observations) M. POLAK Fudacion Roux-Ocefa. Laboratory of Histological and Histopathological Investigations, Buenos Aires (Argentina)

The cells constituting the so-called central and peripheral neuroglia are at present intensively studied. They are being analyzed by numerous methods and techniques, in human and animal material, normal, pathological and experimental. Some investigators with a thorough knowledge of the classical publications of Golgi, Cajal, Hortega and others, have carefully analyzed the structure of neuroglial cells. Others, however, ignoring these studies, when applying histological techniques now out-dated by the silver and gold impregnations, have rediscovered various types of glial cells. Therefore we believe that a summary of the morphological characteristics of the neuroglial elements as seen with the light microscope may be useful. In particular, sections impregnated by the metallic techniques of Golgi, Cajal and Hortega will allow us to distinguish between the different cellular types in great detail. These techniques clearly show the relationship between the neuroglial cells on the one hand, and the nerve cells, their processes, the blood vessels and the meninges, on the other hand. An important advance in knowledge of the morphological and functional interpretation of neuroglial cells was made by Hortega (1919-1921)) when he proved that the ‘third element’ of Cajal was in fact made up by two types of cell: one authentically neuroglic, the other of mesenchymal origin, the oligodendrocyte and the microglial cell. This discovery was partly due to the use of a new histological technique, now called the Hortega method (ammoniacal silver-carbonate). The microglia or mesoglia has been considered by many authors as part of the neuroglia. Glees (1955) adds to this confusion when he says: ‘The origin of neuroglial cells is twofold : some differentiate from neuroectoderm and others from mesoderm. Those of mesodermal origin appear to be of negligible importance except in traumatic reactions in the brain and need not be considered here’. This statement contains two misinterpretations: (1) the consideration that the microglia is part of the neuroglia; and (2) the microglia is of little importance in normal conditions. Hortega clearly explains that ‘there exist two kinds of cells: the true neuroglia and the false neuroglia; the one which has an ectodermal origin, in the same way, the

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same place and the same time as the nerve cells, and the other which has a mesodermal origin. The latter corresponds to the elements which I first described under the title of microglia’. Therefore, we shall not include the microglia in this report, and in referring to neuroglia we shall divide it into two groups: the central neuroglia, made up of ependymoventricular glioepithelial cells, astrocytes and oligodendrocytes; and the peripheral neuroglia made up of gliocytes in the spinal and sympathetic ganglia, Schwann and Remak cells of the nervous fibers and the terminal gliocytes related with the free nerve endings and the capsulated endings. CENTRAL NEUROGLIA

Embryological data The modifications of the ectoderm that lead to the formation of the neural plate, neural canal and neural tube are well known to the embryologists. The epithelium, lining the primitive neural tube, consists of prisma-like cells which rapidly proliferate at the inner limiting membrane, thus resembling a stratified epithelium on account of the different heights at which the nuclei are placed. Among these elements appear spherical cells in mitosis, named germinative cells, from which neuroblasts originate exclusively. His (1889) gave the name of spongioblasts to the primitive epithelial cells from which the ependymal-ventricular epithelium and the remaining neuroglial elements originate. However, some authors deny that the differentiation into neuroblasts and neuroglial elements takes place so early. Schaper (1897) regards the prisma-like cells as undifferentiated neuroepithelial elements, and germinative cells as the same cells in mitosis, giving forth new neuroepithelial cells or undifferentiated apolar cells that will produce new generations of undifferentiated cells or cells differentiated in neuroblasts and spongioblasts through mitotic division. At first, Schaper’s concepts were accepted by many scientists, and Cajal himself, referring to the problem, remarks: ‘We consider it more likely, in agreement with Kolliker and Schaper, that the spherical elements on the way to mitosis may be undifferentiated forms, whose progeny is represented as much by primitive epithelial cells as by neuroblasts. Specificity would occur later in the spongioblast and neuroblast phases’. Later, however, studying the formation of the neuroglia in the early phases of the development of the medulla, Cajal disposed of Schaper’s undifferentiated elements : ‘As our investigations have definitely proved, the elements of the neuroglia are nothing but displaced and transformed epithelial cells’. The germinative cells are transformed into neuroblasts, which, in turn, move from their original site among the spongioblasts to settle between the marginal nuclear zone and the marginal velum going through the following phases of evolution: apolar-, bipolar-, monopolar and young neurocyte. In the final formation of the ependymal and ventricular epithelium, the spongioblast goes through the following stages : primitive spongioblast, primordial epithelium, ramified epithelium and final epithelium. References p. 33/34

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According to Cajal, the neuroglial cells appear in the chick embryo on the 13th day, and in the human embryo in the 3rd month, and their genetic mechanism would be as follows: the cell body leaves the neural duct at different heights, where it undergoes various changes: atrophy of the processes, transversal growth of the soma while short extensions appear that later ramify, and which leads to a greater development of its radial outgrowth. At the beginning the cells are anchored in the outer limiting membrane. This insertion disappears in mammals shortly before birth and the displaced epithelial cells reduce their peripheral expansion, reabsorb their processes and become stellate. Cajal describes two phases in the neuroglial differentiation : (1) displaced epithelial cells whose radial expansions terminate in the pia mater by terminal thickenings (primordial neuroglial cells or astroblasts, according to LenhossGk); and (2) young neuroglial cells, which retract their two radial processes. These two types of cell will develop, according to the zones in which they are present, to become protoplasmic or fibrous astrocytes respectively. As far as the development of the oligodendroglia is concerned, Hortega described two kinds of elements which differentiate from the primitive neuroepithelium : the glioblasts originated from the spongioblasts and the neuroblast originated from the germinative cell. As the former leave the neural tube, some will become astroblasts and others oligodendroblasts. The astroblasts immediately come into contact with mesodermal structures (vessels and meninges) and the oligodendroblasts become intimately related to nerve fibers. These two facts constitute the fundamental morphological principles of the concepts of ‘angiogliona’ and ‘neurogliona’,to which we shall refer later. The astroblasts develop into protoplasmic and fibrous elements. The oligodendroblasts remain associated with nerve fibers. Although the microglia does not belong to the neuroglia, we shall refer briefly to its genesis. The microglial cells, representing the reticulo-endothelial system in the nervous system, originate from the pia mater, particularly from the superior and inferior choroidal tissues. The cells also originate from the adventitial cells of the blood vessels that penetrate into the nervous parenchyma. When an adventitial cell loses its connection with the vascular wall, it changes its shape in order to adapt to the new environment and then it is impossible to differentiate it from other microgliozytee. When a microglial element moves to the wall of a blood vessel, to which it becomes attached, it becomes an adventitial cell. The above-mentioned data allow us to draw up the following scheme: Primitive neural epithelium

F

spongio? ast 4 ependymoventricular glioepithelium

>minative I

glioblast

/

astroblast

microgliocyte

1

microglioblast

astrocyte

t

+

Leptomeniigeal ‘midus’

cell

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Ependy mal-Yentr icular epithelium (Figs. 1-3) On the basis of cytogenetic, morphological and functional criteria, neurohistologists consider that the ventricular and ependymal epithelium belongs to the group of neuroglial elements. The ventricular cavities and the spinal central canal are lined with cubical or cylindrical cells. In some species the apical part of these cells show cilia, for which Hild (1954) demonstrated in tissue cultures the presence of a continuous and coordinated movement in onkdirection. This phenomenon was pointed out i:t situ in-1836.b~Purkinje and simultaneously by Valentin who related-it to the I

Fig. 1. Glial-epithelial cells of ependyma. (Golgi-Hortega staining method.)

Fig. 2. Glial-epithelial cells with ‘feet’ on a blood vessel. (Hortega triple impregnation.) I<&!

ellCeS

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Fig. 3. Tangential cut of the glial-epithelialcells of ependyrna. Small processes of the cells are shown.

(Hortega triple impregnation.)

movement of the spinal fluid towards the brain ventricles. The epithelial cells do not rest upon a basal membrane as can be shown with one of the Hortega staining methods. In histological sections stained according to the Golgi and Hortega method one can observe a number of small processes that penetrate into the nervous tissue. These processes sometimes become associated with blood vessels. In tangential sections one may observe short lateral processes which emerge at the level of the apical part of these cells, and often a few delicate fibrils, which in pathological conditions show up as the so-called ‘fibrillar degeneration’ (Hortega).

Astroglia (Figs. 4-12) Cajal (1914, 1932) gives the most complete description of the morphological characteristics of astrocytes, and, although he does not give an extensive description of the so-called ‘third element’ (apolar cells), he makes it clear in a footnote that ‘the problem of form and physiopathological significance of the enigmatic apolar cells has been brilliantly solved by Hortega with the aid of his ammoniacal silver carbonate

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method in a series of outstanding papers which appeared from 1920 to 1930’. Cajal continues : ‘These investigations controlled by numerous scientists and by myself, ascertain, adding a great number of discoveries, that my third element represents a new factor of the normal texture of the nervous centers. He has also proved that the third element of the grey matter, corresponds to a small cell, often asteriform, very abundant in the nervous centers of mammals (Hortega’s microglia), whilst my third

Fig. 4. Plexus formed by five protoplasmic astrocytes. (Golgi-Hortega staining method.)

Fig. 5. Protoplasmic and intermediate astrocytes arranged around nerve cells, some of which appear to be impregnated. At the bottom an oligodendrocyte may be seen. (Golgi-Hortega method.) References p . 33/34

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Fig. 6 . Bergmann’s cells of the cerebellum. (Golgi-Hortega method.!

Fig. 7. Astrocytes of the intermediatetype with ‘feet’ directed towards blood vessel. (Golgi-Hortega

method.)

element of the white matter constitutes a different cellular species that he calls oligodendroglia. He describes various kinds of this latter glial type. Rio Hortega upholds my supposition that the apolar or cuboid interfascicular cells represent something like a Schwann corpuscle of the central nervous fibers’. This work, together with that published by Rio Hortega in this country in 1941, are essential starting-points for all who wish to investigate the morphological characteristics of the astrocytic elements using optical microscopy, and such characteristics can be satisfactorily obtained if selective techniques are properly applied.

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In this latter study, referring to astrocytes, Rio Hortega says: ‘The last phase in the histological knowledge of astrocitary neuroglia corresponds to Cajal and his school. Our teacher, by making use of his gold sublimate method (one of the most beautiful of histological techniques) was able to obtain in 1913 the brilliant images of protoplasmaticr-and ,fibrous astrocytes which have not been surpassed by any of all those who have tried to usurp his method through useless modifications. Cajal made evident not only the reality of neuroglial forms, but also their most delicate struciures including the granular (urano-formol method) which allowed him to join those who saw a suggestive functional indication in it. As from Cajal’s memorable studies, we no longer speak of astrocytes of long or short radiations in current lexicon, but of protoplasmic and fibrous astrocytes.’ ( a ) Protoplasmic astrocytes (Figs. 4,5 and 7 ) . Since the protoplasmic astrocytes are located primarily in the gray matter they are more difficult to impregnate than the fibrous astrocytes, andit is advisable to fix the tissue as quickly as possible with formol bromide. The cytoplasm of the cells appears to be filled by delicate gliofibrils, which extend into the processes. Some of the processes become implanted in the adventitia of the small blood vessels by means of a perivascular foot (Cajal’s pi6 chupador or sucking foot). Successful impregnations by Cajal’s gold-sublimate method or Hortega’s triple impregnation method will give sufficient information for an architectonic analysis of the neuroglial elements in the gray matter. According to Hortega, we shall distinguish three types of protoplasmic astrocytes : (1) those in intimate contact with nerve cells; (2) an intermediate type; and (3) those in intimate contact with blood vessels. The first group, also called neuronal satellites, is characterized by an intimate relationship with the cell body and the dendrites of neurons. The cell body of these astrocytes is often flattened, adapting itself to the shape of the cell body of the nerve cells. The processes of the astrocytes show a specific orientation towards the neuron. These astrocytes, notwithstanding their special perineuronal arrangement, have one or more processes that are directed towards the blood vessel. The second astrocitary type, interneuronal, has the pattern of the protoplasmic astrocyte as previously described, The vascular satellite (Andriezen’s) astrocytes are elements in which the relationship with the blood vessels is very pronounced. The cell bodies are pressed against the wall of some blood vessel*. In the cerebral cortex the astrocytes are not typically protoplasmic, for they show some processes of a fibrous type and also gliofibrils may be present (Cajal’s intermediate astroglia or Held’s marginal neuroglia). As a special variety of the protoplasmic astroglia we should mention Bergmann’s fibers or cells, which are situated in the molecular layer of the cerebellum. The cell bodies of these elements are located in the Purkinje layer, while the processes run perpendicular through the plexiform layer towards the pia mater. Another variety of the protoplasmic astroglia is the epithelial cell of Muller in the

* Their ovoid or spherical nuclei when stained with aniline dyes can be mistaken for oligodendrocytes. References p . 33/34

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retina. Its morphological and structural characteristics were extensively studied by Cajal with the Golgi method. (b) Fibrous astrocytes (Figs. 8, 9 ) . These neuroglial elements show different morphological characteristics whether the Golgi, Cajal or Hortega method is employed. The Golgi method impregnates astrocytes in which the cell body and the small filiform processes stand out clearly. The ‘sucking-feet’ show up better with the Cajal method, whereas the gliofibrils are better demonstrated with the Hortega method. These astrocytes are observed especially in the white matter. The long processes are frequently oriented in the direction of the leptomeninges. Fibrous astrocytes are also found in the neurohypophysis, the hypophyseal stalk and the optic chiasm. In the neurohypophysis small cells with two or three processes are found, one of the processes sometimes being in close contact with a blood vessel. In the hypophyseal stalk the astrocytes are small with short processes and in the optic chiasm the filiform processes are very long and oriented parallel to the nerve fibers.

Fig. 8. Intermediate and fibrous astrocytes with ‘feet’ directed towards a transversally cut blood vessel. (Golgi-Hortega method.)

( c ) Some.functiona1 aspects of the astrocytes. In 1928, Hortega described the concept of the ‘angiogliona’,summarized in his own words : ‘Each astrocyte is a histophysiological element that acts in relation with the capillaries on the one hand, and in relation with the nerve cells and fibers, on the other’. The ‘angiogliona’ could fulfil mechanical, trophic, secretory and antitoxic functions. Historically, the first function attributed to astrocytes was that of support to neurons and fibers. The glial network would also embrace the synaptic endings. According to De Castro (‘glial mantle’ theory) the astrocytic processes are thus interposed between the synaptic surfaces. This concept has been rejected according to data obtained with the electron microscope. However, it would appear that the so-called

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‘trophic’ function is more important. The astrocytes are interposed between the neurons and the blood vessels. Therefore they are able to extract nutrients from the blood stream, which are then transferred to the neuron. At the same time they would absorb catabolic products from the neuron, and convey them to the blood stream, thus fulfilling an antitoxic function already suggested by Lugaro. Some authors

Fig. 9. Protoplasmic and fibrous astrocytes with short and long processes. Below the blood vessel an impregnated microglial cell is shown. (Golgi-Hortega method.)

Fig. 10. Two small astrocytes with long filiform processes. (Golgi-Hortega method.) Refkrences p. 33/34

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Fig, 11. Two astrocytes of intermediate type with ‘feet’ attached to the vascular adventitia. (GolgiHortega method.)

Fig. 12. Immature astrocytes and astrocytoblasts with few processes. (Cajal gold-sublimate method.)

(Nageotte, 1929; Mawas, 1910) believe that the neuroglia would act as an endocrine gland, since they feel that the special cytoplasmic granulations in the glial elements are true secretion products, similar to those of the interstitial cells of the testicle and the ovary. Rio Hortega thought that the oligodendroglia had a myelogenetic function while Hild has more recently supported the view, based on tissue cultures, that myeline would originate in protoplasmic astrocytes. Based on a very personal interpretation, Bairati feels that there is no structural difference between astrocytes and oligodendrocytes, a view which can hardly be accepted by anyone who has experience with appropriate histological technique.

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Oligodendroglia (Figs. 13-20) In 1918, Hortega observed that the ‘third element’ of Cajal was made up of two kinds of cell : microglia and oligodendroglia. This discovery was mainly attributed to the use of staining solutions containing silver carbonate. Therefore, the interpretation of these elements may be divided into two periods: before and after the application of the silver carbonate method. The method of Golgi did not allow any differentiation in the neuroglial elements except for the classical types: astrocytes with short and lon9 processes.

Fig. 13. At the bottom of the picture numerous oligodendrocytes (type I) in the cerebellar white matter. At the top numerous impregnated elements in the granular layer. (Golgi-Hortega method.)

Fig. 14. Oligodendrocytes of types I and 111. (Hortega-Polak method.) References p. 33/34

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Robertson’s platinum method (1900a) furnished evidence for the existence of minute processes, but did not differentiate the various cell types. His observations correspond to all nonastrocytic elements, i.e. micro- and oligodendroglia. In 1921, Hortega published his first monograph on the oligodendroglia, and in 1928 his well-known monograph entitled : ‘Third contribution to the morphological knowledge and functional interpretation of the oligodendroglia’. The oligodendroglialcells are distributed in the gray matter and in the white matter, in the former as neuronal and vascular satellite cells, and in the latter in relation to myelinated fibers. These cells appear as spherical. polygonal elements with several very delicate processes, which sometimes branch. Sometimes they form whorls that wrap round the nerve fibers. Hortega described 4 different types (I, 11, I11 and IV) of oligodendroglial cells. ‘In the history of oligodendroglia’, Hortega says, ‘there are

Fig. 15. Two oligodendrocytesof types I and I1 with typical long processes. (Golgi-Hortega method.)

three scientists who began its morphological study, and had an opinion about its physiology. Robertson (1900), who described the smallest oligodendrocytes under the name of mesoglia cells; Cajal, who maintained the equivalence of his apolar cell and Schwann cells and Paladin0 who pointed out the intimate relationship between his ‘neuroglial scheletro’ and the medullated fibres’. For this reason, he attached the names of these scientists to the oligodendrocytesI, I1 and 111. He adds: ‘Allow us to make clear something that is related to the priority of the discovery of the types I1 and 111’. Robertson’s mesoglial cells correspond exactly to the smallest type of oligodendrocytes. On the contrary, neither Cajal’s apolar cells, nor Paladino’s ‘neuroglial scheletro’ correspond to the types of oligodendrocytes bearing their names. Type I (Robertson) cells may be well studied in the forebrain, cerebellum and spinal cord and they are arranged around the blood vessels, neurons and fiber tracts. They are small (15 to 20 p), and have a spherical or slightly polygonal cell body from which numerous and delicate processes extend towards the nerve fibers. Type I1 (Cajal) cells are of a

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larger size than the former (20 to 40 p). The cells have a polygonal or cuboid shape and are found only in the white matter. The processes are fewer but thicker than those of type I and are more intimately related with the nerve fibers. Type TI1 (Paladino) cells are found in the cerebral and cerebellar peduncles, the medulla oblongata and the spinal cord. From the cell body 3 or 4 thick processes and some thin ones are sent out to the nerve fibers. The glial processes cover the nerve fibers over a wide area. Type IV (Schwannoid) cells are mono- or bipolar. These cells do not send out branches in the direction of nerve fibers?but they are directly attached to them.

Fig. 16. Top: oligodendrocytes of types I and 11. Bottom: oligodendrocytes of types I1 and 111. (Golgi-Hortega method.)

Some concepts regarding the function of the oligodendroglia. The oligodendrocytes are independent cells like the astrocytes, and two differences are important. Firstly, the oligodendrocytes show a lack of vascular contacts and intracytoplasmic gliofibrils cannot be observed. Secondly, they show an intimate relationship to the nerve fibers. Hortega suggested that these cells have mechanical, trophic and myelogenic functions. Recent studies with the electron microscope have indeed shown that myelin is produced by the oligodendrocytes. Recently in a series of works dedicated to the study of oligodendroglia, Cammermeyer maintains the opinion that the oligodendrocytes tend to orientate in relation to the blood vessels, arranging themselves in juxtaneuronReferences p . 33/34

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Fig. 19. Intersecting processes of a protoplasmatic astrocyte and an oligodendrocyte. (Golgi-Hortega method.)

Fig. 20. Picture showing the differences between a protoplasmatic astrocyte (left) and an oligodendrocyte (right). (Golgi-Hortega method.)

Fig. 17. Oligodendrocytesof types I11 and IV. (Golgi-Hortega method.) Fig. 18. Picture showing the difference between protoplasmic astrocytes and type I oligodendrocytes. (Golgi-Hortega method.) Rcfereiices p . 33/34

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Pig. 21. Two astrocytes in relation to the cell body of a nerve cell and to a blood vessel. (GolgiHortega method.)

a1 groups near the vascular arborizations. The oligodendrocytes would take part, as Cammermeyer suggests, in the intrinsic control of the blood vessels. The juxtavascular oligodendritic apparatus would record nervous, chemical and hemodynamic stimuli and would act upon the blood vessels by means of different effectors. Although the perfusion fixation method, used by the author, is superior to the classical method, we are not of the same opinion regarding the staining techniques for sections previously embedded in paraffin. The embedding of nervous tissue in paraffin and the study of its cellular components by means of aniline techniques is a rather insufficient method. The Golgi and Hortega metallic impregnations show a more complete picture of the oligodendroglia. But just as we maintain that the study of the cellular and fibrous components of the nervous tissue must be carried out fundamentally with the metallic impregnation techniques, we also say that their careful handling is essential. Frequently, in block impregnations with the Golgi method, cellular elements which are incompletely stained may be seen as fibrous of cellular fragments and the spherical bodies which appear orange-colored with this technique have been misinterpreted by Scheibel and Scheibel (1958) as oligodendroglial nuclei, being, in our experience incompletely stained oligodendrocytes. Neither we agree with their ‘synapsis’ between neurons and glial elements. The Golgi technique is also not appropriate for the demonstration of synapses. In summarizing this part we would like to state that only the methods of Golgi, Cajal and Hortega allow the differentiation of the astrocytes from the oligodendrocytes in nervous tissue. The following characteristics are important to note: the shape of the somas, the morphology of the processes, the fibrous structure of the cytoplasm and the relationship of the cells to neurons and blood vessels. Peripheral neuroglia Embryological data. The neuroglia of the sympathetic ganglia have a double origin : the majority originate from cubical cells of the neural crest that develop to become

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cells morphologically similar to the central glioblasts, which in turn mature to become the perisomatic and periexpansional gliocytes. The minority originate from cells in the neural tube and they show modifications similar to those of the neural crest. The perisomatic and periaxonal gliocytes of the spinal ganglia originate from cells of the neural crest. In the early part of this century there were extensive discussions as to whether these cells were of mesodermal or ectodermal origin. In 1924 Harrison carried out experiments on amphibian larvae, clearly establishing the fact that they were of ectodermal origin. The immature cells of the peripheral neuroglia, the lemmoblasts, may be compared to the glioblasts of the central neuroglia. With maturation, the lemmoblast becomes a lemmocyte, accompanying so-called myelinated and nonmyelinated nerve fibers as well as the free and the encapsulated nerve endings. These are called Schwann cells, Remak cells and terminal lemmocytes, respectively. The embryological and histological data have demonstrated that sympathetic and spinal ganglia also contain cellular elements of glial origin. These cells show a number of characteristics similar to those of central neuroglia. The oligodendroglia of type IV in the central nervous system is analogous to the cells of Schwann of the peripheral nerves. In the same way, the oligodendroglia of type I shows features similar to those of the perisomatic gliocytes of the sympathetic and spinal ganglia. Neuroglia of the sympathetic ganglia (Figs. 22, 23) Two fundamental types of gliocytes can be distinguished: the perisomatic and the peridendritic type (Hortega and Prado, 1942). Ordinary staining methods, such as the aniline and silver techniques, only show the nuclei and cytoplasm of these cells.

Fig. 22. Low-power micrograph of a sympathetic ganglion in which the perisomatic and peridendritic glial cells are shown. (Hortega method.) References p . 33134

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Fig. 23. Perisomatic and peridendritic glial cells with their typical morphological characteristics in a sympathetic ganglion. (Hortega method.)

Hortega developed a special modification of the ammoniacal silver-carbonate method to show the processes and other histological characteristics of these cells. The perisomatic glial cells, all of which are endocapsular, are arranged around the neuronal ceU body. The cells have a spherical shape with many processes of various lengths, which form complicated webs around the neurons. Laminar glial cells with reticular cytoplasm are sometimes seen in the same preparations. Tbeperiexpansional glial cells are poor in branches. These cells, often called spirocytes; have one or two branches arranged as spirals around the neurons. From a functional point of view it seems conceivablethat the sympatheticneuroglia is identical to the central glia, taking part perhaps in the metabolic activity of the sympathetic neurons. In the visceral sympathetic plexus, the characteristics of the neuroglia are similar to the ones just described. Neuroglia of the spinal ganglia (Figs. 24, 25) Even though the demonstration of glial elementsin these organs is more difficult than in the sympathetic ganglion, careful studies have shown the following types of cell (Hortega et al., 1942). The perisomatic glial elements have a spherical or polygonal cell body with one or two processes, arranged around the perikarya of the neurons and among the connective capsules. The periaxonal glial elements are primarily found in close association with the nerve fibers. They are more abundant in the glomerular region. These elements could be compared to the oligodendrocytesand the perisomatic to protoplasmic astrocytes.

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Fig. 24. Neurons of a spinal ganglion surrounded by glial elements. (Hortega method.)

Fig. 25. Neuron of a spinal ganglion surrounded by glial elements at high magnification. (Hortega ' method.) References p . 33/34

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Fig. 26. Nerve fibers surrounded by Schwann cells. (Golgi-Hortega method.)

Neuroglin of the nerves and nerve endings (Fig. 26) The myelinated nerve fibers are covered by Schwann cells, with morphological characteristics similar to the Schwann-type oligodendrocytes. Cells of Remak are found in the sympathetic nerve fibers. The encapsulated nerve endings have spherical and ovoid cells with one or two processes which are difficult to stain and which have been called terminal lemmocytes. SUMMARY

Neuroeinbryological introduction. The primitive neural tube is lined by two cellular types : germinal cells and spongioblasts. The former gives origin to neuroblasts, while the spongioblasts give origin to the different types of neuroglia (glioepithelial cells, astrocytes and oligodendrocytes). At the level of the peripheral nervous system (sympathetic ganglia, spinal ganglia, peripheral nerves and nerve endings) the neuroglial cells evolve from the neural crest and possibly from the neural tube. The technical problem. Aniline techniques show only the nucleus and part of the cytoplasm, and are therefore insufficient for the study of neuroglia. This insufficiency has been overcome by several techniques, among which those of Cajal, Hortega and Golgi are invaluable. Definition of terms. Under the term central and peripheral neuroglia, we include those non-neuronal cells of ectodermal origin excluding the microglial cells of mesodermal origin, representing the reticulo-endothelial system of the nervous tissue. Central neuroglia. A description is given of the morphological and structural pattern

CHARACTERISTICS OF CENTRAL A N D PERIPHERAL NEUROGLIA

33

of the glioepithelial cells (ependymal and ventricular), the protoplasmic and fibrous astrocytes with their various ;types and the 4 fundamental types of oligodendrocytes. An analysis is given of the fibrillar differentiation of astrocytes and its relation to blood vessels as well as the relation of oligodendrocyres to nerve fibers. Peripheral neuroglia. The sympathetic and spinal ganglia have been described with respect to the perisomatic and periexpansional glial cells as well as to the cells of Schwann and Remak of the peripheral nerves and the terminal glial elements of the free and encapsulated nerve endings.

REFERENCES ANDRIEZEN, W., (1893); On a system of fiber cells surrounding the blood vessels of the brain of man and mammals and its physiological significance. Int. Monat. Anat. Physiol., 10, 533. BAIRATI, A., (1948); Osservazioni comparate sulle glioarchitectonica. Mem. Accad. SOC.Ist. (Bologna), 9, 613. CAJAL,S . RAMONY, (1910/1911); Histologie du Syst2me Nerveux de I'Homme et des Verribris. Paris, Maloine. CAJAL,S. RAMONY, (1913); Contribucion a1 conocimiento de la neuroglia del cerebro humano. Trab. Lab.Invest. biol., 11, 255-315. CAJAL,S. R A M ~Y,N(1925); Contribution A la connaksance de la nevroglie drebrale et &rebelleuse dans la paralysie generale progressive. Trub. Lab. Invest. biol., 23, 157-216. CAJAL,S. R A M ~Y,N (1932); Etudes sur la nevroglie (macroglie). Trab. Lab. Invest. Biol., 11, 27. CAJAL,S. R A M ~Y,N(1933); Histology. London, Balliere, Tindall and Cox. DE CASTRO, F., (1920); Estudios sobre la neuroglia de la corteza cerebral del hombre y de 10s animales. J . Neurol. Psychopath., 9 , 209. DECASTRO, F., (1946); Sobre el comportamiento y significacion de la oligodendroglia en la substancia gris central y de 10s gliocitos en 10s ganglios nerviosos perifericos. Arch. Hist. Norm. Pat., 3, 317. DE CASTRO, F., (1951); Anatomical aspects of the ganglionic synaptic transmission in mammalians. Translated from Arch. int. Physiol., 59, 479. GLEES,P., (1955) ; Neuroglia, Morphology and Function. Springfield (Ill.), Charles C. Thomas. GOLGI,C., (1873); Sulla struttura della sostanza grigria del cervello. Gazz. med. ital. lombardia, 6. GOLGI,C., (1885-86); Sulla fina Anatomia della Sistema Nervoso. S e e Opera Omnia, Milan, 1903. GOLGI,C., (1894); Untersuchungen iiber denfeineren Bau des Centralen und PerQheren Nervensystems. Jena, Fischer. HARRISON, R. G., (1924); Neuroblast versus sheat cell in the development of peripheral nerves. J . comp. Neurol., 37, 123. HELD,H., (1909) ; Uber die Neuroglia marginalis der menschlichen Grosshirnrinde. Mschr. Psychiat. Neurol., 26, 360. HILD, W., (1954); Histological and endocrinological observations in tissue cultures of posterior pituitary of dog and rat. Tex. Rep. Biol. Med., 12, 474. HIS, W., (1889); Die Neuroblasten und deren Entstehungen im embryonalen Mark. Arch. Anat. physiol., 5, 249-300. HORTEGA, P. DEL RIO, (1916); Contribution a l'etude d'histopathologie de la nkvroglie. Ses variations dans le ramollissement ckrebral. Trab. Lab. Invest. biol., 14, 1-34. P. DEL Rio, (1916); Estructura fibrilar del protoplasma neuroglico y origen de ]as glioHORTEGA, fibrillas. Trab. Lab. Invest. Biol. (Madrid), 14, 269. HORTEGA, P. DEL RIO, (1917); Alteraciones de la neuroglia en la intoxication por pilocarpina. Lab. Rev. Cienc. Biol. Med. ESP.,Barcelona. HORTEGA, P. DEL RIO, (1918); Noticia de un nuevo y facil metodo para la coloracio de la neuroglia y del tejido conjuctivo. Trub. Lab. Invest. biol., 15, 367-378. P. DEL RIO, (1919); El terder elemente de 10s centros nerviosos. Bol. Soc. esp. Biol., 9, HORTEGA, 69-120. HORTEGA, P. DEL RIO,(1920); La microglia y su transformacion en celulas en bastonicjto y cuerpos, granulo-adiposos. Trab. Lab. Invest. biol., 18, 37-83.

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HORTEGA, P. DEL RIO,(1924); Lo que debe enterderse por tercer elemento de 10s centros nerviosos. Bol. SOC.esp. Biol., 11, 33-35. HORTEGA, P. DEL RIO,(1924-25); Le nevroglie et le troisieme element des centres nerveux. Bull. SOC.Sci. mid. biol., Montpellier, 5. HORTEGA, P. DEL RIO, (1925); Participation de la microglia en la formacion de 10s cuerpos amilaceos del tejido nervioso. Bol. SOC.esp. Hist. nat., 27, 127. HORTEGA, P. DEL RIO, (1927); Lesiones elementales de 10s centros nerviosos. Rev. mid. Barcelona, 8, 36-70. HORTEGA, P. DEL RIO, (1928); Tercera aportacion a1conocimiento morfologico e interpretacion funcional de la oligodendroglia. Mem. Real. SOC.esp. Hist. nat., 14, 5. HORTEGA, P. DEL RIO, (1932); Microglia. Cytology and Cellular Pathology of the Nervous System. Bd. 11. Penfield (pp. 483-534). HORTEGA, P. DEL RIO,(1943-45); Ensayo de clasification de las alteraciones celulates del tejido nervioso. 11. Alteraciones de las celulas neuroglicas. Arch. Histol. B. Aires, 2, 5-100. HORTEGA, P. DEL RIO,(1949); Art and Artifice in Histologic Science. Tex. Rep. Biol. Med., 7,363-390. HORTEGA, P. DEL RIO, AND ASUA,F. J., (1921); Sobre fagocitosis en 10s tumores y en otros processos patologicos. Arch. Cardiol. Hematol., 2,161-220. HORTEGA, P. DEL Rfo, POLAK,M., AND PRADO,J. M., (1942); Investigaciones sobre la neuroglia de 10s ganglios sensitivos. Arch. Hist. Norm. Pat., 1, 234. HORTEGA, P. DEL No, AND PRADO,J. M., (1942); Investigaciones sobre la neuroglia de 10s ganglios simpaticos. Arch. His?.Norm. Pat., 1, 83. KOLLIKER, A., (1980); Zur feineren Anatomie des central-Nervensystem. 2nd Beitr. das Ruckenmark. Ztschr. wiss. Zool., 51. KOLLIKER, A., (1896); Handbuch der Gewebelehre des Menschen. Bd. II,6. Auflage. Leipzip, Wilhelm Engelmann. M. W., (1895); Das Mervensystem im Lichte neuester Forschungen. Berlin. LENHOSS~K, LUGARO, E., (1907); Sulle funzioni della nevroglia. Riv. Pat. nerv. ment., 12. MAWAS,P., (1910); Note sur la structure et la signification glandulaire probable des cellules n6vrogliques du systeme nerveux central des vertebres. C . R. SOC.Biol. (Paris), 49. J., (1929); Phenomenes de skretion dans le protoplasma des cellules nkrogliques de la NAGEOITE, substance grise. C. R. SOC.biol. (Paris), 68, 1068. PALADINO, G., (1892); De la continuation de la nkvroglie dans le squelette myelinique des fibres nerveuses et de la constitution pluricellulaire du cylindraxe. Arch. ital. Biol., 19, 26. W., (1924); Oligodendroglia and its relation to classical neuroglia. Brain, 47, 43W52. PENFIELD, POLAK,M., (1958); El valor de las tknicas de impremacion metklica y resultados de su aplicacibn en el estudio de la histologia normal y patoI6gica. Arch. Hist.Norm. Pat., 6, 261. POLAK,M., (1956); Sobre una variante de la tecnica de Rio Hortega para la impregnacion de celulas reticuloendoteliales normales y patol6gicas, micro y oligodendroglia etc. Arch. Hist.Norm. Par., 6, 220. J., (1836); Uber Flimmerbewegungen im Gehirn. Miillers Arch., 289. PURKINJE, ROBERTSON, W. F., (1897); The normal histology and pathology of the neuroglia. J. ment. Sci., 43, 729. ROBERTSON, W. F., (1900a); A microscopic demonstration of the normal and pathological histology of mesoglia cells. J. ment. Sci., 46, 733-752. W. F., (1900b); A Textbook of Parhology in Relation to Mental Diseases. Edinburgh, ROBERTSON, William F. Clay. SCHAPER, A., (1894); Die morphologische und histologische Entwicklung des Kleinhirn der Teleostier. Morph. Jb. SCHAPER, A., (1897); Die fruhesten Differenzierungsvorgange im Central-nervensystem. Arch. En?wickl.-Mech. Org., 5, 81-132. SCHEIBEL, M. E., AND SCHEIBEL, A. B., (1958); Neurons and neuroglia cells as seen with the light microscope. Biology and Meuroglia. W. F. Windle, Editor. Springfield, Thomas (p. 5). VALENTIN, G., (1836); Fortgesetzte Untersuchungen iiber die Flimmerbewegungen. Repert. Anal. Physiol., 1, 148.

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Enzyme Histochemistry of Neuroglia R E I N H A R D L. F R I E D E Mental Health Research Instituie and Department of Pathology, The University of Michigan, Ann Arbor, Mich. (U.S.A.)

(A) H I S T 0 C H E M I S TR Y 0F 0 L I G 0 DEN D R 0 G L I A

( I ) Enzyme content of oligodendroglia Histochemical studies have shown marked activity of oxidative enzymes in oligodendroglia (Wolfgram and Rose, 1959; Friede, 1961b, 1962b, and others). The activity is in the cytoplasm and processes, but never in the nucleus. In comparison with other elements of white matter, oligodendroglia have more oxidative enzymatic activity than normal astrocytes, axons or the endothelia of blood vessels. ( I I ) Types of oligodendroglia cells Hortega described 4 types of oligodendroglia based on the mode of ramification of their processes. Type IV resembles Schwann cells; these cells are attached to large axons. Type I11 is characterized by husky processes which form rings or spirals around axons. Histochemically, types IV and I11 oligodendroglia cells have a very intense enzymatic reaction; they are found in the cranial nerve motor roots, certain spinal tracts, the longitudinal fascicle, and the pyramidal tracts. Hortega types I and I1 oligodendroglia cells have less enzymatic activity than types I11 and IV and are found in other fiber tracts, particularly in the cerebral and cerebellar hemispheres (Friede, 1961b). Recognition of these types, and of their characteristic distribution among tracts is of importance for the interpretation of ‘changes’ in oligodendroglia cells. (111) Satellite cells Under favorable conditions, it can be shown that neuronal satellite cells contain marked cytochrome oxidase activity (Roizin, 1951). In our experience, satellite cells have not usually been sharply delineated from each other, but they form an indistinct ‘cloudy’ region of oxidative enzymatic activity around the neuron (Friede, 1962a). Because the intensity of enzymatic reaction in them is about equal to that in the surrounding neuropil, satellite cells are difficult to distinguish, except in those regions where there is very little neuropil.

(IV) A comparison of various oxidative enzymes in oligodendroglia Comparing the relative intensity of the reactions in oligodendroglia of enzymes References p . 46/47

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involved in glycolysis, hexosemonophosphate shunt, and citric acid cycle, one notes that oligodendroglia have a relatively strong reaction for enzymes involved in hexosemonophosphate shunt and glycolysis and a weak reaction for citric acid cycle enzymes. In particular, glucose-6-phosphate dehydrogenase is extremely intense in oligodendroglia cells in white matter (Romanul and Cohen, 1960; Schiffer and Vesco, 1962; Friede et al., 1963b) and in] satellite cells (Schiffer and Vesco, 1963). Comparing several enzymes, the relative intensity of reaction in oligodendroglia cells decreases in approximately the following order : glucose-6-phosphate dehydrogenase, lactic dehydrogenase, DPN-diaphorase, malic dehydrogenase, succinic dehydrogenase, and cytochrome oxidase. This gradient of activity among the enzymes in oligodendroglia cells is in correlation with the gradients of activity for these enzymes between gray and white matter as reported in the biochemical studies of Buell et al. (1958) and Robins et al. (1957). The correlation of histochemical and biochemical findings confirms the conclusion by Pope and Hess (1957) that the magnitude of an enzymatic gradient between gray and white matter is mainly a reflection of the enzymatic properties of oligodendroglia. Evidently, oligodendroglia have a relatively better supply of the enzymes involved in the hexosemonophosphate shunt or glycolysis than those of the citric acid cycle. ( V ) Increased enzyme reaction in glia during myelination Myelination of fiber tracts is preceded by a marked increase of enzymatic activity in the glial cells (Friede, 1957, 1961b). This enzymatic change correlates with the period of glial proliferation at the beginning of myelination, a time at which oligodendroglia differentiate from undifferentiated precursor glial cells. Proliferation and increased enzymatic activity slightly precede the visible formation of myelin sheaths and persist during the period of active formation of sheaths. The increase of enzymatic activity in glia has also been observed during myelination in tissue culture (Yonezawa et al., 1962). ( B ) H I S T O C H E M I S T R Y OF A S T R O C Y T E S

( I ) Enzyme supply of normal and reactive astrocytes Normal astrocytes have very little supply of oxidative enzymes, as compared with oligodendroglia, nerve cells, or ependyma. This is seen in microscopic studies of histochemical preparations from any part of the brain (Ogawa and Zimmermann, 1959), and also by systematic studies of selected regions which contain almost exclusively astrocytes, such as the anterior medullary velum or the subependymal glia layer (Friede, 1962b). One exception to the generally low supply of oxidative enzymes in astrocytes may be noted : the reaction for glutamate dehydrogenase is relatively intense, staining the astrocytes almost like a silver impregnation. In contrast to normal astrocytes, hypertrophic (reactive, swollen) astrocytes exhibit an extremely intense reaction for all oxidative enzymes. In human material the intensity of enzymatic reaction in reactive astrocytes often exceeds that of any other cell type present in the section. No other cell type in the CNS has been found capable of such extreme changes of enzymatic activity.

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(II) Selectivity of enzymatic changes in astrocytes The development of hypertrophy of astrocytes can be studied histochemically by comparing the intensity of enzymatic reaction in astrocytes and oligodendroglia cells in white matter at various distances from a focus of disease (infarct, tumor), or by making such comparison in brains with different degrees of astrocytic reactivity. The same findings are observed in both types of study; the enzymatic reaction is always increased in reactive astrocytes, and the degree of hypertrophy and enzyme increase usually parallels the local intensity of edematous changes. The enzymatic reaction in oligodendroglia does not noticeably change with increasing intensity of the edema, up to the time when these cells disappear from the tissue. Astrocytes, in contrast, exhibit a gradual increase of enzymatic activity (Fig. 1 A and B) and survive beyond the time at which oligodendroglia disappear. Throughout the period of enzyme increase, astrocytes can be distinguished from oligodendroglia in enzyme preparations counterstained with chromalum gallocyanin and in adjacent sections treated with silver impregnation. Astrocytes are identified by their typical shape, and by their vascular processes with footplates (Fig. 1A). Also, a comparison of the relative frequency of these cell types in the given area is suggestive of whether or not identification has been correct. These studies give no support to

Fig. 1. (A) Slight staining of an astrocyte(arrow) anti its vascular process; the intensity of the reaction is less than that it: oligodendroglia;cerebral white matter, DPN-diaphorase.(B) Edema near arterioscleroticinfarct; excessive reaction in swollen astrocytes (arrows); no changes in oligodendroglia. References p . 46/47

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Fig. 2. Reactive astrocytes in the neuropil of gray matter. The vascular processes (arrow) of the cells are clearly demonstrated by enzymatic activity. Lactic dehydrogenase.

TABLE I PATHOLOGICAL CONDITIONS I N WHICH INCREASE OF OXIDATIVE ENZYMATIC ACTIVITY H A S BEEN OBSERVED I N REACTIVE ASTROCYTES

Collateral edema of brain tumors Multiple sclerosis Experimental cyanid encephalopathy Experimental cold injury Experimental anoxia Cerebral infarcts Diffuse sclerosis Amyotrophic lateral sclerosis; scars; near tumors Collateral edema of brain tumors Collateral edema of brain tumors Experimental brain wounds Alzheimer’s disease In and near brain tumors In and near brain tumors In and near brain tumors Multiple sclerosis

Friede, Friede, Van Houton and Friede, Rubinstein et al., Colmant, Friede, Nelson et al., Osterberg and Wattenberg, Schiffer and Vesco, Miiller and Nasu, Kreutzberg and Peters, Friede and Magee, SchSer and Vesco, Smith,

1958 1961a 1961 1962 1962 1962a 1962 1962a 1962 1962 1962 1962 1962 1963

Rubinstein and Sutton, Ibrahim and Adams,

1963 1963

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the concept that hypertrophic astrocytes are derived from oligodendroglia (Koenig and Barron, 1962). Distinction of normal astrocytes from oligodendroglia is easier in human material, than in material from other species. This is particularly true in the hemispheres where usually a very uniform population of oligodendroglia is seen. In the brain stem, gradations of the enzymatic reaction in the various types of oligodendroglia render a comparison of oligodendroglia with astrocytes more difficult than in the hemispheres. The intense enzymatic reaction of hypertrophic astrocytes is the same in white matter and gray matter. In the latter, the enzymatic activity of reactive astrocytes often exceeds the staining of the adjacent neuropil or of nerve cells (Fig. 2). (III) Pathological conditions which cause astrocytic hypertrophy That hypertrophy of astrocytes is accompanied by marked enzymatic changes has been reported by all workers in this field. Pertinent literature has been arranged in a table showing a variety of neuropathological conditions under which enzymatic changes in hypertrophic astrocytes have been observed (Table I).

( I V ) Relative participation of various enzymes in the response of hypertrophic astrocytes Comparative histochemical studies of the relative increase of various enzymes in hypertrophic astrocytes have been done by Rubinstein et al. (1962), and Osterberg and Wattenberg (1962a). Our observations are basically in agreement with their findings. Comparison of various oxidative enzymatic reactions in hypertrophic astrocytes indicates that an increase can be observed earlier for those enzymes involved in glycolysis or hexosemonophosphate shunt than for enzymes involved in the citric acid cycle (e.g. succinic dehydrogenase). Among the enzymes studied, lactic dehydrogenase is the most sensitive indicator of early reactive changes in astrocytes, followed by :DPN-diaphorase, TPN-diaphorase, succinic dehydrogenase, cytochrome oxidase. The comparison of intensity of various histochemical reactions among each other is difficult and in making such comparisons, one should keep in mind that any histochemical technique is more dependable for demonstrating gradations of an enzyme among cells and regions in a given section than for comparison of various enzymes among each other. It must also be recognized that the general intensity of a histochemical reaction depends on various factors of methodology; this does not usually affect the gradient of activity between cells in a given section, but it is difficult to compare the intensity of various enzymatic reactions among each other. E.g. most enzymes are subject to some solubilization and leakage from the section, the extent of which is dependent on the thickness of the section and other factors. Leakage of some enzymes is prevented by fixation in which case fixation time, fixation temperature, and extent to which fixative is rinsed out of the section after cutting are important factors. Also, incubation time and minor variations of the composition and pH of the media affect the intensity of a reaction (Friede et al., 1963a). Thus slight differences in methodology can lead one observer to find a weak reaction whereas another does not see any reaction in the same area. Also, knowledge of normal gradations of a specific References p . 46/47

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enzyme among cells helps one to determine whether the enzymatic activity in a certain cell type has changed from normal. For example, we have observed in the brain of the rhesus monkey (Friede and Fleming, 1963) that the reaction for lactic dehydrogenase in normal astrocytes varies among regions ; those in the superficial portion of the molecular layer of the cerebral cortex and Bergmann’s glia cells in the cerebellum normally show a distinct reaction for lactic dehydrogenase. In all other areas of the brain, there is very little or no lactic dehydrogenase activity in astrocytes. ( V ) Special or regional types of astrocytic hypertrophy (a) Subpial astrocytes. Subpial astrocytes respond enzymatically in the same way as the astrocytes in gray and white matter. When these cells are reactive, their processes, which are directed toward the pial surface where they terminate in footplates, are demonstrated by an intense enzymatic reaction. The footplates often form small tent-shaped elevations at the pia, containing an intense enzymatic reaction. (b) Membrane of molecular layer covering infarcts. In cortical infarcts, the superficial portion of the molecular layer usually survives, forming a thin membrane which covers the cavity of the infarct. This membrane in which hypertrophic astrocytes abound (Friede, 1962a) has a much higher level of enzymatic activity than is normal for this portion of the molecular layer. This observation is probably significant for two reasons: first, membranes covering infarcts provide a unique opportunity to obtain reasonably pure samplings of hypertrophic astrocytes for metaboIic studies; second, since normal cellular composition in this layer is almost exclusively astrocytic, it seems unlikely that hypertrophic astrocytes are derived from any other cell type than normal astrocytes. (c) Bergmann’s glia cells. It would be of great interest to know whether Bergmann’s glia cells in the cerebellum respond enzymatically like astrocytes or like oligodendroglia. As yet, we have not observed increased enzyme in Bergmann’s glia in a variety of human pathological material, including various types of cerebellar degeneration. However, in encephalitic guinea-pig brain, we have seen Bergmann’s glial processes and footplates at the pial membrane with a markedly increased reaction for several oxidative enzymes (Fig. 3). These few observations suggest that Bergmann’s glial cells also are capable of adaptive enzymatic response to a pathological situation. In studies of at least 12 species we have never seen such increased enzymatic activity in Bergmann’s glial processes and footplates of normal animals. (d) Miiller’s cells in the retina. Observations by Kuwabara and Cogan (1960) indicate that Muller’s glia cells in the retina respond to injury with an increase of oxidative enzymes, particularly lactic dehydrogenase. This suggests that Muller’s cells also may be in the same general category as astrocytes. (e) Neoplastic astrocytes. Enzyme histochemical studies of gliomas have been reported by Friede (1958), Mossakowski (1962), Schiffer and Vesco (1962), Smith (1963), and Rubinstein and Sutton (1963). Marked variances of enzyme reaction were noted among individual cells but opinions vary as to an overall increase or decrease of the reactions. Correlations between the type of tumor and the distribution

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Fig. 3. Pathological staining of Bergmann’s glial fibers and their footplates in guinea-pig cerebellum. This reactivity is not seen in normal material from various species.

Fig. 4. Minimal reaction of DPNcliaphorase in Alzheimer’s glia cells (arrows) in a case of hepatolenticular degeneration. References p. 46/47

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of several oxidative enzymes in astrocytes were described by Rubinstein and Sutton (1963). (VI) Failure of development of astrocytic enzyme changes (a) Lesions in immature brain. Experimental lesions in the immature rat at any time prior to 10 days of age do not produce reactive gliosis with the typical hypertrophy and increased enzyme. This may indicate an inability of the immature glia cell to respond or an absence of postulated inducing substances, or both (Osterberg and Wattenberg, 1962b and 1963). (b) Alzheimer’s glia in Wilson’s disease. At this point, I would like to report another observed exception to the rule of hypertrophic astrocytes having an increased enzyme supply. A 13-year-old boy had the symptoms, laboratory findings, and clinical diagnosis of Wilson’s disease. Neuropathologically, the diagnosis was confirmed by an advanced bilateral degeneration, necrosis, and scar formation in putamen and nucleus caudatus and characteristic histological changes. In this case, the majority of Alzheimer’s glial cells showed a weak reaction for DPN-diaphorase in the voluminous cytoplasmic bodies, so that they were just barely discernible in the tissue. The cell processes also showed little enzymatic reaction (Fig. 4). A few typical hypertrophic astrocytes with a strong enzymatic reaction were also seen near the scar tissue in the basal ganglia and were scattered among the Alzheimer’s glial cells. This observation suggests that the enzymatic response is not inherently linked with cytoplasmic hypertrophy. Another explanation might be that the low enzyme supply in Alzheimer’s glial cells is merely a function of their age; however, this seems unlikely since Osterberg and Wattenberg (1962a) showed that hypertrophy and enzyme increase persist in astrocytes for as long as 4 years after the injury. (VII) Causes of hypertrophy of astrocytes Exactly which factors trigger the enzymatic response of astrocytes are unknown. Edema represents one rather universal phenomenon under which reactive enzymatic changes in astrocytes occur. Osterberg and Wattenberg (1962b, 1963) suggested that enzymatic changes in astrocytes may be triggered by breakdown products of the myelin sheath, as suggested by the development of reactive astrocytes in Wallerian degeneration of central tracts and near plaques of multiple sclerosis. Our observations, however, indicate that the extent of astrocytic response is independent of the extent of destruction of myelin in any given lesion. Changes in reactive astrocytes near or in small cortical infarcts where no white matter is involved (Friede, 1962a, Figs. 1-4) are usually more developed than those seen near demyelinating plaques (Friede, 1961a; Ibrahim and Adams, 1963). Recently we have used tissue cultures of newborn rat cerebral cortex for studying the effect of variation of the ionic composition of the environment on enzymatic activity of astrocytes (Friede, 1964). It was found that the oxidative enzymatic activity (succinic dehydrogenase, DPN-diaphorase, and several other enzymes) of astrocytes is influenced by the concentration of sodium in their environment. Elevation of sodium elicited an increase of oxidative enzymatic activity in astrocytes, while as-

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Fig. 5. Intense reaction of succinic dehydrogenase in astrocytes grown for 8 days at elevated Na concentration (205 mequiv/l Na+).

trocytes in cultures grown at low sodium concentration did not show any enzymatic response (Fig. 5). Of the salts used, only sodium chloride could elicit such a response at concentrations which conceivably can exist in a living organism. However, this response is not specific for sodium. Lithium and magnesium salts (probably also calcium) did elicit a similar response, but only when excessive unphysiological concentrations were used. Potassium chloride in excessive unphysiological concentrations elicited only a weak and often questionable response. Astrocytes were the only cells which responded this way, and it appeared that the footplates were particularly involved in the response, since enzyme increased first in the footplates and later in the perikarya. The ‘low’ sodium concentrations used in our cultures were in the range of that reported for normal brain, while the increase of sodium necessary to produce enzymatic changes in astrocytes in culture was approximately in the magnitude of the increase of sodium reported for edematous brain tissue. Thus, we have found that the NaCl concentrations used in tissue culture to elicit enzymatic increase in astrocytes were comparable with the concentration range in pathological brain tissue, where similar enzymatic changes are found in astrocytes in situ. It was concluded that astrocytes are metabolically involved in the maintenance of the ionic environment of the central nervous system, particularly in regard to the active transport of sodium. The conclusion that an increased rate of transport of sodium required an increased energy supply in these cells is well in agreement with current concepts of sodium transport. References p. 46/47

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A W O R K I N G HYPOTHESIS O N G L I A L F U N C T I O N

Based on observations recorded in the preceding text, and others quoted below, we would like to propose a working hypothesis regarding the physiological role of neuroglia cells. It seems that astrocytes and oligodendroglia have significantly different physiological functions. Astrocytes probably belong to a group which includes several other types of glial cells, all having the common feature of processes and footplates which link the vascular and meningeal surfaces of nervous tissue to the interior. Astrocytes, Miiller’s cells in the retina, Bergmann’s glia in the cerebellum, embryonal spongioblasts and embryonal ependymal cells, the ‘tanycytes’ (Horstmann, 1954)which send one long process to the surface of the brain, and the various types of glia in insects and other invertebrates apparently belong in this group. Presumably these cells are engaged in the maintenance of a constant environment for the nerve cells, especially in regard to the ionic composition of tissue fluids (de Robertis et al., 1960; Katzman, 1961; Katzman and Wilson, 1961; Giacobini, 1962; and Koch et al., 1962). Probably, maintenance of the environment also includes the removal of solid waste products (e.g. lipofuscin; Friede, 1962~).In vertebrates, these cells have less oxidative enzyme supply than oligodendroglia. Apparently they have a special tendency to contain glycogen, e.g. in subependymal glial tissue and especially, the glia of invertebrates (Oksche, 1961). Oligodendroglia represent a later phylogenetic acquisition ; invertebrates and some primitive vertebrates do not have oligodendroglia cells. We would like to propose that the oligodendroglia cells represent ‘auxiliary metabolic units’ (Friede and Van Houton, 1962), necessary for maintenance of normal metabolism throughout the extreme morphological extensions of the nerve cell and also for particular functional demands put on the neuron. Instead of simple hypertrophy of the perikaryon or bizarre elongations of its nucleus, the nerve cell has individual ‘auxiliary units’ which attach at sites of special metabolic needs. (a) Special metabolic needs may be conditioned anatomically by the extreme extension of the cell processes ; this would explain the correlation observed between axonal length and number of satellite cells (Friede and Van Houton, 1962) as well as the increase in the glia-index (number of glia cells per one nerve cell) with increasing size of the brain (Friede, 1954; Hawkins and Olszewski, 1957; Friede, 1963). (b) Special metabolic needs may result from the formation of special ‘apparatus’, such as the myelin sheath. Evidently, the perikaryon of a nerve cell could not possibly provide metabolically for the synthesis of the myelin sheath of the axon, which represents many times the volume of the perikaryon. This conclusion would be in keeping with neuron-glia relationship during myelination (see above). (c) Special metabolic needs, likewise, may result from extreme functional activity; this could explain the numerical increase of satellite cells of motor neurons during forced motor activity (Kulenkampff, 1952), and also the subtle relationships of neuronal and glial metabolism shown by Hydtn and coworkers (1960, 1962). The concept of ‘auxiliary metabolic units’ implies that there are no over-all, inherent characteristics of the oligodendrogliaper se, but rather a dynamic equilibrium

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controlled by the needs and supply of that portion of the neuron to which the oligodendroglia cells are attached. For example, Hamberger (1963) showed metabolic differences between vascular and neuronal glia. This concept is further supported by the fact that there is an inverse relationship of oxidative enzymatic activity in oligodendroglia and axons in the various fiber tracts of the brain; tracts with great enzymatic activity in axons have little activity in oligodendroglia, and vice versa (Friede, 1961b). It seems very likely that these cells have a symbiotic relationship with a neuron and acquire those functions which are required by the immediate metabolic needs of the neuron. Therefore, any search for inherent or permanent metabolic properties of oligodendroglia will probably give inconsistent results. SUMMARY

A survey of the histochemical distribution of oxidative enzymes in neuroglia is given. Oligodendroglia normally contain more oxidative enzymatic activity than astrocytes. Among the individual enzymes involved in the hexosemonophosphate shunt and citric acid cycle, oligodendroglia are particularly rich in the former (particularly glucose-6-phosphate dehydrogenase) but have comparatively little activity of the citric acid cycle enzymes. This suggests a predominance of glucose shunt metabolism in oligodendroglia. Normal astrocytes have less enzymatic activity than oligodendroglia cells. Astrocytic hypertrophy, however, is accompanied by an extreme increase of oxidative enzymes. Various experimental studies were done in search for factors which induce this metabolic change in astrocytes. The effect of environmental ion concentration on the enzymatic activity of astrocytes was investigated in tissue cultures of rat cerebral cortex. It was found that the oxidative enzymatic activity (succinic dehydrogenase, DPN-diaphorase, and several other enzymes) of astrocytes depended on the concentration of NaCl in the environment. This response was not specific for NaC1, but was also elicited by MgC12 and LiC1; the response was less consistent, and often questionable for KC1. However, only NaCl could elicit enzymatic changes in astrocytes at concentrations known to be present in a living organism. Astrocytes were the only cells which responded this way; it appeared that the footplates were particularly involved in the response since increase of enzymes occurred earlier in the footplates than in the perikarya. It was concluded that astrocytes are metabolically involved in the maintenance of the ionic and osmotic environment of the central nervous system, particularly in regard to the active transport of sodium. Preliminary experiments suggest that astrocytes respond in a similar manner in vivo if exposed to NaCI. ACKNOWLEDGEMENT

This work was supported by U.S. Public Health Grant B 3250. References p . 46/47

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REFERENCES N. R., CHANG,M. W., AND KAPPHAN, J. I., (1958); The BUELL,M. Y., LOWRY,0. H., ROBERTS, quantitative histochemistry of the brain. J. biol. Chem., 232, 979-993. COLMANT, H. J., (1962); Histochemical detection of various enzymes in selective neuronal necrosis of rat brain. Proc. ZVth Znt. Congr. Neuropath. Stuttgart, 1, 89-94. DE ROBERTIS, E., GERSCHENFELD, H. M., AND WALD,F., (1960); Ultrastructure and function of glial cells. Structure and Function of the Cerebral Cortex. D. B. Tower and J. P. Schade, Editors. Amsterdam, Elsevier (p. 69-80). FRIEDE,R. L., (1954); Der quantitative Anteil der Glia an der Cortexentwicklung. Acta anat., 20, 230-296. FRIEDE,R. L., (1957); Die histochemische Reifung des Kleinhirnes der Ratte, dargestellt durch das Verhalten der Succinodehydrogenase. Arch. Psychiat. Z. Neurol., 196, 196-204. FRIEDE,R. L., (1958); Histochemischer Nachweis von Succinodehydrogenase in Biopsien von menschlichem Hirngewebe. Virchows Arch. path. Anat., 322, 216-223. R. L., (1961a); Enzyme histochemical studies in multiple sclerosis. A.M.A. Arch. Neurol., 5, FRIEDE, 43343. R. L., (1961b); A histochemical study of DPN-diaphorase in human white matter; with some FRIEDE, notes on myelination. J. Neurochem., 8, 17-30. FRIEDE, R. L., (1962a); An enzyme histochemical study of cerebral arteriosclerosis with some data on the pathogenesis of periarterial scars. Acta neuropath., 2, 58-72. FRIEDE,R. L., (1962b); Cytochemistry of normal and reactive astrocytes. J. Neuropath. exp. Neurol., 21,471-478. FRIEDE,R. L., (1962~);The relation of the formation of lipofuscin to the distribution of oxidative enzymes in the human brain. Acta neuropath., 2, 113-125. FRIEDE, R. L., (1963); The relationship of body size, nerve cell size, axon length and glial density in the cerebellum. Proc. nut. Acad. Sci., 49, 187-193. FRIEDE,R. L., (1964); The enzymatic response of astrocytes to various ions in vitro. J. Cell Biol., 20, 5-15. FRIEDE, R. L., AND FLEMING, L. M., (1963); A mapping of the distribution of lactic dehydrogenase in the brain of the rhesus monkey. Amer. J. Anat., 113, 215-234. L. M., AND KNOLLER, M., (1963a); A quantitative appraisal of enzyme FRIEDE,R. L., FLEMING, histochemical methods in brain tissue. J. Histochem. Cytochem., 11, 231-245. M., (1963b); A comparative mapping of enzymes FRIEDE,R. L., FLEMING,L. M., AND KNOLLER, involved in hexosemonophosphate shunt and citric acid cycle in the brain. J, Neurochem., 10, 263-277. FRIEDE,R. L., AND MAGEE, K. R., (1962); Alzheimer’s disease. Neurology, 12,213-222. FRIEDE,R. L., AND VANHOUTON,W., (1962); Neuronal extension and glial supply; functional significance of glia. Proc. nar. Acad. Sci., 5 , 817-821. GIACOBINI, E., (1962); A cytochemical study of the localization of carbonic anhydrase in the nervous system. J. Neurochem., 9,169-178. HAMBERGER, A., (1963) ; Difference between isolated neuronal and vascular glia with respect to respiratory activity. Acta physiol. scand., 58, Suppl. 203. HAWKINS, A., AND OLSZEWSKI, J., (1957); Glia nerve cell index for cortex of the whale. Science, 126, 76-77. HORSTMANN, V., (1954); Die Faserglia des Selachiergehirns. Z. Zellforsch., 39,588-617. P., (1960); Differences in the metabolism of oligodendroglia and nerve cells HYDBN,H., AND LANGE, in the vestibular area. Regional Neurochemistry. S. Kety and J. Ekes, Editors. Proceedings of the IVth International Neurochemistry Symposium. New York, Pergamon (p. 190-199). HYDBN,H., AND LANGE,P., (1962); A kinetic study of the neuron-glia relationship. J. Cell Biol., 13,233-237. HYD~N H., , AND PIGON,A., (1960); A cytophysiological study of the functional relationship between oligodendroglial cells and nerve cells of Deiters’ nucleus. J. Neurochem., 6, 57-72. IBRAHIM, M. Z. M., AND ADAUS,C. W. M., (1963); The relationship between enzyme activity and neuroglia in plaques of multiple sclerosis. J. Neurol. Neurosurg. Psychiat., 26, 101-1 10. KATZMAN, R., (1961); Electrolyte distribution in mammalian central nervous system: are glia high sodium cells? Neurology, 11, 27-36. KATZMAN, R., AND WILSON,C. E., (1961); Extraction of lipid and lipid cation from frozen brain tissue. J. Neurochem., 7 , 113-127.

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KOCH,A., RANK,B., AND NEWMAN, L., (1962); Ionic content of neuroglia. Exp. Neurol., 6, 186-200. KOENIG,H., AND BARRON, K. D., (1962); Reactive gliosis - a histochemical study. 38th Annual Meeting, American Association of Neuropathology, 1962. J. Neuropath. exp. Neurol., 22, 336339. KREUTZBERG, G . ,and PETERS, G., (1962); Enzymhistochemische Beobachtungen beim experimentellen Hirntrauma der Ratte. Extrait du livre jubilaire du Dr. Ludo van Bogaert. Actu med. belg., p. 454462. KULENKAMPFF, H., (1952) ; Das Verhalten der Neuroglia in den Vorderhornern des Ruckenmarks der weissen Maus unter dem Reiz physiologischer Tatigkeit. Z. Anat. Entwick1.-Gesch., 116, 304-312. KUWABARA, T., AND COGAN,D. G., (1960); Tetrazolium studies on the retina. J. Histochem. Cytochem., 8, 214-224. MOSSAKOWSKI, M. J., (1962); The activity of succinic dehydrogenase in glial tumors. J. Neuroputh. exp. Neurol., 21, 137-147. M ~ ~ L L EW., R , UND NASU,H., (1962); Enzymhistochemische Untersuchungen an Gliomen. Nafurwissenschafien, 21, 496497. NELSON,E., OSTERBERG, K., BLAW,M., STORY,J., AND KOZAK,P., (1962); Electron microscopic and histochemical studies in diffuse sclerosis. Neurology, 12, 896909. OGAWA, K., AND ZIMMERMANN, H., (1959); The activity of succinic dehydrogenase in the experimental ependymoma of C3H mice. J. Histochem. Cytochem., 7 , 342-349. OKSCHE,A., (1961); Der histochemische nachweisbare Glykogenaufbau und -abbau in den Astrocyten und Ependymzellen. 2. Zellforsch., 54, 307-361. OSTERBERG, K., AND WATTENBERG, L., (1962a); Oxidative histochemistry of reactive astrocytes. A.M.A. Arch. Neurol., 7 , 211-218. OSTERBERG, K., AND WAITENBERG, L., (1962b); Inductive factors in gliosis. Proc. SOC.exp. Biol. Med., 111,452455. OSTERBERG, K., AND WATTENBERG, L., (1963); The age dependency of enzymes in reactive glia. Proc. SOC.exp. Biol. Med., 113, 145-147. POPE,A., AND HESS,H. H., (1957); Cytochemistry of neurones and neuroglia. Metabolism of the Nervous System. D. Richter, Editor. London, Pergamon Press (p. 72-86). ROBINS, E., SMITH,D. E., AND JEN,M. K., (1957); The quantitativedistribution of eight enzymes of glucose metabolism and two citric acid cycle enzymes in the cerebellar cortex and subjacent white matter. Progress in Neurobiology. II. Ultrastructure and Cellular Chemistry of Neural Tissue. H. Waelsch, Editor. Hoebner (p. 205-7.12). ROIZIN,L., (1951); Comparative morphologic and histometabolic studies of nerve cells in brain biopsies and operations. J. Neuropath. exp. Neurol., 10, 177-189. ROMANUL, F., AND COHEN,R. B., (1960); A histochemical study of dehydrogenases in the central and peripheral nervous systems. J. Neuropath. exp. Neurol., 19, 135-136. RUBINSTEIN, L. J., KLATZO,I., AND MIQUEL,J., (1962); Histochemical observations on oxidative enzyme activity of glial cells in a local brain injury. J . Neuropath. exp. Neurol., 21, 116-137. RUBINSTEIN, L. J., AND SUTTON,C. H., (1963); Histochemical observations on oxidative enzyme activity in tumors of the nervous system. 39th Annual Meeting; American Association of Neuropathologists, 1963, Atlantic City (N.J.). J. Neuropath. exp. Neurol., 23, 196-197. SCHIFFER, D., AND VESCO,C., (1962); Contribution to the histochemical demonstration of some dehydrogenase activities in the human nervous tissue. Acta neuropath., 2, 103-1 12. SCHIFFER, D., AND VESCO,C., (1963); Histochemical observations about the pattern of tetrazolium reduction, with different substrates, in glia cells of normal and pathological human nervous tissue. J. Histochem. Cytochem., 11, 335-341. SMITH, B., (1963); Dehydrogenase activity in reactive and neoplastic astrocytes. Brain, 86, 89-94. VANHOUTON, W. H., AND FRIEDE, R. L., (1961); Histochemical studies of experimental demyelination produced with cyanide. Exp. Neurol., 4,402412. WOLFGRAM, F., AND ROSE,A. S., (1959); The histochemical demonstration of dehydrogenases in neuroglia. Exp. Cell Res., 17, 526-530. YONEZAWA, T., BORNSTEIN, M. B., PETERSON, E. R., AND MURRAY, M. R., (1962); A histochemical study of oxidative enzymes in myelinating cultures of central and peripheral nervous tissue. J. Neuropath. exp. Neurol., 21, 479487.

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Functional Implications of Structural Findings in Retinal Glial Cells ARNALDO LASANSKY Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires (Argentina)

The present report summarizes some observations on the fine structure of retinal glial cells, as well as data on the extracellular space of the retina which might be of significance in relation to the problem of the function of glial cells. In selecting this tissue for study we hoped that at least some of the conclusions would apply to the central nervous system at large, since the retina by origin, structure and function is a sheet of gray matter. However, although this concept is valid with respect to the general organization of the tissue, the retinal glial cells are endowed with some morphological features that set them apart from glial cells in other parts of the central nervous system. These differences, which are as interesting as the similitudes, will be pointed out during the following discussion. On the other hand, the retina offers some definite advantages for electron microscopic studies of glial cells. In the first place, its somewhat schematic architecture permits an easier identification of the cellular elements. In addition the retina provides a thin layer of tissue, which is ideally suited for diffusion studies, without the need to section cell processes. Finally, the functional state of the retina can be estimated during an experiment by measuring the magnitude of the electrical response to light stimulation (ERG). The interest in the function of the neuroglia has been greatly stimulated by the fact that considerable refinements in electrophysiological, electron microscopical and microchemical techniques have facilitated a direct experimental approach to the problem. In general, research on the hypothetical roles of the glial cells has been directed along two main lines. One of them is concerned with a possible function of glial cells in metabolically supporting the neurons. Several suggestive data have been gathered in this area by means of cytochemical techniques (Hydtn and Pigon, 1960; Hydtn and Lange, 1962; Hamberger and Hydtn, 1963), the evidence being based mainly on apparently complementary enzymatic systems of nerve and glial cells. In the retina we have reported some observations which were interpreted as suggesting a transfer of energy-rich materials from glial to visual cells (Lasansky, 1961). However, actual transfer of substances has not yet been demonstrated and therefore defihite morphological or functional evidence of such a metabolic role of retinal glial cells is still lacking. Consequently in the following we will refer mainly to those aspects of the retinal fine structure which might have relevance to the second of the two

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mentioned lines of research. This is concerned with the possibility that glial cells may mediate exchanges between the neuron and the body fluids. The interest of many investigators was focused in this direction mainly as a consequence of electron microscope observations reporting on the very small size of the extracellualr space in the central nervous system (Wyckoff and Young, 1956; Dempsey and Wislocki, 1955; Luse, 1956; Farquhar and Hartmann, 1957; Schultz et al., 1957; Gerschenfeld et al., 1959). The narrow gaps of about 200 A between the cells were considered to be occupied by lipidic components of the plasma membranes and not adequate to serve as diffusion routes (Sjostrand, 1958, 1961, 1962). Since some kind of interstitial space seemed to be required in order to supply the nerve cells with oxygen, metabolites and ions, it appeared reasonable to assume that the glial cells might represent a functional substitute for such space. Thus, the electron microscopic data seemed to indicate that solutes must traverse glial cytoplasm in order to reach the neurons (Maynard et al., 1957; Gerschenfeld et al., 1959). In addition, it was found necessary to discuss the possibility that the glial cytoplasm could represent the outside of the excitable membrane (Sjostrand, 1958, 1961). Since the need to endow the glial cells with the functional attributes of an extracellular space arose mainly as a consequence of assuming that the intercellular gaps in nervous tissue do not permit free diffusion of solutes, it seemed important to test the correctness of this view by direct experimental means. It must be pointed out that the morphological evidence appeared to stand against a considerable amount of physiological data indicating the existence of an extracellular compartment in brain and retina (Allen, 1955; Ames and Hastings, 1956; Woodbury et al., 1956; Davson and Spaziani, 1959; Rall and Patlak, 1962). We shall now direct our attention to the retinal aspects of the problem to see to what extent they can contribute in elucidating some of the issues under discussion. The fine structure of the Miiller cells The main glial elements of the vertebrate retina are the cells first described by Miiller (1851) and which are known to constitute the framework containing all other retinal cells (Cajal, 1904; Polyak, 1941). Miiller cells are generally regaided as modified astrocytes (Polyak, 1956). They are elongated cells oriented perpendicularly to the plane of the retinal layers and extend from the outer to the inner surface of the neural retina. Because at the level of the light microscope Miiller cells resemble regularly spaced columns which project slender processes between the other retinal cells, it has been believed by some authors that the main function of Muller cells is to provide mechanical support and electrical insulation (Polyak, 1956). Our studies on the fine structure of Muller cells have been performed mainly on the toad retina, which is an advantageous material for in vitro experiments of the type described below. Furthermore, the amphibian retina has been a classical subject of physiological studies and a great amount of information is available on the type and localization of the electrical phenomena which it generates (Granit, 1947; Brindley, 1960; Tomita et al., 1960). Rdermces p . 70-72

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Fig. 1. Electron micrograph of the inner surface of the toad retina showing the inner end of a Miiller cell (G). The numerous channels (c) are formed by infoldings (arrows) of the plasma membrane. Several mitochondria (m) and numerous dense granules Cg) are observed within the cytoplasm of the Miiller cell. The plasma membrane of the Miiller cells at the inner surface of the retina is separated from the vitreous humour (h) by a basement membrane (b). v = small vesicles within the Miiller cell. Fixed in a buffered osmium tetroxide solution. Embedded in Epon. Stained with uranyl acetate and lead hydroxide.

The Muller cells of the toad retina appear at the level of the electron microscope as rather thick processes intervening between the retinal neurons. Within these processes numerous meandering paired membranes are observed (Figs. 1, 2, 3 and 4) which represent infoldings of the plasma membrane of the Miiller cells (Lasansky and Wald, 1962). It is therefore evident that the clear spaces (- 300 A wide) between

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Fig. 2. Electron micrograph of a Muller cell process (G)at the inner synaptic layer of the toad retina. c = Muller cell channels. g = dense granules in the Muller cell. s = synaptic endings. i = intercellular spaces. Preparative procedures as in Fig. 1.

these paired membranes could be considered as spaces between thinner processes of the Miiller cells. In the toad retina the Muller cells are at a distance of about 10 p from each other. Consequently it can be assumed that usually all the thin processes within a thick branch belong to a single Muller cell. An exception to this may occur at the points where neighboring Miiller cells come into contact, such as the inner surface of the retina, where the processes may represent interdigitations of adjacent cells. From a morphological point of view, infoldings of the plasma membrane or interspaces between thin processes appear both to be adequate ways to term the paired References p. 70-72

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Fig. 3. Electron micrograph of a Muller cell process (G) at the outer nuclear layer of the toad retina. c = Muller cell channels. g = dense granules. m = Miiller cell mitochondria. v = small vesicles in the Miiller cell. p = perikaryon of a photoreceptor cell. Preparative procedures as in Fig. 1 .

membranes found within the Miiller cells. In the following, however, we will refer to them as Muller cell channels since they represent diffusion pathways (see below) that permeate the whole extent of the Muller cells and communicate the intercellular spaces of the retina with the ocular cavity. As shown by Fig. 4 the channels appear occasionally occluded at the level of the outer nuclear layer by the presence of tight junctions. These tight junctions are probably analogous to the close contacts between glial cells described in other parts of the central nervous system by Peters (1962). Their significance in the retina is unknown.

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Fig. 4.Electron micrograph of a Miiller cell process (G) at the limit between the outer nuclear and outer synaptic layers of the toad retina. c, g, v and m = Muller cell channels, granules, vesicles and mitochondria respectively. t = tight junction in a Muller cell channel. i = intercellular spaces. s == synaptic ending of a photoreceptor cell. p = photoreceptor cell. Preparative procedures as in Fig. 1.

Channels similar to those in Miiller cells have been observed in the Schwann cells of the squid giant fiber (Villegas and Villegas, 1963) and in satellite cells in toad spinal ganglia (Rosenbluth, 1963) and molluscan ganglia (Gerschenfeld, 1963). Their presence has been reported also in glial cells of the brain (Luse, 1956; Dempsey and Luse, 1958), but this observation was not confirmed thereafter (Farquhar and Hartmann, 1957; De Robertis and Gerschenfeld, 1961). It is possible that the channels are related with some general function of the glial cells, but at present it is difficult to References p . 70-72

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conjecture which such a function might be. It could be assumed that they represent devices to facilitate the access of metabolites for the neurons, but then it is difficult to understand why the Muller cell channels are closed at the outer surface of the neural retina (see below) where an important part of the nutrients for the retinal neurons arrives through the pigment epithelium. Another hint might be given by the resemblance existing between the Muller cell channels and the infolding of the plasma membrane found in cells engaged in water and ion transport (Rhodin, 1954; Pease, 1956; Maxwell and Pease, 1956; Scott and Pease, 1959). It could then be hypothesized that Muller cells are also capable of pumping ions across the retina. However, there are morphological and physiological data which would seem to speak against such a transport process. First, as will be shown later, there are no definite diffusion barriers at either surface of the neural retina. Therefore if the Muller cells do perform an active ion transport, they could hardly be expected to maintain a concentration gradient of any diffusible ion across the neural retina. Furthermore, there are no electrical manifestations, such as a sizable steady potential, of an ion transport across the neural retina. As shown by Noel1 (1953,1963) most of the steady potential across the whole retina is generated by the pigment epithelium which appears to be the site of an active ion transport. A useful lead in analyzing the possible function of the Miiller cell channels might be given by the fact that Miiller cells in mammalian retinae do not have such a welldeveloped system of channels (Lasansky, 1962). However, in this case the Muller cell processes are much thinner and more numerous than in the toad retina. This disposition results in a very extended surface of the Muller cells in the mammalian retina. Therefore the channels in the toad retina could represent a device for similarly increasing the Muller cell surface relatively to that of the nerve cells, perhaps in order to facilitate hypothetical exchanges between both types of cells. For instance, the channels could facilitate the diffusion of COZ away from the neurons and its penetration into the glial cells where it would be hydrated to bicarbonate (Ashby et al., 1952). The activity of carbonic anhydrase in glial cells has been recently estimated by Giacobini (1962) as being 120 times higher than in nerve cells from the nucleus of Deiters. The content of carbonic anhydrase in the retina is very high (Van Goor, 1948) but the cytological localization of the enzyme is unknown. Another possibility is that the large surface of Muller cells might contribute in speeding the removal of the potassium, leaving the neurons as a consequence of bioelectrical activity. A role of glial cells in removing potassium from the intercellular spaces was suggested by Horstmann and Meves (1959) but no definite evidence in support of this view has been obtained so far. On the contrary, the fact that the channels in the Schwann cells of giant nerve fibers constitute the only pathway communicating the periaxonal space with the interstitial spaces, appears to cause a delay in the diffusion of potassium away from the bioelectrically active membrane (Frankenhaeuser and Hodgkin, 1956). Besides the channels, Miiller cells exhibit other structures which might be of importance to assess their functional abilities. In Figs. 1,2 and 3 the presence ofnumerous dense particles is observed within the Muller cell cytoplasm. As shown by Fig. 2

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these granules are particularly abundant within Muller cell processes at the inner synaptic layer. No special effort has been made so far to investigate the chemical nature of the granules. Their high electron opacity is due to the staining with uranyl acetate and lead hydroxide and they resemble the ribosomes found in other cells, although in Muller cells the granules are not associated with endoplasmic reticulum membranes. However, the fact that Muller cells in amphibian retina are not basophilic (Wislocki and Sidman, 1954) makes uncertain whether the granules should be identified as ribosomes. On the other hand, the existence of glycogen within Muller cells has been demonstrated by histochemical means (Shimizu and Maeda, 1953) and therefore it seems likely that the dense particles represent glycogen granules (Revel et al., 1960). Nevertheless further efforts are needed to characterize chemically these granules because of the widely differentfunctional implications such characterization would carry. As was expected mitochondria are present within the Muller cells of the toad retina (Figs. 1 , 3 and 4), but they are very peculiar in that they have very few cristae (Lasansky, 1961). They are found throughout the whole length of the Muller cell in contrast to what has been observed in other retinae, where the Muller cell mitochondria are confined to the outer and inner endings of this cell (Villegas, 1960; Wald and De Robertis, 1961). The mitochondria1 membranes appear to be the site where the enzymes related to oxidative phosphorylation are embedded (Lehninger, 1961). It is then not unlikely that the scarcity of cristae in their mitochondria indicates that Muller cells in the toad retina have a predominantly anaerobic metabolism. Numerous small vesicles are observed within the Muller cell (Figs. 1, 3 and 4) usually arranged in rows parallel to the membranes of the channels and occasionally attached to them. These vesicles were interpreted previously (Lasansky, 1962) as indicating a process of pinocytosis comparable to that suggested by De Robertis and Bennett (1954) in other glial cells. Yet, recent observations of Rosenbluth in spinal ganglia (1963) would seem to insinuate that at least some of the vesicles might originate as a consequence of the breakdown of the paired membranes of the channels during the fixation of the tissue. Of considerable importance in discussing the probable functional properties of Muller cells is the analysis of the fine structure of the so-called ‘outer and inner limiting membranes’. Fig. 1 shows that on the inner surface of the retina the only structure intervening between the inner ends of Muller cells and the vitreous humour is a basement membrane about 500 A thick. This basement membrane together with the plasma membrane of Muller cells and the clear space in between, constitutes what, at the level of the light microscope, is seen as a limiting membrane. The Muller cell channels communicate unrestrictedly with the space separating the basement membrane from the plasma membrane of Miiller cells (Fig. 1). At the outer surface of the neural retina the situation is quite different since there is no continuous layer representing the electron microscope counterpart of the ‘outer limiting membrane’. Instead, what is seen at this level is a stratum of terminal bars (Sjostrand, 1958; Wald and De Robertis, 1961; Lasansky and Wald, 1962). It is therefore proposed that the expression ‘outer limiting membrane’ be replaced by the more adequate one of junctional layer. References p . 70-72

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Fig. 5. Electron micrograph at the outer surface of a toad retina showing a glial junction with its two components: the tight junction or occluding fascia (t) and the adhering zonule (a). G, c and v = Muller cell cytoplasm, channel and villi respectively. j = intercellularjunction. Preparative procedures as in Fig. 1 .

Two main types of cellular attachments are found at the junctional layer of the toad retina. A first type is that joining the two membranes of the Muller cell channels; it will be designated as glialjunction. The structure of glial junctions closely resembles that of the junctional complexes described by Farquhar and Palade (1963) in a variety of epithelia, although glial junctions do not have a desmosome. Therefore glial junctions have only two components which according to Farquhar and Palade will be referred to as the occluding fascia or tight junction and the adhering zonule. The occluding fasciae or tight junctions are found in the whole extension of the junctional layer at the points where the plasma membrane of the Muller cells invaginates to form channels (Figs. 5, 6 and 10). At those places the adjoining membranes come close together and the clear space between them is reduced to a width of 80 A. If the tissue is fixed in permanganate (Luft, 1956) a dense line is observed bisecting the clear space (Fig. 7). This finding indicates the obliteration of the channel since the intermediate layer is formed by the fusion of the outer leaflets of the plasma membranes. An identical arrangement has been described by several previous workers

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Fig. 6. Junctional layer of a toad retina. t = occluding fascia of a glial junction. a = adhering zonule of a glial junction. j = intercellularjunction. 0, c and v = Miiller cell cytoplasm, channel and villi, respectively. p = inner segment of a photoreceptor. Preparative procedures as in Fig. 1.

in other cellular junctions (Farquhar and Palade, 1963). An additional feature exhibited by the occluding fasciae of glial junctions is that the adjoining membranes are very densely stained (Figs. 5, 6 and 8) probably reflecting a local difference in the chemical organization of the plasma membrane. As seen in Figs. 8 and 9 the glial tight junctions are not continuous belts; thence the use of the term ‘fascia’ to designate them. It is also noticed that the discontinuities are very limited in extent when compared with the length of the occluding fasciae in a direction parallel to the retinal surface. The second component of glial junctions is the adhering zonule (Figs. 5, 6 and 10). This is located internally to the occluding fasciae. The paired membranes, which are References p . 70-72

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Fig. 7. Electron micrograph of a toad retina at the junctional layer. The occluding fasciae (t) of the glial junctions exhibit a five-layered appearance as a consequenceof the fusion of the outer leaflets of the plasma membranes.j = intercellular junctions. f = Miiller cellvilli formingthe ‘fiber baskets’. Fixed in potassium permanganate. Unstained.

also very densely stained, are separated by a wider space (W 200 A). The more conspicuous feature of the adhering zonule is the increased density of the adjacent cytoplasm (Figs. 5 and 6). The other type of junctional structure at the outer surface of the neural retina is found joining the plasma membrane of visual cells to the plasma membrane of Muller cells. These junctions are therefore related to the intercellular spaces and will be designated as intercellular junctions. The intercellular junctions do not have an extended area of tight junction comparable to that in glialjunctions (Figs. 7,lO and 12). The adhering zonule is the main or the only junctional component at the intercellular junctions. This adhering zonule is similar in structure to the adhering zonule in glial junctions, but the intermembrane space measures 110 to 130 A. Since the intercellular spaces in Epon (Luft, 1961) embedded toad retina are approximately 200 A wide (Figs. 2 , 4 and 8) it is evident that the intercellular junctions constitute a definite bottle-neck. In some instances intercellular junctions were observed between adjacent photoreceptor cells (Fig. 11). These junctions are formed only by an adhering spot. As stated before, Muller cells in mammalian retinae do not have an elaborated

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Fig. 8. Oblique section of the junctional layer of the toad retina. Several tight junctions (t) are ob served in a tangential plane. A discontinuity in a tight junction is marked by an arrow. G = Mulle cell cytoplasm. p = inner segments of photoreceptors. j = intercellular junction. i = intercellula space. f = Muller cell villi. Preparative procedure as in Fig. 1.

system of channels as in toad retina. Consequently in the mammalian retina there are no glial junctions at the junctional layer, but the intercellular junctions are identical to the intercellular junctions in the toad retina (Fig. 12). In Figs. 5, 8, 9 and 10, Miiller cell villi are seen interspersed between the inner segments of the photoreceptors. These villi arise from the outer ends of Muller cells to constitute the ‘fiber baskets’ of the classical literature (Polyak, 1941). Within the villi, rows of vesicles are frequently encountered (Figs. 9 and 10). These vesicles are only very seldom attached to the plasma membrane and their significance .is unknown. The routes for diffusion in the retina

In the preceding account reference has been made to the channels of the Miiller cells References p . 70-72

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Fig. 9. Oblique section of a glial junction (a). At the point marked with an arrow the occluding fascia is absent. On the right side an intercellular junction (j) is observed. Preparative procedures as in Fig. 1. G, c, v and m = Muller cell cytoplasm, channel, villi and mitochondria respectively.

as diffusion pathways. It has also been implied that the intercellular spaces communicate with them and therefore are also open for diffusion. However, as stated in the introduction, there were reasons to doubt that these narrow intermembrane gaps could constitute ‘true’ spaces. In order to determine whether or not there was a fundamental disagreement between the evidence provided by the electron microscope and the physiological observations indicating the existence of an extracellular space in the central nervous system, we analyzed the histological distribution of ferrocyanide within the retina (Lasansky and Wald, 1962). This substance has been used to measure the size of the extracellular space in brain (Allen, 1955) because ordinarily it does not penetrate into cells (Weed, 1923). Ferrocyanide was selected as tracer substance because its location can be detected at the level of the light and electron microscopes

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Fig. 10. Electron micrograph of the junctional layer of a toad retina. Notice the absence of an occluding fascia at the intercellular junction on the right (j). G = Miiller cell cytoplasm. c = Miiller cell channels. v = vesicles within the Miiller cell villi. p = inner segments of photoreceptors. t = occluding fascia at a glial junction. a = adhering zonule of glial junctions. Preparative procedures as in Fig. 1.

by means of a precipitation technique involving its reaction with iron or copper respectively. Therefore isolated toad retinae were immersed during variable intervals (usually several minutes) in a saline solution containing ferrocyanide and then fixed in the presence of ferric or cupric ions and prepared for microscopic examination. The details of the experimental and preparative techniques have been mentioned elsewhere (Lasansky and Wald, 1962). The electroretinogram was usually recorded before and after the experiments. It showed variable degrees of preservation depending on the length of the experiment and the concentration of ferrocyanide. However, the distribution of ferrocyanide was not related to the maintenance of the retinal function as revealed by the magnitude of the electroretinographic response. Rpferences p . 70-72

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Fig. 11. Adhering spot (as) between the inner segments of two photoreceptors (p). j = intercellular junctions. a = adhering zonule of a glial junction. bb = basal body. G = Muller cell cytoplasm. Preparative procedures as in Fig. 1.

At the level of the light microscope ferrocyanide, detected as Prussian blue staining, is found within the Muller cells and also diffusely distributed across the retina (Fig. 13). Usually the Prussian blue deposits are more abundant at the inner retinal layers, but in other instances the full length of the Muller cells is stained. About 5 min of immersion of the retina in the ferrocyanide medium suffice to bring about the typical pattern of Prussian blue distribution. This was expected since the equilibration time for inulin in the retina is 3 min (Ames and Hastings, 1956). At the level of the electron microscope ferrocyanide, detected as copper ferrocyanide deposits, is found in two locations : (1) within the channels of the Muller cells (Fig. 14) in such way explaining the staining of Muller cells at the level of the light

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Fig. 12. Electron micrograph at the junctional layer of a ratretina. Theintercellularjunctions (j) are similar to those in toad retina. G = Miiller cell process. p = photoreceptor cells.

microscope. Therefore ferrocyanide does not penetrate into Miiller cells as light microscopy appears to suggest, since the lumen of the Muller cell channels represents extracellular space; (2) within the intercellular spaces (Fig. 15), which is the cause of the background staining at the level of the light microscope. Thus, the extracellular space of the toad retina is defined as formed by an intercellular and a glial compartment. It is also demonstrated that such narrow intermembrane gaps as the intercellular spaces and the Miiller cell channels represent very effective diffusion pathways. The probable significance of the channels as routes for diffusion was discussed above. With respect to the intercellular spaces, it seems logical to assume that if ferrocyanide can penetrate within them in a relatively short time, chlorine, potassium and sodium should encounter no difficulty in doing the same. Therefore, it can be concluded that the surface of the neurons is exposed to an extracellular medium and that the cell membrane of the neurons is the bioelectrically active membrane required by current ideas on the origin of the nerve impulse (Hodgkin, 1951, 1958). It should be pointed out that the same conclusion has been reached for the giant axon using entirely different techniques (Villegas et al., 1962). One aspect of the intercellular gaps in central nervous system that is somewhat puzzling, particularly after it is recognized that they are ‘true’ spaces, is the fact that their width is very constant even in conditions of severe edema (Gerschenfeld et al., 1959). This could be explained by postulating the existence within these spaces References p. 70-72

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Fig. 13. Toad retina immersed in a saline solution containingferrocyanideand fixed in formaldehyde with ferric ammonium sulphate. The inner processes of Muller cells are stained by Prussian blue. There is also a diffuse background staining. i = inner limiting membrane (Lasansky and Wald, 1962). (By courtesy of the J . Cell Bid.)

of a hydrophilic cement (Villegas and Villegas, 1963) such as the ground substance postulated by Hess (1953). Our observations on the penetration of ferrocyanide of course do not deny nor do they support the existence of such an intercellular cement. In fact, it would not be very surprising if the neurons and glia cells were shown to be lined by a mucoid coat since this seems to be a rather generalized feature of cells (Bennett, 1963). However, we want to advance the notion that the stability of the intercellular spacing can be explained without resorting to a hypothetical cementing substance. In fact, the narrow intercellular spaces in the central nervous system bring the cells within the range of adhesion forces which are known to be of several types (Pethica, 1961). In particular it seems important to take into account in this case the role of surface energy, which can be extremely high at such close ranges (Weiss, 1960). As discussed above, the postulated absence of a functionally significant extracellular space in the central nervous system introduced not only the problem of interpretation of bioelectrical phenomena, but also the need of assuming that metabolites and ions should traverse the glial cytoplasm in passing from the blood to the neurons. This last concept also would seem to be dispelled by the preceding observations. However, this is not necessarily true considering that although the intercellular spaces provide a direct route to the neurons, the access of solutes into the intercellular spaces could be regulated by the glia cells. This possibility is suggested by Gray’s (1961) finding that the plasma membranes of adjacent astrocytic ‘sucker-feet’ are joined by tight junctions. Thus, the intercellular spaces in the immediate vicinity of cerebral

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Fig. 14. Electron micrograph of a toad retina showing the presence of copper ferrocyanide deposits within the Miiller cell channels (c) near the inner surface of the retina. The retina was immersed in a ferrocyanidecontaining solution and then fixed in a mixtureof osmiumtetroxide and copper sulphate. h = remnants of vitreous humour attached to the retina. Embedded in methacrylate. Unstained.

capillaries are sealed. This disposition would appear to be a device to force water, electrolytes, glucose and so forth to traverse the glial cytoplasm before entering the intercellular spaces. Granting this, the astrocytes would mediate the exchanges between blood and nervous tissue and would be in a position to regulate the composition of the extracellular fluid. In order to investigate whether Miiller cells might behave like the cerebral astrocytes, it seemed necessary to establish if the intercellular spaces of the neural retina are open in all their extension. The areas concerned were obviously both surfaces of the neural retina. At the inner surface, the Miiller cell channels (which in turn lead to the intercellular spaces) appear to be wide open (Fig. 1) and there is no structural reason to suspect a diffusion barrier at this level. This point was further corroborated by investigating the penetration of ferrocyanide when a ferrocyanide containing solution is flushed into the ocular cavity in the excised toad eye (Lasansky and Wald, 1962). In such cases the intraretinal distribution of ferrocyanide is the same as when the References p . 70-72

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Fig. 15. Electron micrograph of a toad retina at the bipolar cell layer. Notice the presence of copper ferrocyanide deposits within the intercellular spaces (i). N = nucleus of a bipolar cell. m = mitochondria within bipolar cells. b = bipolar cell process. G = thin Miiller cell process. Experimental and preparative procedures as in Fig. 11 (Lasansky and Wald, 1962). (By courtesy of the J. CeN Biol.)

isolated retina is immersed in the solution. Consequently there are no obstacles for a free exchange of solutes between the retina and the vitreous humour. Nevertheless, a substantial fraction of the nutrients for the retinal neurons, particu-

a.c. preamplifier

Fig. 16. Diagram of the chamber used in the ferrocyanide experiments to analyze the permeability of the junctional layer. The retina is extended across an opening of 3 mm in diameter. The front face of the half-chamber on the left of the figure is recessed t o a depth of 200 p. The front face of the half-chamber on the right is lightly vaselinated. There is no significant lateral diffusion as tested with dyes and ferrocyanide. The electroretinogram is recorded by means of platinum wires.

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larly for those in the outer layers, arrives in the retina from the chorioidal vessels. When these solutes traverse the pigment epithelium they face the junctional layer which, as suggested by previous work on junctional structures (Farquhar and Palade, 1963), could constitute a diffusion barrier. However, as shown by Farquhar and Palade, this role of junctional complexes appears to be due to the presence of the tight junction component. As shown above, the intercellular junctions of the retina do not display any extensive area of tight junction and it would be doubtful whether they can prevent the penetration of solutes within the intercellular spaces. On the other hand, glial junctions do have the morphological substratum to act as diffusion barriers, although the tight junctions are discontinuous and the barrier is probably only

Fig. 17. Electron micrograph of the junctional layer of a toad retina. Observe the copper ferrocyanide precipitate within the intercellular spaces (i) a t the level and beyond the junctional areas. Ferrocyanide deposits are found also within a Miiller cell channel (c). Only the outer surface of the retina was exposed to the ferrocyanide-containing solution (see text). G = Miiller cell process. p = inner segments of photoreceptors. f = Miiller cell villi. Fixed first in a solution containing glutaraldehyde and copper sulphate and then further fixed in osmium tetroxide-copper sulphate. Embedded in Epon. Stained with uranyl acetate. After this procedure the cell membranes are not clearly seen and the characteristic electron opacity observed in conventionally fixed junctional complexes is also not manifest. All of the electron opacity along the intercellular spaces in this photograph is due to ferrocyanide precipitation. Copper ferrocyanide is not a crystalline material but it precipitates as a colloidal aggregate. The appearance of the deposits changes according t o pH, concentration and so forth. Consequently sometimes the deposits may have an amorphous appearance as in this photograph, or a particulate one as in Fig. 14, References p. 70-72

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partial. These assumptions have been tested again resorting to ferrocyanide, this time investigating whether this substance can penetrate into the neural retina at the level of the junctional layer. In a previous study on ferrocyanide distribution (Lasansky and Wald, 1962) it was stated that this substance appeared not to penetrate at the outer surface of the neural retina. In that occasion however, we did not have the means to submit this hypothesis to a rigorous test. Such a situation has been achieved with the following experiments and the earlier assumption proved to be incorrect. To expose to ferrocyanide only the outer surface of the neural retina, a special chamber was designed (Fig. 16) in which the isolated retina separates two pools of saline solution, the pool corresponding to the outer retinal surface containing in addition ferrocyanide. The toad retina stands quite well this treatment as an electro-

Fig. 18. Electron micrograph of the junctional layer of a toad retina showing the presence of copper ferrocyanide deposits within the intercellular spaces (i) at the level and beyond the junctional areas. p = inner segments of photoreceptors. G = Muller cell process. v = Muller cell villi. Experimental and preparative procedures as in Fig. 17.

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retinogram can be elicited at any time during the experiments (usually no longer than 20 min). The light microscopy reveals the same distribution of the Prussian blue deposits as in Fig. 13. Therefore ferrocyanide traverses the junctional layer, although in a somewhat slower fashion since it takes about 8-10 min to bring about the maximal intensity of staining. The pathway for ferrocyanide penetration was investigated at the level of the electron microscope. In Figs. 17 and 18 the copper ferrocyanide precipitate is observed within the intercellular spaces at the level and beyond the junctional areas. The usual appearance of the junctions is not seen in this micrograph because the retina has been fixed in glutaraldehyde (Sabatini et al., 1963). For a control preparation see Fig. 19. In Fig. 17 copper ferrocyanide deposits are also found within a Miiller cell channel, probably due to penetration at points of discontinuity of the occluding fasciae.

Fig. 19. Control preparationfor Figs. 17 and 18. The retina was immersed in a saline solutionwithout ferrocyanide and then fixed and processed as in Fig. 17. Notice the absence of dense deposits as those in Figs. 17 and 18 within the intercellular space at an intercellular junction (j). G = Muller cell cytoplasm. v = Muller cell villi. p = photoreceptor cell. Refwences p . 70-72

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It is then evident that the junctional layer does not prevent diffusion and that solutes having a similar or smaller particle size than ferrocyanide do not need to traverse the Muller cell cytoplasm in order to enter the intercellular spaces at the outer border of the neural retina. Actually the contrary proposition would seem more likely, since the fact that most of the access to the Muller cell channels is obliterated by tight junctions appears as giving priority to the intercellular spaces as the points of entrance within the neural retina of ions and metabolites coming across the pigment epithelium. The logical conclusion to be drawn from the preceding observations is that the composition of the extracellular fluid in the neural retina is to a great extent determined by the surrounding environment. Therefore the maintenance of the retinal homeostasis would appear to be mainly dependent on the activity of the ciliary body and pigment epithelium. There is a considerable amount of information on the physiology of the ciliary body which appear to support this possibility (Davson, 1956). On the other hand, the knowledge on the probable role of the pigment epithelium in actively mediating exchanges between retina and blood is not so widespread, although it has been extensively analyzed by Noell (1953, 1963). The pigment epithelium cells are very much connected from an embryological point of view to ependymal cells and some of their functional features might be similar to those of glia cells (Wald, 1958; Noell, 1963). It is then suggested that studies on this somewhat neglected retinal layer might prove to be useful in providing valuable information on the physiology of the neuroglia. SUMMARY

(1) The fine structure of the Muller cells of the retina is analyzed. The system of channels formed by the infoldings of the plasma membrane and the junctional structures forming the socalled ‘outer limiting membrane’ are given particular consideration. (2) The pathways for diffusion across and within the retina are investigated by using ferrocyanide as a tracer for electron microscopy. It is concluded that ions and metabolites are not forced to traverse glial cytoplasm in order to reach the retinal neurons. ACKNOWLEDGEMENTS

This work was supported by research grants from the Consejo Nacional de Investigaciones Cientificas y TCcnicas de la Repliblica Argentina and the U.S. Air Force Office of Scientific Research (656-64). REFERENCES

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Observations on Penetration of Serum Proteins into the Central Nervous System I. KLATZO, H.

W ~ S N I E W S K AI N D

D. E. S M I T H

Section of Neuropathology, Surgical Neurology Branch, National Institute of Neurological Diseases and Blindness, Bethesda 14, Md. (U.S.A.)

The entry and movement of serum proteins in the nervous system, under normal and abnormal conditions, can be studied by the application of proteins conjugated with either fluorescent or radioactive markers. Such observations may provide significant data on various aspects of permeability of brain ‘barriers’, as well as on the functional properties of the glia intimately involved in either passage or transport of substances through the nervous tissue. The present observations are derived from experiments on cats in vivo and on isolated chick and rabbit choroid plexuses using human and bovine albumin and y-globulin labeled with fluorescein isothiocyanate, 1251 and 13lI. The permeability of the normal cerebral vasculature to serum proteins was ascertained by injecting cats intravenously with fluorescein labeled albumin (FLA) and fluorescein labeled y-globulin (FLGG). Under the fluorescence microscope the localization of the green fluorescent protein conjugates throughout the brain, with the exception of the ‘special‘ areas described below, remained confined to the lumina of the blood vessels without any evidence of protein entry into either the endothelial cells or the underlying structures (Klatzo et al., 1962). In ascertaining the distribution of the fluorescent proteinconjugates with regard to the structures of the vessel wall, a useful landmark is frequently presented by the location of the adventitial cells and pericytes

Fig. 1. A small artery from the cerebral hemisphere of a normal cat injected intravenously with FLA. The green fluorescence of FLA is seen to be confined to the lumen of the vessel. Arrows indicate vascular wall cells containing bright orange autofluorescentpigment. Fluorescence microphotograph. References p . 87/88

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which are clearly visible due to the bright orange autofluorescent pigment in their cytoplasm (Fig. 1). With respect to the 'special' areas, the choroid plexus revealed the presence of fluorescent conjugates in the connective tissue stromal fibers and in the region of the basement membrane below the choroidal epithelium. A similar vascular permeability relationship with regard to conjugates was also observed in the area postrema, pineal gland, and hypothalamus. In these areas, FLA and FLGG were seen to penetrate outside of the vascular lumina and to outline the adventitial connective tissue fibers with green fluorescence. In no instance, however, was there any evidence of further penetration into the perivascular brain parenchyma. Observations on the penetration of proteins from the blood vessels into the brain tissue under abnormal conditions were derived from experiments in which the normal physiological vascular permeability was altered by various types of injury. These experiments included: (1) the breakdown of the blood-brain barrier in one hemisphere by unilateral intracarotid injection of a hypertonic glucose, and (2) the production of edema by application of cold to the exposed cerebral cortex. The intracarotid injections were carried out according to the technic described by Broman and Olssen (1956) and Steinwall(1958). Anesthesizedcats were tracheotomized to allow for artificial respiration if required during the experiment. Fifty ml of 30 % glucose was injected during 50 to 60 sec, into the cannulated common carotid artery in a cranial direction. The FLA was given intravenously (25 ml of 8.6 % albumin) either 10 min preceding or at various time intervals following the intracarotid injection. The animals were sacrificed within half an hour after receiving the fluorescent protein conjugate. Formalin-tixed, frozen sections were examined under the fluorescence microscope. The details of the fluorescence optics used have been reported elsewhere (Klatzo et a[., 1962). Microscopic observations under the fluorescence microscope revealed that 30 % glucose produced an abnormal vascular permeability in the ipsilateral hemisphere, but this disturbance was only of a short duration. Thus, the abnormal permeability was demonstrable in the cats which were given FLA preceding the hypertonic glucose injection and in those animals in which the injection of FLA was carried out not longer than 8 min after the intracarotid introduction of the glucose. In the sections of the brains with disturbed vascular permeability, the majority of the blood vessels in the affected hemisphere revealed a normal pattern, the presence of the protein conjugate being strictly confined to a vascular lumen. In some areas, however, the blood vessels showed evidence of increased permeability of two distinct degrees: (1) penetration of FLA extended through all the component layers of a vascular wall, but not beyond into the nervous parenchyma and (2) penetration of FLA was visible outside of the adventitial confines in the surrounding brain parenchyma. In the vessels of the first mentioned. variety, the green fluorescence in a vessel wall appeared-to be predominantly outlining what electron microscopists describe as the basement membrane system (Hager, 1961), i.e. a homogeneous substance which forms a distinct layer under the endothelium, separates the cells of the media, and provides a limiting layer against the surrounding glial cells. The vessels of this type showed a sharp demarcation between the green fluorescence in the described vascular structures and

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Fig. 2. A small artery from the cerebral hemisphere on the side of the hypertonic glucose injection. The green fluorescence of FLA outlines various component elements of the arterial wall. The orange pigment containing cell (arrow) lies within the confines of the green fluorescentstructures.There is no visible penetration of FLA into the surrounding brain parenchyma. Fluorescence microphotograph.

the dark-blue autofluorescence of the surrounding nervous parenchyma (Fig. 2). In the vessels which showed penetration of FLA outside of the adventitial structures, the spread of the green fluorescencein the white matter resembled a pattern previously described as the 'mottled appearance' (Klatzo et al., 1962), whereas in the grey matter it appeared in the form of circumscribed perivascular green fluorescent exudates. In order to ascertain some dynamic aspects of protein conjugate movement once it penetrated from blood vessels into the nervous parenchyma, increased vascular permeability was induced by application of cold according to the procedure previously described (Klatzo et al., 1958, 1962). In the present investigations, this procedure was slightly modified by applying the metal plate cooled to -65" for 10 sec to the exposed cortex of the supramarginal gyrus in cat. Approximately 30 ml of FLA solution (containing 8.6 % of bovine albumin) were injected either immediately preceding the cold injury or after a certain time following the operation. Three series of cats (Fig. 3) were sacrificed at various time intervals in groups consisting of 2 to 3 animals. In several cats, instead of FLA albumin labeled with 1251 (1 % human albumin* specific radioactivity of 350 ,uC/ml) was injected intravenously at 1 mC/kg of body weight. Gross observations under the U.V. lamp revealed, in the first series of cats which were injected with FLA preceding the cold injury, a progressive development of an area of green fluorescence bearing a direct time relationship to the duration of the cold lesion. Thus, cats sacrificed 5 min after the cold injury showed the bright green fluorescence limited to a narrow, saucer-shaped zone in the superficial cortical layers of the injured gyrus. In the animals sacrificed 30 min after the cold injury, the green fluorescence descended to occupy the upper half of the white matter of the injured gyrus. One hour after the cold lesion the green fluorescence was seen to have spread further down through the white matter reaching the base of the injured gyrus. In cats sacrificed later than 2 h, the green fluorescence progressively extended through the white matter of the adjacent gyri reaching its maximal area at 24-48 h. The extension of the green fluorescence at various time intervals is diagramatically represented in Fig. 3. ___

*

Nuclear Consultants Corp., Chicago.

References p.IS7/SS

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Fig. 3. Diagramatic representationof the dynamic movement of FLA in 3 series of cats subjected to cortical cold injury. The times stipulated on the left side of each diagram represent the total duration of the lesion from the time of operation until sacrifice. The times given on the right denote the duration of the intravenously injected FLA in the circulation before sacrifice. In the first series of cats the protein tracer was injected directly before the operation.

In the second series of cats, FLA was injected intravenously at various time intervals following the cold injury and the animals were sacrificed 1 h after the FLA injection. The gross observations revealed that, independent of the duration of the lesion, the spread of FLA within 1 h was similar and corresponded roughly to the picture observed in cats injected with FLA preceding the cold injury and sacrificed 3 hour later (Fig. 3). In order to ascertain further the dynamics of the FLA spread through the edematous brain tissue, the cats of the third series were killed 24 and 48 h after the induction of the cold lesion with FLA being injected at various time intervals prior to sacrifice. As can be seen from the diagramatic representation of the results in Fig. 3, the spread of the green fluorescence within the first 4 h after FLA injection was slower in animals with previously induced lesions than it was in the animals which received FLA preceding the cold injury (see diagrams 24 h-2 h, 24 h-4 h versus 2 h-2 h, 4 h-4 h). The areas of green fluorescence were similar in size in the cats in which FLA was present in the blood circulation for either 10 or 24 h independent of whether FLA was given either directly before induction of the cold lesion or at certain time intervals after the application of the cold lesion (see diagrams 10 h-10 h, 24 h-24 h versus 24 h-10 h, 48 h-24 h). The fluorescence microscopic observations on the brain sections of the cats sub-

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jected to the cold injury have been extensively described elsewhere (Klatzo and Miquel, 1960; Klatzo et al., 1962) and, therefore, only the findings which are of direct significance for the interpretation of the above described experiments will be emphasized. The sections derived from the cats of the first series, which received FLA prior to the induction of the cold lesion, revealed an extravascular presence of the protein conjugate in an area corresponding topographically to the extent of the green fluorescence observed grossly under the U.V. lamp. In all time intervals studied, the blood vessels in the region of the cortical lesion were seen to be surrounded by circumscribed, intensely green fluorescent exudates. The edematous white matter showed predominantly a pattern of green fluorescence which has been previously described as the ‘mottled appearance’ (Klatzo et al., 1962). The blood vessels included in this area showed only occasionally some denser accumulations of the green fluorescent material. Discrete, intracytoplasmic green fluorescent inclusions were conspicuous in the glia cells in the later stages of the lesion. The ‘mottled’ green fluorescence in the white matter appeared frequently to be more intense in the vicinity of the Ufibers. As a rule, the area of the green fluorescence appeared at its periphery as a solid front usually demarcated in a straight line against the blue autofluorescent white matter. This straight-line demarcation was neither altered by the crossing blood vessels nor were any green fluorescent perivascular exudates demonstrable in the white matter outside of the continuous area of the green fluorescence. By staining the sections used for the fluorescence observations, it was possible to ascertain that in the cats injected with FLA preceding the cold injury the histologically recognizable edema in the white matter occupied an area similar to the green fluorescence. The fluorescence microscopic observations derived from the cats injected with FLA at various time intervals following the cold injury (second and third series) revealed basically similar features. The leakage of FLA from the injured cortical blood vessels was evident even in the animals sacrificed 48 h after the operation. The area of green fluorescence in the edematous white matter showed similar straight borders of demarcation. The blood vessels within the green fluorescent area and outside of it in the edematous white matter failed to show any evidence of exudation. Only occasional blood vessels within the fluorescent area showed some accumulation of the conjugate in the perivascular spaces and in the macrophages.

Fig. 4. An artery close to the pial surface of the cerebral hemisphere. FLA was introduced into the subarachnoidspace. The green fluorescenceoutlines structures similar to those in Fig. 2. Fluorescence microphotograph. References p . 87/88

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Fig. 5. A cortical area in the injured gyms of the cat which had been injected intravenously with lZ5I-labeledalbuinin 2 h before sacrifice and 22 h after the induction of the cold lesion. An intense radioactivity is seen surrounding a blood vessel. Autoradiograph, counterstained with hematoxylin and eosin, x 250.

Fig. 6. The same gyrus as in Fig. 5. Deeper portions of the white matter below the ‘solid front’ extension of the radioactive conjugate. A blood vessel in the markedly edematous white matter exhibits the radioactivity con6ned to the lumen. Autoradiograph, counterstained with hematoxylin and eosin, x 320.

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The autoradiographic observations with 1251-labeled albumin were derived from groups of cats which were injected with the radioactive conjugate 1 h and 2 h before their sacrifice at 24-h interval after the induction of the cold injury. One group of cats received 1251 albumin 24 h after the operation and was sacrificed another 24 h later (total duration of the lesion: 48 h). The radioautographs revealed a distribution pattern of radioactive albumin similar to the FLA pattern which was observed in the brains of animals injected with the fluorescent conjugate and sacrificed at the corresponding time intervals (Fig. 5). The feature in the radioautographic observations which appeared to be especially emphasized was that the blood vessels in the edematous white matter below the ‘solid front’ of conjugate extension from the cortex showed no increased permeability to injected albumin: the radioactivity being confined to the lumina of the blood vessels (Fig. 6). For observations on the penetration of serum proteins into the brain tissue from the cerebrospinal fluid (CSF), cats were perfused intraventricularly with fluorescent and radioactive albumin and y-globulin. The inflow of a perfusate was provided by an intraventricular cannula introduced into the left lateral ventricle whereas the outflow needle was inserted into the lumbar subarachnoidal space (Klatzo et al., 1963). FLA (6.5 % protein) and FLGG (7.4 % protein) were perfused for 1 h at the rate of 0.1 ml/min; 6 ml of 1251 human albumin and bovine y-globulin (500 pC/total volume of perfusate) were perfused at a similar rate. In several cats, the perfusion with the fluorescent protein conjugates was carried out directly post-mortem. The results from these studies indicated some significant differences between the particular conjugates with regard to their penetration into the various brain structures surroundingthe CSF‘pathways. From the subarachnoid space, FLA and FLGG failed to show any direct ,penetration through the pial lining into the underlying brain parenchyma. Both protein conjugates appeared to extend for some depth along the blood vessels entering the brain from the subarachnoid space (Fig. 7). It was interesting

Fig. 7. Pial surface of the cat brain after the introduction of FLA into the CSF. The subarachnoidal space is filled with the fluorescent conjugate which is also seen in the wall of a blood vessel extending into the cortex. There is no evidence of a direct penetration of FLA through the pial lining. Fluorescence microphotograph. References p . 87/88

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to note that the distribution of the green fluorescence in such blood vessels (Fig. 4) resembled closely that observed in some vessels after intracarotid injection of hypertonic glucose where the abnormal FLA penetration from the lumen was confined to the vascular walls (Fig. 2). From the ventricles and CSF pathways lined by the ependyma, the passage of FLA and FLGG differed with regard to the underlying anatomical structures. FLA spread considerably into the periventricular grey matter, but only to a very limited degree in the white matter underneath the ependymal lining. No passage of FLA was observed into either the area postrema or the infundibular region. The penetration of the FLGG through the ependyma was restricted regardless of whether grey or white matter was involved. The choroid plexus revealed stromal uptake of FLA in occasional villi, whereas no such uptake was observed with FLGG. In cats which were perfused with the fluorescent protein conjugates postmortem, the penetration of both FLA and FLGG into the periventricular tissue was restricted with regard to both the grey and the white matter, involving at most only a narrow zone directly underlying the ependyma. To test whether the differences between grey and white matter with regard to the penetration of FLA were related to the regional differences in the ependymal lining or to the inherent properties of the underlying structures, particularly neuroglia, an attempt was made to destroy the ependymal lining by intravital ventricular perfusion with 2 % buffered osmic acid introducing the outflow catheter into the aqueduct. A few days after this procedure, the cats were subjected to the standard perfusion with FLA. Observations under the fluorescence microscope revealed that in spite of the destruction of the ependymal lining and the narrow zone of underlying tissue by osmic acid, the differences between the grey and white matter with regard to the depth of FLA penetration remained preserved. The radioautographic studies on cats perfused intraventricularly with 1251-labeled albumin and y-globulin revealed no evidence of direct penetration of both radioactive serum proteins through the pial lining (Fig. 8). The periventricular spread of both con-

Fig. 8. An area corresponding to that in Fig. 7 with 125I-albumin introduced into the CSF showing similar features of albumin distribution. Tissue piocessed by freezedrying. Radioautograph,counterstained with hematoxylin eosin, x 450.

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Fig. 9. The area of the floor of the third ventricle in the cat subjected to intraventricular perfusion with 125I-labeled albumin. There is a marked difference in the radioactivity exhibited by the periventricular grey matter (upper portion of the picture) and by the white matter of the optic tract (lower portion). Radioautograph, counterstained with lux01 blue and cresyl violet, x 420.

jugates was similar and much more pronounced in grey than in white matter (Fig. 9). There was no penetration into the area postrema and infundibular region. In the animals perfused with 1251-labeled albumin, the choroid plexus showed radioactivity in occasional villi and particularly in those villi located in proximity to the ependymal junction (Fig. 10). In order to elucidate further the nature of passage of conjugates into the choroid plexus, observations were carried out on isolated chick choroid plexus incubating this organ in various solutions of protein conjugates in vifro (Smith ef al., 1964). The viability of such a plexus was ascertained from the characteristic rapid movement of the cilia and the penetration of conjugates was evaluated either under the fluorescence microscope or in the radioautographs. The isolated newborn chick plexuses incubated in fluorescent conjugates showed a conspicuous difference between the penetration of FLA and FLGG. Isolated plexuses from the lateral ventricles, when incubated immediately in 0.1 % FLA in a balanced References p . 87/88

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Fig. 10. Choroid plexus from the third ventricle of the cat perr'used intraventricularly with 1251labeled albumin. The radioactivity is seen to be localized in the stroma. Radioautograph, counterstained with luxol blue and cresyl violet, x 480.

Fig. 11. Whole mount of a living chick choroid plexus after incubation in 0.1 % FLA for 15 min showing an intense accumulation of the fluorescent conjugate in the stroma of a villus. Fluorescence microphotograph.

salt solution (Tyrode's) for 15 min and mounted in toto for observations under the fluorescence microscope, showed an intense accumulation of the FLA in the stroma with the choroidal epithelium remaining blue autofluorescent (Fig. 11). Incubations

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Fig. 12. Diagramatic representationof the effect of unlabeled serum proteins on the uptake of FLA and FLGG by the isolated chick choroid plexus. The white areas in the diagram correspond to the green fluorescence observed in the sections prepared by the freeze-drying procedure and viewed under the fluorescence microscope.

in the FLGG failed to show any uptake of the conjugate by the choroid plexus. The ability of the chick choroid plexus to take up FLA was shown to develop between the 9th and 13th day of embryonic life. Thus, in the choroid plexuses from chick embryos 9 days old or younger, no stromal uptake of FLA could be demonstrated. The passage of FLA into the stroma was inhibited at low temperatme (4")and in incubations with FLA solutions which contained metabolic inhibitors such as potassium cyanide (10-4 M ) , sodium fluoride (2 x 10-2 M ) , and sodium azide ( 5 x 10-3 M ) . An intriguing effect was obtained when the incubations in the fluorescent protein conjugates were carried out with the addition of unlabeled proteins. The incubations in a mixture of FLGG and unlabeled globulin produced a striking FLGG uptake by the choroidal epithelium with some passage of the fluorescent conjugate into the stroma, whereas the admixture of unlabeled albumin to FLGG failed to produce any effect. The addition of unlabeled globulin to FLA also resulted in the epithelial uptake of this conjugate. The observations on the effect of unconjugated proteins are summarized diagramatically in Fig. 12. The difference in the uptake between albumin and y-globulin was also evident in radioautographic observations on the isolated chick choroid plexus using lZ5I-labeledconjugates. The radioautoReferences p . 87/88

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Fig. 13. Isolated chick choroid plexus incubated for 15 min in 1251-labeledbovine albumin (0.1 % protein solution, specific radioactivity 100 pC/ml) showing strong radioactivity in the stroma. Tissue processed by the freeze-drying. Radioautograph, counterstained with hematoxylin and eosin, x 420. Fig. 14. Isolated chick choroid plexus incubated for 15 min in 1251-labeledbovine y-globulin (0.1 % protein solution, specific radioactivity 100 pC/rnl) showing radioactivity localized predominantly in the choroidal epithelium. Tissue processed by freeze-drying. Radioautograph, counterstained with hematoxylin and eosin, x 350.

graps revealed the stromal upstake of l”I-labeled albumin with the epithelium I emaining virtually free of radioactivity (Fig. 13), whereas 125I-globulin appeared to be frequently localized in the epithelial layer (Fig. 14). DISCUSSION

The present observations prompt some speculation on certain aspects of vascular permeability in the brain, as well as on the mechanisms inherent in the nervous tissue which accounted for the described penetration of serum proteins both from the blood circulation and from the CSF. With regard to the intracarotid injection of hypertonic glucose, it was interesting to note two distinct degrees of increased vascular permeability to serum proteins, i.e. with or without the spreading of a protein conjugate into the adjacent nervous parenchyma. Our fluorescence microscopic observations revealed that, in blood vessels larger than capillaries and endowed with structural elements intervening between endothelium and glia, the serum proteins may penetrate the endothelial layer and spread into the vessel wall including the adventitia, but still be prevented from entry into the surrounding nervous parenchyma and particularly into the glial compartment. As a counterpart of this finding one may consider a common observation from descriptive human neuropathology pertaining to the presence of dense vascular cuffings

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without an apparent passage of hematogenous inflammatory cells into the adjacent nervousLparenchyma.These morphological observations thus imply that the passage of either serum proteins or of hematogenous cellular elements from the blood circulation is impeded at two levels: one presented by the vascular endothelium and the other located between the adventitia and the glia. Observations on the dynamic aspects of serum protein movement in an area of brain edema indicate that the extravasated serum proteins may migrate for a considerable distance after leaving the blood vessels, using the white matter as their preferential pathway. Although the extravascularly located protein tracers in the area of edema could possibly be derived from locally increased vascular permeability to serum proteins, the following observations strongly suggest that we are dealing with a direct spread of conjugates from the site of the cortical injury: (1) in cats injected with the fluorescent conjugate preceding the cold lesion the area occupied by FLA appeared extending from the site of the injury in a direct relationship to the time elapsed, (2) the peripheral demarcation of the area of green fluorescence was in a straight-line fashion being not modified by the vasculature in this region, and (3) the vessels in the edematous white matter outside of the ‘solid front’ of a conjugate failed to reveal an increased permeability to the injected protein tracer (Fig. 6). The observation that cats injected at various stages of edema 1 h before sacrifice (second series) showed an area of the green fluorescence uniform in size which was smaller than was observed in animals injected with FLA preceding the cold injury and sacrificed after 1 h, can most likely be explained by the assumption that the leakage of proteins from the injured cortical vessels was most intense within the first hour after injury, i.e. allowing for the escape of a protein tracer in larger amounts than in the following hours. Similarly, this could also account for the slower spread of the conjugate into the edematous white matter during the first 4 h following the injection (third series). Since the temporal sequence of progressive infiltration of the white matter can also be demonstrated after administration of sodium fluorescein in place of fluorescent proteins (unpublished observations), it can be assumed that observations obtained with trypan blue or some other dyes in such experiments would yield results comparable to those reported in our studies. Thus, it should be kept in mind that an area stained by various blood-brain barrier indicators, which are allowed to circulate in the blood for only a limited time, may not completely delineate the whole area affected by an increased vascular permeability. The striking predilection of extravasated serum proteins for spreading in the white matter constitutes one of the most intriguing questions in the problem of cerebral edema. Our present observations merely suggest that this predilection cannot be explained by the lower threshold of permeability to serum proteins inherent in the blood vessels of the white matter since these vessels were shown not to be permeable to protein tracers even in the area of established edema. Our observations on the passage of serum proteins from the CSF pose several questions equally difficult for interpretation. These are primarily concerned with the described difference in passage from the ventricles between FLA and FLGG, as well as with the difference with regard to FLA penetration into the grey versus the white References p . 87IR8

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matter. The relatively more intense uptake by the periventricular grey matter has been recorded with regard to histamine (Draskoci et al., 1960), bromphenol blue (Feldberg and Fleischhauer, 1960), radiochromium and some organic acids (Edstrom and Steinwall, 1961), with a prevailing impression stated by the authors that an active transport mechanism might be involved in this phenomenon. Our observations on the ventricular perfusion with FLA did not provide any data directly supporting active transport, except perhaps for the fact that the periventricular penetration into the grey matter was absent in the animals which were perfused with FLA post-mortem. On the other hand, in the observations on the isolated chick choroid plexus, the possibility of an active transport mechanism appeared to be strongly favored by the effect of lowered temperature and various metabolic inhibitors on the stromal uptake of FLA. Also, an active transport mechanism in the isolated choroid plexus has been reported with regard to organic acid dyes (Rall and Sheldon, 1961), 1311 (Becker, 1961 and Welch, 1962a) and thiocyanate (Welch, 1962b). The reason for the inability of FLGG to penetrate into either the periventricular grey matter or into the isolated chick choroid plexus must be related to some physico-chemical property of this conjugate. It was interesting to note that this inability of FLGG to enter the isolated choroid plexus was overcome in incubations with addition of unlabeled globulin (see Fig. 12) which could possibly be responsible for stimulation of a pinocytotic uptake of the fluorescent conjugate. Our observations with both fluorescent and radioactive conjugates indicate that the serum proteins under physiological conditions do not penetrate into the brain tissue directly through the pial lining. The existence of such a barrier to serum proteins, although denied in the studies of Bowsher (1957) and Lee and Olszewski (1960), is supported by the experiments of Tschirgi (1950) in which trypan blue dye bound to serum proteins was found not to penetrate the cortex, as well as by our investigations using the freeze-drying procedure, which should have reduced to a minimum any possible movement of a tracer during either the fixation or further processing of the tissue. With regard to assessment of the role played by the glia in the penetration of serum proteins into nervous tissue, important information is still lacking as to whether the spread of serum proteins either from the injured blood vessels or from the ventricles takes an intra- or extracellular route. On the other hand, there are indications that the encounter between serum proteins and glia has some profound effects on the latter. Thus, it appears that the presence of serum proteins in the area of edema may stimulate a iapid and intense formation of the Holzer astrocytic fibrils (Rubinstein et al., 1962). In the same study it was also shown that in a similar situation the astrocytes react with an increased enzynatic activity which persists for a surprisingly long time after the edema has subsided. The uptake of fluorescent protein conjugates in the form of discrete intracytoplasmic inclusions has been previously related to a pinocytotic mechanism on the basis of our observations in the tissue culture (Klatzo and Miquel, 1960). Recently, Raimondi et al. (1962), in their electron-microscopic studies on cerebral edema, described globules of protein density demonstrable in the cytoplasm of the perivascular glial cells. Since these protein globules correspond

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in size and distribution to the fluorescent protein inclusions, their common nature appears to be likely. It is obvious that none of these protein droplets in the glial cells represent primary pinocytotic vesicles. On the other hand, they could easily correspond to the lysosomal structures which, according to interpretation of Novikoff (1960), are the end product of the transformation of pinocytotic vesicles associated with concentration of ingested material and its enzymatic digestion. Although the transport itself of serum proteins by astrocytes and oligodendroglia could not be actually demonstrated in our studies, an apparent migration of microglial cells laden with protein conjugates from the edematous white matter towards the pial and ependymal surfaces (Klatzo et al., 1962) as well as the intense uptake of protein tracers by microglia in the subarachnoid space (Klatzo et al., 1963) indicate the importance of this type of glia with regard to the transport and removal of soluble substances in the brain. Even though the described observations provide only a few clues concerning the interaction between serum proteins and glia, it might be expected that further studies on this subject, utilizing modern approaches such as electron microscopy, will significantly contribute to the understanding of the basic mechanisms underlying the function and reactivity of glia. SUMMARY

1. Penetration of albumin and y-globulin, labeled with fluorescent and radioactive markers, into the brain tissue was studied under various experimental conditions. 2. Two distinct degrees of an increased permeability of cerebral vessels to fluorescein labeled albumin (with or without the penetration of the protein conjugate into the nervous parenchyma) were demonstrable after the intracarotid injection of hypertonic glucose. 3. Observations on the local cold injury to the cerebral cortex demonstrated an extensive migration of extravasated protein tracers using the white matter as their preferential pathway. This movement of protein conjugates paralleled the progression of edema, whereas the blood vessels in the area of already established edema in the white matter failed to show increased permeability to protein conjugates. 4. Observations on the passage of protein conjugates from the CSF suggested that serum proteins do not penetrate directly through the pial lining into the brain parenchyma. On the other hand, passage of protein conjugates, with the preferential involvement of the periventricular grey matter, was demonstrable from the ventricles. 5. Observations on protein penetration into the isolated chick choroid plexus indicated the presence of an active transport mechanism which may be responsible for the stromal uptake of fluorescein labeled albumin. 6. The role of glia in relation to the penetration of serum proteins into the nervous tissue is briefly discussed. REFERENCES BECKER, B., (1961); Cerebrospinal fluid iodine. Amer. J . Physiol., 201, 1149-1151. D., (1957); Pathways of absorption of protein from the cerebrospinal fluid: autoradioBOWSHER, graphic study in the cat. Anat. Rec., 128, 23-39.

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BROMAN, T., AND OLSSEN,O., (1956); Technique for pharmaco-dynamic investigation of contrast media for cerebral angiography. Effect on the blood-brain barrier in animal experiments. Acta radiol., 45, 96-100. DRASKOCI, M., FELDBERG, W., FLEISCHHAUER, K., AND HARANATH, P. S. R., (1960); Absorption of histamine into the blood stream on perfusion of the cerebral ventricles, and its uptake by brain tissue. J. Physiol., 150, 50-66. EDSTROM, R., AND STEINWALL, O., (1961); The blood-brain-barrier phenomenon - the relative importance of permeability and cellular transport mechanisms. Acta psychiat. scand., 37, 1-21. FELDBERG, W., AND FLEISCHHAUER, K., (1960); Penetration of bromphenol blue from the perfused cerebral ventricles into the brain tissue. J. Physiol., 150, 451462. HAGER,H., (1961) ; Elektronenmikroskopische Untersuchungen iiber die Feinstruktur der Blutgefasse und perivascularen Raume irn Saugetiergehirn. Acta neuropafh., 1, 9-33. KLATZO, I., AND MIQUEL, J., (1960); Observations on pinocytosis in nervous tissue. J. Neuropath. exp. Neurol., 19,475487. KLATZO, I,, MIQUEL, J., FERRIS,P. J., PROKOP, J. D., AND SMITH,D. E., (1964); Observations on the passage of fluorescein labeled proteins (FLSP) from the cerebrospinal fluid. J. Neuropafh. exp. Neurol., 23, 18-35. KLATZO, I., MIQUEL, J., AND OTENASEK, R., (1962); The application of fluorescein labeled serum proteins (FLSP) to the study of vascular permeability in the brain. Acta neuropath., 2, 144-160. KLATZO, I., F’IRAUX, A., AND LASKOWSKI, E. J., (1958); The relationship between edema, blood-brain barrier and tissue elements in a local brain injury. J. Neuropathol. exp. Neurol., 17, 548-564. LEE,J. C., AND OLSZEWSKI, J., (1960); Penetration of radioactive bovine albumin from cerebrospinal fluid into brain tissue. Neurology, 10, 814-822. NOVIKOFF, A. B., (1960); Biochemical and staining reactions of cytoplasmic constituents. Developing Cell Systems and their Control. D. Rudnick, Editor. New York, Ronald Press Company (pp. 167-203). RAIMONDI, A. J., EVANS,J. P., AND MULLAN, S., (1962); Studies of cerebral edema 111. Alterations in the white matter: an electron-microscopic study using ferritin as a labeling compound. Acfa neuropathol., 2, 177-197. RALL,D.P., AND SHELDON, W., (1961); Transport of organic acid dyes by the isolated choroid plexus of the spiny dogfish S. Acanthias. Biochem. Pharmacol., 11, 169-170. RUBINSTEIN, L. J., KLATZO, I., AND MIQUEL, J., (1962); Histochemical observations on oxidative enzyme activity of glial cells in a local brain injury. J. Neuropath. exp. Neurol., 21, 116-136. SMITH, D. E., STREICHER, E., MILKOVIC, K., AND KLATZO, I., (1964); Observations on the transport of proteins by the isolated choroid plexus. Acta neuropath., 3, 372-386. STEINWALL, O.,(1958); An improved technique for testing the effect of contrast media and other substances on the blood-brain barrier. Acta radiol., 49,281-284. TSCHIRGI, R. D. (1950); Protein complexes and the impermeability of the blood-brain barrier to dyes. Amer. J. Physiol., 163, 756. WELCH,K., (1962a); Active transport of iodide by choroid plexus of the rabbit in vitro. Amer. J. Physiol., 202, 757-760. WELCH, K., (1962b); Concentration of thiocyanate by the choroid plexus of the rabbit in vitro. Proc. SOC.exp. Biol. ( N . Y.), 109, 953-954.

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Astroglial Reactions to Ionizing Radiation : with Emphasis on Glycogen Accumulation* J. M I Q U E L

AND

W. H A Y M A K E R

National Aeronautics and Space Administration, Ames Research Center, Moffett Field, Calif. (U.S.A.)

Particle radiation has recently proved a highly useful tool and has taken its place with X-radiation as a means of investigating glial function. It was only 6 years ago that cyclotron-generated monoenergetic particles were first directed to the cerebral cortex of an experimental animal, and a discrete pseudolaminar lesion produced (Malis et ul., 1957). Energy absorption by the tissue was 4 to 5 times greater at this level than in the transirradiated cortex, allowing the effects of different doses in the same cortex to be observed. Our own investigations in this field were begun in the late 50’s in collaboration with Dr. Cornelius Tobias, of the University of California in Berkeley. One of the members of our group, Dr. Igor Klatzo, fixed, as a matter of routine, some a-particle irradiated brain material in Rossman’s fluid. It was in this way that the presence of an abundance of glycogen was discovered. The purpose of this presentation is to describe our results to date on the effects of a-particle and X-radiation on astroglia and blood vessels. The knotty problem of the effects of particle irradiation on oligodendroglia has been discussed in another paper by members of our group (Estable-Puig et al., 1963). MATERIAL AND M E T H O D S

X-Radiation data. A total of 505 rats of both sexes were used in this study. Of these, 185 rats (Sprague-Dawley strain), 4 to 6 weeks old and weighing 100 to 200 g, received X-radiation to the head. The animals were reared on a complete laboratory stock diet. For studies on the effects of X-rays on the immature brain, 8 rats, 8 days of age, were used. The source of radiation was a constant-potential X-ray therapy unit with the following characteristics: 250 kVp, 15 mA, 0.25 mm Cu and 1.0 mm Al filters. The dose rate in air (at a target-to-skin distance of 35 cm) was 115.4/min. The midline tissue dose was estimated at approximately 105 r/min (or 98 rad/min). As described elsewhere (Miquel et al., 1963), the irradiation was performed in groups of 4 animals each, under light nembutal anesthesia. The heads, extending from beneath lead shields,

*

Dedicated to Prof. W. Scholz on the occasion of his 75th birthday (15 december 1964).

References p . 112-114

90

J. MIQUEL AND W. H A Y M A K E R

TABLE I W H O L E B R A I N X - I R R A D I A T I O N : N U M B E R OF R A T B R A I N S S T U D I E D A T V A R I O U S TIME I N T E R V A L S AFTER I R R A D I A T I O N A N D TEC HN IQU ES U S E D

Number of brains studied

Radiation dose (r) 150 300 600

4 10 4 4 4 12 12

1200 2400

3000 3000

63

3000

4 4 4 4 4 24

3600 4800 6000 7200

8 12 4 4*

8500 10,000 10.000

10,000 20,000 m.000

Time of sacrifice (h)

PAS

24 24 24 24 24 5 12 24 24 24 24 24 24

4 10 4 4 4 4 4 16 4 4 4 4 4

Glycogen

Lactic acid determination

Lactic acid in anoxic brain

8

8

8 8 47

5

8

12 24 24

8 8

4 4

Control 71

20

35

8

8

Total

98

122

16

16

*

256

The animals exposed to 40,000 r had convulsions shortly after irradiation and were dead 24 h later. TABLE I1 NUMBER OF

Number of brains 3 3 3 3

3 Control 3 Total

18

X-IRRADIATED BRAINS

OF 8-DAY-OLDR A T S A N D TECHNIQUE USED

Radiation dose ( r )

Time of sacrifice ( h )

600

24

1200 3000 6Ooo

24

12,000

24 24 24

PAS 3 3 3

3 3 3 18

were close to the center of the radiation field. The source was situated above the head, irradiating its dorsal aspect. Both cerebrum and cerebellum were irradiated in some of the rats, while in the others, in which the cerebellum and brain stem were shielded, irradiation was limited to the cerebrum. Following irradiation with doses ranging

91

A S T R O G L I A L R E A C T I O N TO R A D I A T I O N

from 150 to 40,000 r, the animals were sacrificed at different time intervals (Tables I and 11). For irradiation of a localized field in the brain, the heads were exposed to X-rays under two lead shields placed 5 mm apart. Under these slit-beam conditions, a dose of approximately 10,OOO r was delivered to a small rectangular-shaped field of the brain. After irradiation the animals were sacrificed at the time intervals shown in Table 111. a-Particle radiation data. Methods of irradiation with a-particles, which were delivered by the 60-inch cyclotron in Berkeley, have been described elsewhere (Janssen et al., 1961; Klatzo et al., 1961). The particles had an energy of 12 MeVper nucleon. TABLE I11 X -IRRADIAT IO OF NA

LOCALIZED A R E A OF THE R A T CEREBRUM: N U M B E R OF A N I M A L S S T U D I E D A N D T E C H N I Q U E S U S E D

Control Total

Number of brains studied

Radiation dose (r)

Time of sacrifice (h)

NaFl

PAS

2 2 2

10,Ooo

24 48 72

2 2 2

2

4

2

2

10

8

4

10,000 10,000

The surface dose to the brain was 6000 rad and the peak dose was 30,000 rad. Dose rate during the exposure was approximately 10,000 rad/min to the brain surface. The cyclotron aperture through which the particle beam passed was 14.3 mm in diameter. This aperture was of sufficient size to allow irradiation of most of the dorsal surface of the cerebrum and cerebellum. A total of 92 rats (Long-Evans strain) of both sexes were used. They were 3 weeks of age and weighed approximately 60 g. With the animals under nembutal anesthesia, the scalp was incised in the midline and reflected, and the animals were mounted on a holder and their heads were placed at a distance of 5 mm from the end window of the ionization chamber. Following exposure the animals were sacrificed at time intervals ranging from 5 min to 5 months (Table IV). The 60-inch cyclotron not being available during the entire course of the experiments, some of the brains were exposed to an a-particle beam from the Heavy Ion Linear Accelerator (HILAC). The particles had an energy of 10.4 MeV/nucleon and the dose rate during the exposure of the brains was approximately 24,000 rad/min. The aperture through which the particles passed was 10 mm in diameter, sufficient to irradiate most of the dorsal surface of the cerebrum. A total of 105 Tats (SpragueDawley strain) were used in this study. The animals were 3 weeks of age and weighed approximately 60 g. They were exposed to doses ranging from 500 to 48,000 rad and were sacrificed at various time intervals thereafter (Table V). Studies of the blood-brain barrier mechanism (BBBM) with the use of sodium fluorescein as indicator. For gross observations of altered functioning of the BBBM, References p. 112-114

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J. M I Q U E L A N D W. H A Y M A K E R

cyclotron-irradiated and 6 control rats were injected intravenously, approximately 24 h before sacrifice, with 1.0 ml of 10% sodium fluorescein in buffered saline. On TABLE IV R A T B R A I N I R R A D I A T E D W I T H a - P A R T I C L E S FROM T H E 6 0 - I N C H C Y C L O T R O N (6000 rad S U R F A C E D O S E ; 30,000 rad P E A K D O S E ) : N U M B E R O F B R A I N S S T U D I E D A T V A R I O U S TIME I N T E R V A L S A N D T E C H N I Q U E S U S E D

Number of brains studied

Time of sacrifice

2 2 2 2 2 5 7 10 8 8 9 5 4 3 2 4 2 1 3 3 1 5 1 1

5 min 33 l h 3 6 12 24 48 72 4 days 5 6 7 8 10 11 12 13 14 18 24 36 4 months 5

NaFI

3 2 4 4 5 3 3

1

2 2 2 2 1

1 1

PickworthLepehne

FLA

1 2 2 1 2 1 1

3 4 5 5 5 5 4 3 2 2 2 1 1 2 3 1 2 1

Control 18

4

4

2

Total 110

18

47

53

3 3 1 2 3 3 3 2 2 2

PAS 2 2 2 2 2 2 3 5 3

3 3 1 2

2 1

1 4

8

34

48

the other hand, for the study of permeability disturbances in the brains of rats exposed to a-particle radiation from the HILAC, 1 ml of 10% sodium fluorescein was injected intraperitoneally 1 h prior to sacrifice. The animals were killed by decapitation and the dorsal surface of the brains and coronally sectioned blocks were observed in a dark room under U.V. light. Histological and histochemical studies. For observations on vascular permeability to serum proteins, bovine albumin was conjugated with fluorescein isothiocyanate, as previously described (Klatzo et al., 1962). The fluorescein-labeled albumin (FLA) used was in 8 % concentration in buffered saline. Irradiated and control animals were injected intravenously with 2 ml of the solution at 24 h before sacrifice. Details of the technique for microscopic observation of the brain sections under U.V. light are given elsewhere (Klatzo and Miquel, 1960).

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ASTROGLIAL REACTION TO RADIATION

TABLE V R A T B R A I N S I R R A D I A T E D W I T H a - P A R T I C L E S FROM T H E

B E R K E L EH YI L A C :

N U M B E R O F B R A I N S S T U D I E D A T D I F F E R E N T TIME I N T E R V A L S AFTER EXPOSURE A N D TECHNIQUES USED

Number of brains studied

Radiation dose (rad) *

Time of sacrifice

11 3

500 lo00

48 h 48

3 3 3 2 2 1

1500

24 48 72 14 days 30 60

3 6 2

3000

1 1

24 h 48 72 14 days 30

NaFl

Cajal

PAS

11 3

2 2 1

3 3 3 2 2 1

3 3 3 2 2 1

1 2

3 1 2

3 6 2 1 1

3 3

1 1

1

1

60

4 2 3 3 3 2

4

2 3 3 3 2

24 h 48 72 14 days 30 60

3 3 3 2 2

3 3 3 3 2 2

3 7 3 3 2 2

2 2 3 3 2 1

2 4 3

2

2

1 1 1

1 1 1 1

1 1

Control 6

6

3

6

Total 1 1 0

63

77

111

24 h 48 72 14 days 30

4 8 3 3 3 2

3 7 3 3 2 2

12,000

2 4 3 3 2 1 2 1 1 1 1

24 h 48 72 14 days 30 60 48,000

24 h 48 72 30 60

2 3 3

2 1 1

8 3 3 3

2

3

2 1

1 1

* These represent surface doses; peak doses, about 1 mm deep to the brain surface, were 5 times higher. Re$eerencm p . 112-114

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J. M I Q U E L A N D W. H A Y M A K E R

Brains of some of the irradiated and control rats were perfused intravascularly through the heart, by saline followed by Rossman’s fluid (approximately 300 ml per animal). The other animals were sacrificed by decapitation at various time intervals following irradiation (Tables I-V). After brain removal, tissue blocks were placed in the following fixatives : 10% formol saline, formalin ammonium bromide, and Rossman’s fluid. The tissue fixed in 10% formol saline was used in fluorescein conjugated protein and Pickworth-Lepehne studies, that fixed in ammonium bromide, for Cajal gold chloride impregnation of astrocytes, and the tissue fixed in Rossman’s fluid, for the periodic acid Schiff (PAS) procedure. To obtain selective demonstration of the glycogen, dimedon* was used as a blocking reagent prior to PAS staining, as recommended by Bulmer (1 959). For histochemical analysis of carbohydrates, sections from Rossman-fixed brains, in addition to being used for PAS staining, were subjected to enzymatic digestion with a- and p-amylase and to additional stains and procedures which have been described elsewhere (Klatzo et al., 1961). Chemical analysis. For chemical analysis of the irradiated and control brains, the animals, after fasting for 24 h, were sacrificed by immersion in liquid nitrogen. A manual bone saw was used to cut through the frozen head, with one cut posterior to the eyes, another at the ear openings, and a thiid at an intermediate distance between the two. The central part of the head was then removed in two pieces through use of a prechilled metal chisel and the pieces were placed on a Petri dish cooled with dry ice. The frozen brain was separated from the surrounding tissue and weighed quickly on the cooled pan of a torsion balance. Glycogen isolation was performed on the frozen brain by the procedure devised by Kerr (1938), and the analysis was performed with the use of glucose oxidase, as previously reported (Miquel et al., 1963). Lactic acid analysis was carried out as follows :The frozen brains were homogenized in a ‘Teflon’-pestle homogenizer in 10% trichloroacetic acid (TCA). The homogenate was made up to 50 ml with TCA and the lactic acid determinations done in an aliquot by the Barker-Summerson method (Hawk et al., 1954). To study the effects of radiation on anaerobic glycolysis, rats receiving 10,000 r of X-rays to the head were used, with 16 rats serving as controls. The rats were decapitated and the brains left in situ at room temperature for 15 min. Then the cerebrum was removed and homogenized in TCA, and the lactic acid analyzed as just described above. RESULTS

All particle radiation doses in the following are indicated as surface doses unless otherwise specified. Vascular changes. In Pickworth-Lepehne preparations of brains exposed to 6000 rad of a-particle radiation, vascular dilatation was first observed at 48 h (Janssen et al., 1962). Hemorrhages, noted in about one-third of the brains, were first evident

*

5,5-Dimethyl-l,3-cyclohexanedione. Matheson, Coleman and Bell, Nonvood, Cincinnati, Ohio

(USA.).

ASTROGLIAL REACTION TO RADIATION

95

at 72 h, predominantly in the lower part of the ‘Bragg zone’ (Fig. 1). At 4 days, vessels of capillary proportion within the band were severely damaged and some had a swollen basal membrane. At 10 days postradiation, small hemorrhages were noted, particularly in parts of the cerebral cortex adjacent to the interhemispheric fissure (Fig. 2). At 18 days they were rare both in the cerebrum and cerebellum. On the other hand, vascularization in the ‘zone’ was considerably reduced, and the larger vessels passing through the ‘Bragg-peak band’* showed varicose dilatations (Fig. 3). At 4 months postexposure, capillary density in the ‘Bragg zone’ was relatively increased owing to tissue atrophy (Fig. 4). BBBM disturbances. Observations with NaFI. In the brains exposed to 6000 rad of a-particles from the cyclotron the earliest time interval at which NaFl penetration was noted in the brain parenchyma was at 72 h. Examination of coronally sectioned brains under U.V. light revealed faint green fluorescence in the cerebral cortex and an intense fluorescence in the underlying white matter. In the brains of animals sacrificed subsequently the fluorescence gradually faded and was last seen at 14 days after irradiation. To a-particles from the HILAC, penetration of NaFl into the cerebral parenchyma was noted 24 h after exposure to 12,000, 24,000 and 48,000 rad. On the other hand, in the brains receiving 3000 or 6000 rad, penetration of the indicator was observed at 72 h. In the brains receiving 6000 rad or more, permeability was still altered at 14 days, but in 30 days no permeability change was noted. In the experiment concerned with the effects of local exposure of the cerebrum to 10,000 r X-radiation, the brain tissue showed faint fluorescence in the irradiated area at 24 h. A tthe 48- and 72-h stages the fluorescence in the exposed area was intense. BBBM disturbances. Observations with FLA. Grossly, the earliest parenchymal penetration by FLA was observed in 2 of the 4 rats sacrificed at 72 h after irradiation (Fig. 5). Thereafter the fluorescence faded gradually and was last seen at 13 days. The first evidence of vascular penetration by FLA was noted at 48 h. At this time period small FLA globules lay just outside vessel walls. At 72 h the perivascular globules were more numerous in the cortex and many neuroglial cells in the underlying white matter contained intracytoplasmic FLA inclusions in profusion. The protein was also observed in the white matter in the form of irregular aggregates in what appeared to be extracellular spaces. At later postradiation stages the amount of fluorescent material in the white matter and cortex, underwent progressive reduction. Extravasated protein was last seen at 36 days. In the animals sacrificed at 4 and 5 months after irradiation the fluorescent protein was contained within the blood vessels as in the control (non-irradiated) rats.

* Damage to cellular elements occurs in accordance with the degree of energy transfer to tissue as expressed by the Bragg curve. The energy at the greatest depth of particle penetration in the tissue, corresponding to the peak of the Bragg curve, was, in the present experiments, about 5 times greater than at the surface of the brain. As used in the present article, the term, Bragg zone, refers to the part of the cerebral or cerebellar cortex in which cellular damage was demonstrable, and the term, Bruggpeak band, to the region in the deepest part of the Bragg zone in which destruction of cellular elements was maximal. Rsfwences p . 112-114

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Figs. 1 4 . Cerebrum, a-particle radiation at 6000 rad (cyclotron). Pickworth-Lepehne stain.

A S T R O G L I A L R E A C T I O N TO R A D I A T I O N

97

Fig. 5. Rat cerebrum at 72 h after irradiation with 6000 rad of a-particles (cyclotron). The rat was injected intravenously with fluorescein-conjugated albumin. The cerebral cortex fluoresced a faint green, while the white matter in an area extending down to about the middle of the brain on the right side had an intense fluorescence. (From Klatzo et al., 1961).

Observations on CajaI gold chloridepreparations. Observations on HILAC-irradiated brains were highly similar to those described by Klatzo et al. (1961), and by Janssen et al. (1961) in rat brains exposed to a-particles from the 60-inch cyclotron (Table IV). The initial change in astrocytes, consisting in an increase in the intensity of impregnation, was observed at 48 h after exposure to 3000 rad and more. By 72 h the astrocytes in the ‘Bragg zone’ had become hypertrophic (Figs. 6 and 7). In the irradiated part of the cerebellum these changes were prominent both in the cortex and the intrafolial white matter. During the 3rd or 4th day, astrocytes in the ‘Bragg-peak band‘ were undergoing disintegration and the few that remained were obviously damaged. In the cortex both above and below the ‘Bragg-peak band’ many astrocytes were hypertrophic (Fig. 8). The reaction was very conspicuous in the part of the cortex adjacent to the interhemispheric fissure (Fig. 9). In later stages (up to 5 months after irradiation) hypertrophic and oddly formed astrocytes persisted along the lower edges of the ‘zone’. Histochemical studies on the glycogen accumulation in the irradiated brain. In the non-irradiated control rats, histochemically demonstrable glycogen was limited to PAS-positive granules in the processes of the subependymal cells of the walls of the lateral and third ventricles. In brains exposed to cyclotron generated a-particles at 6000 rad, glycogen accumulation in the cortex was visible in PAS preparations at the 12-h postexposure stage Fig. 1. 72 h after irradiation, showing hemorrhages within and adjacent to the ‘Bragg-peak band’, x 60. Fig. 2.10 days after exposure. Cerebral cortex, showing hemorrhages confined mainly to the vicinity of the interhemispheric fissure, x 60. Fig. 3. 18 days after exposure. Cerebral cortex. Irregular varicose dilatations in larger blood vessels passing through the band, x 90. Fig. 4. 4 months after irradiation. The ‘band‘ is still visible as a narrow pale strip. In the adjacent atrophic cortex the vessel density is increased, x 120. (From Klatzo et al., 1961). References p. 112-114

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Figs. 6-9. Cerebral cortex, a-particle radiation a t 6M)o rad from cyclotron. Cajal gold chloride impregnation. Fig. 6. Normal rat cortex, illustrating an area corresponding in location to the ‘Bragg zone’, x 500. Fig. 7. 72 h after irradiation. Area in the ‘zone’ showing early astroglial activation, X 500. Fig. 8. 5 days postexposure. All astrocytes are hypertrophic. Those just beneath the ‘band’ (in the lower part of the photograph) have a vertically oriented process, while those located more superficially (in the irradiated region) have no special orientation, x 220. Fig. 9. 8 days after irradiation. Cerebral cortex adjacent to the interhemispheric fissure, showing striking astrocytic hypertrophy in the region just beneath the ‘Bragg-peakband‘, x 176.(From Klatzo et al., 1961).

A S T R O G L I A L R E A C T I O N TO R A D I A T I O N

99

(Klatzo et al., 1961). In rats sacrificed at 48 h the amount of histochemically defined glycogen had increased sharply and was found maximal when comparison was made with preparations from animals which were sacrificed at later stages. Glycogen granules gradually decreased in number and at 36 days after irradiation they were no longer present. In the HILAC a-particle irradiated cerebrum, PAS staining revealed glycogen accumulation at 12 h after exposure to 3000 rad or more. At 48 h after irradiation, glycogen increase was also observed in the brains receiving 1500 rad. In general, the glycogen content at 48 h was proportional to the radiation dose. In animals exposed to 24,000 and 48,000 rad, glycogen was seen in abundance, e.g. in the hippocampus (Figs. 10-12). The corpus callosum also exhibited some glycogen in glial cells. In addition to the presence of glycogen granules, the histological picture was dominated by an edematous process in the white matter (Figs. 13-14) and, in the periphery of the area of maximal damage, by abundant mitotic figures (Fig. 15). At 14 days after exposure the glycogen content of the irradiated brains was still abnormally high, but at 30 days, no glycogen granules were observed. X-irradiated brains showed glycogen accumulation at doses of 1200 r or more. As in particle-irradiated brains, glycogen content appeared to be roughly proportional to the dose. In brains exposed to 3000 r, glycogen granules were seen as early as 5 h postexposure, with maximal accumulation at 12 to 24 h. On the following days the amount of glycogen decreased markedly, and at 6 days postexposure, no PAS-positive, amylase-extractable granules were visible. In regard to glycogen distribution, it differed markedly in the particle- and Xirradiated brains owing to differences in size of brain area irradiated, and radiation intensity. By 48 h after exposure to a-particle radiation, glycogen granules had accumulated on both sides of the ‘Bragg-peak band’, which itself was devoid of histochemically demonstrable glycogen (Fig. 16). In the cerebellum, glycogen granules were congregated in the lowermost part of the molecular layer adjacent to the granular layer, where the ‘Bragg-peak band’ was recognizable by the presence of pyknotic granule cells. In X-irradiated brains, on the other hand, the heaviest glycogen accumulations were in the hippocampus and rhinencephalon (Figs. 17 and 18). Glycogen granules were also present in the cerebral cortex (Fig. 19), geniculate bodies, corpus striatum and globus pallidus, and in moderate amount in the subcortical white matter. No granules were found in more centrally located structures, such as the thalamus. In the cerebellum, glycogen accumulation was most abundant in the Bergmann-cell layer. Glycogen granules were also present in the molecular layer but they were never observed in the granular layer or in the white matter (Figs. 20-23). As to cytological localization in irradiated areas, glycogen accumulation was never seen in nerve cells. On the other hand, histochemically demonstrable glycogen was plentiful in astrocytes (Figs. 24-26). In addition, abundant glycogen granules were found at random in the neuropil (Fig. 19) or were concentrated around blood vessels. Glycogen granules were also seen in reticulo-endothelial elements of the leptomeninges in later postradiation stages (Fig. 27). Here and there they were observed in macroReferences p . 112-114

100

-.

J. MIQUEL ANDLW. HAYMAKER

Fig. 10. Hippocampus at 48 h after a-particle irradiation at 24,000 rad (HILAC). In contrast with the glycogen accumulations in the hippocampus, no glycogen granules are present in the underlying corpus callosum. PAS-hematoxylin, x 150.

Fig. 11. Hippocampus at 48 h after exposure to a-particlesat 24,000 rad (HILAC). Abundant glycogen is present in astroglial cytoplasm. PAS-hematoxylin, x 150.

A S T R O G L I A L R E A C T I O N TO R A D I A T I O N

101

Fig. 12. Hippocampus at 48 h after a-particle irradiationat 24,000 rad (HILAC). Glycogen is concentrated in glial cells, and is not present in the neuronal perikaryon. PAS-hematoxylin, x 625.

Fig. 13. From cerebrum of a rat at 48 h after exposure to 24,000 rad of a-particles (HILAC). Edematous white matter below a well preserved cortex. PAS-hematoxylin, x 260. References p . 112-114

102

J. M I Q U E L A N D W. H A Y M A K E R

Fig. 14. From cerebrum of a rat at 48 h exposure to 24,OOO rad of a-particles (HILAC). Edematous white matter. PAS-hematoxylii, x 260.

Fig. 15. Mitotic figure in cerebral cortex at 48 h after exposure to 24,OOO rad of a-particles (cyclotron). PAS-hematoxylin, x 600.

phages, but whether they were present in oligodendroglia or in microglia could not be said with certainty. In cortex irradiated with the slit-beam, glycogen granules were seen scattered in the exposed area at the 24-h stage. No observations at later stages were made. The cerebrum of the control (non-irradiated) immature brains was free from glycogen granules except around the ventricles and, in heavy concentration, in the

ASTROGLIAL REACTION TO RADIATION

103

Fig. 16. Cerebral cortex at 48 h after a-particleirradiation at 6000 rad (cyclotron). Glycogen granules are abundant on both sides of the ‘Bragg-peak band’, which itself shows only faint PAS staining, indicating a low carbohydratecontent. PAS-hematoxylin, x 250 (From Klatzo et al., 1961). TABLE VI E F F E C T O F V A R I O U S X-RAYD O S E S O N T H E G L Y C O G E N C O N T E N T O F T H E R A T B R A I N A T 24 h *

Number of animals used

Dose ( r )*

8 8

10,000

Glycogen content** (mg glucose/100 g brain)

71.6 f 6.14 126.6 f 16.2

5000

48.7 f 10.7

Control

*

Surface doses are indicated

**

Mean values f S.D.

TABLE VII B R A I N G L Y C O G E N A T V A R I O U S TIME I N T E R V A L S AFTER X - I R R A D I A T I O AT N 3000 r

Number of Time of animals used sacrifice ( h )

8 8 8

* Referencesp. 112-114

5 24 48

Mean values f S.D.

Glycogen content * (mg glucose/IOOg brain)

58.7 f 5.1 72.5 f 5.4 62.7 f 4.3

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Figs. 17 and 18. Olfactory tubercle at 24 h after X-irradiation at 20,000 r. Fig. 17. Concentric distribution of glycogen is to be seen in the outer layer of the tubercle. None is present in the myelinated central core. PAS-dimedon-hematoxylin, x 41. Fig. 18. DPN-diaphorase in the olfactory tubercle of a control (non-irradiated) rat. The distribution of this enzyme, which is a good index of the aerobic metabolism, very closely parallels that of the glycogen deposits appearing after irradiation, as shown in Fig. 17, x 41.

choroid plexuses. Following exposure to X-ray doses of 3000 r or more, glycogen granules were seen scattered in other regions of the brain as in the older rats. Chemical glycogen studies in the irradiated brain. In general, quantitative chemical analyses have confirmed the conclusions drawn from histochemical studies. A real increase in glycogen content has been demonstrated by chemical analysis both in

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Fig. 19. Cerebral cortex at 24 h after 6000 rad X-irradiation.Numerousglycogen granulesare scattered in the neuropil. PAS-hematoxylin, x 210.

a-particle and X-irradiated brains. In brains X-irradiated at 3000 r the increase was approximately 40 % at 24 h. Accumulation of glycogen in accordance with radiation dose and a tendency to fall at the 48-h stage have been confirmed by chemical analysis (Tables VI and VII). The mean value for lactic acid content of the normal rat brain was 20.9 mg per 100 g of brain (S.D. f 4.0). Under anoxic conditions, at 15 min after decapitation, the mean values rose to 83.6 mg (S.D. 2.9) and 97.7 mg (S.D. f 8.4) per 100 g for normal and irradiated brain, respectively. DISCUSSION

Our observations of histological changes in the irradiated brain agree in general with those of numerous investigators who have described disturbances of the BBBM and/or gliosis following irradiation (Arnold and Bailey, 1954; Arnold et al., 1954; Clemente and Holst, 1954; Clemente and Richardson, 1962; Scholz et al., 1962). On the other hand, to our knowledge, this is the first report of the presence of numerous mitotic figures (Fig. 15) in rat brains exposed to particle radiation. Recently, Altman (1963) using autoradiographic techniques, has observed that the rat brain has a low rate of glial proliferation, and from histological studies of irradiated brains it has been recognized that the functions of astroglia are suppressed for a time, and that luxuriant proliferation then occurs. On the other hand, we have seen as many as 5 mitotic figures in a single microscopic field of the cerebrum of a rat exposed to a-particle radiation at 24,000 rad. We were unable to identify the glial species undergoing this stimulation of References p . 112-1 I4

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Figs. 20-23. Cerebellum. Fig. 20. From the cerebellum of a control (non-irradiated)rat. Histochemicallydemonstrableglycogen is absent. PAS-dimedon-hematoxylin, X 41. Fig. 21. Cerebellum at 24 h after X-irradiation with 20,000 r, showing glycogen accumulation in the molecular layer. PASdimedon-hematoxylin. Fig. 22. The same section as in Fig. 21, at higher magnification. Glycogen granules are confined to the Bergmann and molecular layers, X 41. Fig. 23. Demonstration of DPN-diaphorase in the cerebellum of a control (non-irradiated) rat. Intense respiratory activity is to be noted in the granular and molecular layers, X 41.

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Figs. 24 and 25. Glycogen in the cytoplasm of hippocampal astrocytes in a brain exposed 48 h previously to 24,000 rad of a-particles (HILAC). PAS staining includes the astroglial processes and reveals glycogen condensation at the site of an end-foot on a capillary. PAS-dimedon-hernatoxylin, x 375.

Fig. 26. Corpus callosum in a brain exposed 48 h previously to 24,000 rad a-particles (HILAC). The glycogen appears to be concentrated in neuroglial cells with oval, comparatively large hypochromatic nuclei (probably astrocytes). PAS-dirnedon-hernatoxylin, x 375.

mitotic activity. In every instance the cytoplasm of cells in mitosis was rich in glycogen. Bullough (1952) has postulated that the energy supply for mitosis in the mouse epidermis is derived from glycogen and from glucose and its derivatives. Our observations suggest that glial cells in the irradiated region also store glycogen before engaging in mitotic activity. Cells of the subependymal matrix contained such an abundance of glycogen normally that it was difficult to say whether or not it was increased following irradiation. In this connection it is of interest that subependymal matrix cells in the wall of the lateral ventricle of the rat show enduring mitotic activity (Altman, 1963; Smart and Leblond, 1961). The puzzling glycogen deposits which these cells possess in the normal brain might therefore be linked with the mitotic process. Regarding permeability studies, in our experience NaFl is a sensitive indicator of BBBM disturbances. It seems that intravenous injection of the NaFl (Van Dyke et al., 1962; Klatzo et al., 1962) has no definite advantage over the intraperitoneal route of administration as used in the present study. Electron-microscopic studies by Hager et al. (1962) have shown that astrocytic and References p . 112- I14

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Fig. 27. Meninges of rat brain exposed to 12,000 r X-rays and sacrificed at 24 h. Abundant glycogen inclusions are present in the reticulo-endothelial cells. PAS-hematoxylin, x 400.

other cellular damage in irradiated cerebral cortex is not associated with an increase in size of the intercellular gap. Our studies with FLA revealed a decided difference in the reaction of cortical and white matter parenchyma as far as BBBM disturbances were concerned. Extravasated protein in the cortex was usually apparent in droplets confined to the immediate vicinity of the blood vessel, as if an impregnable wall of cellular membranes prevented its diffusion. By contrast, in the white matter the FLA was widespread, reaching far beyond the irradiated area. Furthermore, glia participated actively in the uptake of the extravasated serum proteins. Recent electronmicroscopic studies have confirmed the presence of intercellular gaps and they have provided evidence of pinocytotic uptake of materials from edematous white matter (Gonatas et al., 1963; Raimondi et al., 1962). The accumulation of glycogen granules in the neighborhood of the 'Bragg-peak band' bore a remarkable similarity to glycogen localization in stab wounds, as described by Friede (1954) and by Shimizu and Hamuro (1958). It appears that glycogen accumulation is a constant feature of the brain reaction in a variety of localized brain injuries :stab wounds, heat) DeEstable andEstable-Puig, 1963),and from intracerebrally injected plasma (Fig. 28). Glycogen granules have also been observed at the periphery of some human brain tumors (Oksche, 1961). Under all these conditions, glycogen accumulates in an area of morphologically well-preserved cells in the vicinity of a necrotic zone which itself is totally devoid of glycogen. On the other hand, in the X-irradiated brain, glycogen accumulation has been observed in the absence of any pathological change as demonstrated by routine histological stains. A further point is that in the X-irradiated brains the glycogen granules disappeared more rapidly than in the brains exposed to a-particles. Thus, abnormal glycogen deposits

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Fig. 28. Glycogen granules in a cerebral-cortical area close to the site of injection of 0.1 ml of rat serum. Glycogen deposits are numerous. PAS-hematoxylin, x 210.

were absent in the X-irradiated animals at 6 days, while in the animals exposed to a-particles they persisted for as long as 24 days after irradiation. This difference might be due to the difference in energy transfer per unit volume, which is very high in the ‘Bragg-peak band’ (resulting in severe tissue damage in this location) and comparatively low in the X-irradiated brain. That glycogen accumulation occurs in glial cells under pathological conditions in human brains was recognized some 50 years ago by Casamajor (1 9 13). More recently, Friede (1954) also observed glycogen in glial cells, which he sometimes identified as astrocytes. In our opinion, the cells in which glycogen accumulates following brain irradiation are mostly identifiable as astrocytes. Given the metabolic peculiarities of astroglia (Friede, 1962; Pope, 1958), it would not be surprising if the accumulation of glycogen in the irradiated brain were an exclusive property of the astroglia. The contrast between the scattered distribution of abundant glycogen and the less frequent concentration of glycogen in astrocytic cytoplasm, as observed by us, has been noted also by Friede (1954) in a study of stab wounds. Friede held the view that the two configurations represent consecutive steps in a dynamic process of glycogen concentration. We have never observed glycogen accumulation in nerve-cell perikarya in the irradiated brain. Electron-microscopic studies appear essential to ascertain whether some glycogen granules scattered in the neuropil are in nerve-cell processes. Interpretation as to the mechanism of glycogen accumulation in the irradiated brain is made difficult by the fact that the role of the glycogen in the normal central nervous system is still largely unknown. References p . 112-1 14

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Palladin (1956) was of the opinion that glycogen is a highly important constituent of the brain in that it is responsive to functional changes in the CNS. In investigations utilizing 14C,Prochorova (1963) reached the conclusion that glycogen participates actively in the biochemicalprocesses of nervous tissue. According to her observations, glycogen turnover in the cerebrum exceeded by 50 to 100 times that of lipids, and the rate of glucose incorporation into glycogen was significantly higher than in liver and muscle. With regard to the pathological accumulation of glycogen in the irradiated brain the following possible mechanisms can be considered : (1) liberation of carbohydrates in the injured tissue and its subsequent uptake by glial cells, (2) increase in permeability of the BBBM to glucose, (3) inhibition of the process of anaerobic glycolysis, and (4) inhibition of aerobic glycolysis. Regarding the jirst possibility, it can be assumed that disintegrating cells in the region of maximal radiation damage release their normal glycogen content and structural carbohydrates. Subsequently these substances would be concentrated in the astrocytes by the process of pinocytosis. Lending some plausibility to this hypothesis was the pallor of the neuropil in the ‘Bragg-peak band’ in sections stained by PAS method, signifying a reduction in the tissue-bound carbohydrates. This hypothesis is, however, not supported by our observation in the X-irradiated brain, that glycogen accumulates in significant quantities in the apparent absence of cellular breakdown. As to the second alternative, changes in permeability of the BBBM might be the basis of the glycogen accumulation. In tackling various phases of this problem, Geiger et al. (1954) reached the conclusion that glucose was taken up by the brain by means of an active transport mechanism which was regulated by some hepatogenic factor present in the circulating blood (Alweis and Magnes, 1958). More recent investigations suggest that the transport of sugars is controlled by permeability mechanisms inherent in the cytoplasmic membrane or by the presence of binding properties possessed by the cellular matrix. At any rate, permeability phenomena are closely associated with metabolic processes. Furthermore, Ungar and Psychoyos (1963) expressed the opinion that energy is required not for the transport of sugars but for their exclusion from the cell. In the case of the brain this regulation of the glucose uptake would be a function of the BBBM. That radiation may increase the penetration of glucose into cells has been shown by Wesemann et al. (1962). In effect, at 12 h after local X-irradiation of the small intestine with 1200 r, penetration of glucose into cells was increased as compared with initial values. At 44 h after exposure, on the other hand, penetration was decreased. One may speculate that radiationinduced metabolic disturbances will result in a reduction of the energy available for preventing the entry of glucose into astrocytes. The glucose surplus would then be transformed into glycogen, which is the normal carbohydrate reserve in animal tissue. The third possible basis of glycogen accumulation is inhibition of the process of anaerobic glycolysis, which appears to be intense in astroglia (Friede, 1962). Since glyceraldehyde dehydrogenase has been identified by Krebs (1956) as one of the pacemakers in the glycolytic process, if it was inactivated in vivo by radiation, glycogen accumulation might occur. Almost complete inhibition in vitro of phosphoglyceraldehyde dehydrogenase following exposure to 500 r X-radiation has been observed.

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If this enzyme was inactivated in vivo by radiation, glycogen accumulation might occur. That this is not the case was demonstrated by our analysis of lactic acid content in anaerobic brain. More lactic acid was formed in the irradiated than in the control animals, providing evidence of the existence of an active glycolytic process. This observation agrees with the result reported by Golubtsova et al. (1960). Lactic acid, formed upon incubation of irradiated cerebellar tissue of the chick, was increased as compared with the control values. The conclusion reached was that local irradiation of the cerebellum with 500 and 7000 r had deranged the oxidative pathway of glucose metabolism while the glycolytic pathway was preserved. The fourth alternative is that of inhibition of aerobic glycolysis associated with a lowered metabolic rate. This was considered by Friede (1954) and by Shimizu and Hamuro (1958) as the most likely mechanism by which glycogen accumulated in the region of stab wounds. The experimental evidence is, however, conflicting. Oxidative processes in the brain of experimental animals have been investigated in vivo by Snezhko (1960) by means of polarogaphic records of free oxygen content in tissue. After 1000 to 3000 r X-irradiation of the head of rabbits, the oxygen tension in the cerebral tissues increased drastically. Since no parallel was found with hemodynamic shifts or with pulmonary respiration frequency, it was concluded that the rise in the oxygen content of the brain was indicative of a reduction in oxidative processes. On the other hand, Hall et al. (1963) reported no significant changes in mouse brain respiration with doses as high as 19,000 r. Further, in vitro studies on slices of irradiated brain have revealed a decrease in both aerobic and anaerobic glycolysis (Egaiia, 1962). Further experiments will be needed to map radiation-induced metabolic disorders which have their expression in glycogen accumulation in astroglia. One may, however, speculate that the initial step in the radiation lesion is a disorganization of tissue ultrastructure resulting in disturbances in the ntracellular distribution of the metabolic intermediaries and coenzymes (Dose, 1962). Finally, a few points may be raised as to the significance of the glycogen studies for widening our knowledge of glial function. The assumption that the astroglial cell functions as a compartment behaving like the extracellular spaces of other tissues has been made by De Robertis and Gerschenfeld (1961). Moreover, Tschirgi (1958) wondered whether astrocytes may not contain ‘the metabolic machinery and the functional capacity to regulate solute transfer’. Further, Palay (1958) thought that an extracellular glucose flow in the space of 200-300 A between the brain cells was unlikely. Friede (1954) and Oksche (1961) have postulated that neuroglial cells have an essential function in carbohydrate metabolism of the CNS. The accumulation of glycogen, apparently exclusively in astrocytes, provides further support for this view. Moreover, the distribution of glycogen in the irradiated brain supports the assumption that some astroglial cells are metabolically linked with the neurons. In effect, in the irradiated cerebrum the heaviest accumulation of glycogen occurs in the hippocampus. Since this region is one of the most richly vascularized in the brain (Lierse, 1961), and a congruity between brain metabolism and capillary population has been shown by Campbell (1939), the nerve cells in the hippocampus probably have a very intense aerobic metabolism. Glycogen accumulation has also been observed in other References p . 112-114

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regions showing very intense oxidative activities, namely the peripheral region of the olfactory tuberculum (Figs. 17 and 18) and the molecular layer of the cerebellum (Figs. 20-23). The localization of glycogen in astroglial cells situated in the vicinity of neurons with a high metabolic rate as just described, further suggests that these astroglial cells are indeed trophocytes. Under normal conditions the glucose could be phosphorylated in the astroglia, as recent investigations by Friede (1962) suggest. The glycogen accumulation in astrocytes after irradiation might be the expression of a stoppage in a normally occurring flow of carbohydrates from blood vessels to nerve cells by way of astroglia. That such an interruption of the normal metabolic supply of the nerve cell may result in serious functional deficit of the brain is illustrated by the relations between glycogen accumulation in brain tissue and alterations in the EEG (Friede, 1956; Nair et al., 1963). SUMMARY

Ionizing radiation has proved to be a highly useful tool in experimental pathology for the study of astroglial reactions. Shortly after exposure of the brains of rats to X-ray or a-particles, disturbances in vascular permeability, glial mitosis and edema of the white matter have been observed. Moreover, glycogen accumulation has been demonstrated in the astroglial cells. The glycogen increase was observed in the absence of morphological changes in the brain cells and was maximal in the gray matter. The disturbances in the glycogen metabolism appear to be associated with a derangement of the aerobic metabolism of the astroglia-neuron metabolic unit. REFERENCES ALLWEIS,C., AND MAGNES, J., (1958); The uptake and oxidation of glucose by the perfused cat brain. J. Neurochem., 2, 326-336. ALTMAN, J., (1963); Autoradiographic investigation of cell proliferation in the brain of rats and cats. Anat. Rec., 145, 573-592. ARNOLD,A., AND BAILEY, P., (1954); Alterations in the glial cells following irradiation of the brain in primates. A.M.A. Arch. Pathol., 57, 383-391. A., BAILEY, P., AND LAUGHLIN, J. S., (1954); Effects of betatron radiation on the brain of ARNOLD, primates. Neurology, 4, 165-178. BROWNSON, R. H., SUTER,D. B., AND DILLER,D. D., (1963); Acute brain damage induced by low dosage X-irradiation. Neurology, 13, 18 1-191. W. S., (1952); The energy relations of mitotic activity. Biol. Rev., 27, 133-168. BULLOUGH, BULMER, D., (1 959); Dimedon as an aldehyde blocking reagent to facilitate the histochemical demonstration of glycogen. Stain Technol., 34, 95-98. CAMPBELL, A. C. P., (1939); Variations in vascularity and oxydase content in different regions of the brain of the cat. Arch. Neurol. Psychiat., 41, 223-242. L., (1913); uber das Glucogen im Gehirn. Nissl-Alzheimer Histof. Histopathol. Arb., 6, CASAMAJOR, 52-72. CLEMENTE, C. D., AND HOLST,E. A., (1954); Pathologic changes in neurons, neuroglia and bloodbrain barrier induced by X-irradiation of heads of monkeys. A.M.A. Arch. Neurol. Psychiat., 77, 66-79. CLEMENTE, C. D., AND RICHARDSON, R. E., JR., (1962); Some observations on radiation effects on the blood-brain barrier and cerebral blood vessels. Response of the Nervous System to Zonizing Radiation. T. J. Haley and R. S. Snider, Editors. New York, Academic Press (pp. 411-428). DE ESTABLE, R., AND ESTABLE-PUIG, J. F., (1963); Personal communication to the authors.

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DE ROBERTIS, E., AND GERSCHENFELD, H. M., (1961); Submicroscopic morphology and function of glial cells. International Review of Neurobiology. C . C . Pfeiffer and J. R. Smythies, Editors. New York, Academic Press (pp. 1-65). DOSE,K., (1962) ; Zum Mechanismus der Glykolysehemmung in Aszitestumorzellen durch Rontgenstrahlen. 11. Auslosung der Glykolysehemmung durch unspezifischeZellgifte. Struhlentherapie, 119, 419424. EGARA,E., (1962); Aerobic and anaerobic metabolic studies of CNS exposed to internal irradiation (S2P). Effects of Ionizing Radiation on the Nervous System. Vienna, IAEA (pp. 267-283). J. F., TOBIAS,C., AND HAYMAKER, W., (1963); Degeneration and regeneration of ESTABLE-PUIG, myelinated fibers in the cerebral and cerebellar cortex following damage from ionizing particle radiation. Acta neuropath., 4, 175-190. FRIEDE,R., (1954); Die Bedeutung der Glia fur den zentralen Kohlenhydratstoffwechsel. Zbl. a&. path. Anat., 92, 65-74. FRIEDE,R., (1956); Uber Beziehungen zwischen histochemischen Glycogenbefunden und der Hirnwellenfrequenz im EEG an einem Material von menslichen Biopsien. 2.ges. Neurol. Psychiat., 194, 213-237. FRIEDE,R., (1962); The cytochemistry of normal and reactive astrocytes. J. Neuropath. exp. Neurol., 21, 47 1478. GEIGER,A., MAGNES, J., TAYLOR, R. M., AND VERALLI, M., (1954); Effect of blood constituents on uptake of glucose and on metabolic rate of the brain in perfusion experiments. Amer. J. Physiol., 177, 138-149. E. V., AND SAFRONOVA, M. I., Cited by N. N. Livshits (1960). GOLUBTSOVA, A. V., MOISEYENKO, Physiological effects of nuclear radiation on the central nervous system. Advances in Biological and Medical Physics. C . Tobias and J. H. Lawrence, Editors. New York, Academic Press (p. 227). GONATAS, N. K., ZIMMERMAN, H. M., AND LEVINE,S., (1963); Ultrastructure of inflammation with edema in the rat brain. Amer. J. Pathol., 42, 455469. HAGER,H., HIRSCHBERGER, W., AND BREIT,A., (1962); Electron microscope observations on Xirradiated central nervous system in the Syrian hamster. Response of the Nervous System to Zonizing Radiation. T. J. Haley and R. S. Snider, Editors, New York, Academic Press (pp. 261-275). HALL,J. C., GOLDSTEIN, A. L., AND SONNEBLICK, B. P., (1963); Recovery of oxidative phosphorylation in rat liver mitochondria after whole body irradiation. J. biol. Chem., 238, 1137-1140. HAWK,P. B., OSER,B. L., AND SUMMERSON, W. H., (1954); Practical Physiological Chemistry. New York, McGraw-Hill (p. 625). JANSSEN, P., KLATZO, I., MIQUEL, J., BRUSTAD, T., BEHAR, A., LYMAN, J., HENRY, J., TOBIAS, C., AND HAYMAKER, W., (1961); Pathologic changes in the brain from exposure to a-particles from a 60-inch cyclotron. Response of the Nervous System to Ionizing Radiation. T. J. Haley and R. S. Snider, Editors. New York, Academic Press (pp. 383409). KERR,S. E., (1938); The carbohydrate metabolism of the brain. Isolation of glycogen. J. biol. Chem., 123, 443449. KLATZO,I., AND MIQUEL,J., (1960); Observations on pinocytosis in nervous tissue. J. Neuropath. exp. Neurol., 19, 475487. KLATZO,I., MIQUEL,J., AND OTENASEK, R., (1962); The application of fluorescein labeled serum proteins (FLSP) to the study of vascular permeability in the brain. Acta neuropath., 2, 144-160. KLATZO,I., MIQUEL,J., TOBIAS, C., AND HAYMAKER, W., (1961); Effects of a-particle radiation on the rat brain including vascular permeability and glycogen studies. J. Neuropath. exp. Neurol., 20, 459483. KREBS,H. A., (1956); The effects of extraneous agents on cell metabolism. CIBA Found. Symp. lonizing Radiations and Cell Metabolism. G. E. W. Wolstenholme and C. M. O’Connor, Editors. Boston, Little Brown (pp. 92-105). LIERSE,W., (1961); Die Kapillarabstande in verschiedenen Hirnregionen der Katze. Z. Zellforsch., 54, 199-206. MALIS,L. J., LOEVINGER, R., KRUGER, L., AND ROSE,J. E., (1957); Production of laminar lesions in the cerebral cortex by heavy ionizing particles. Science, 126, 302-303. W., (1963); Glycogen changes in XMIQUEL,J., KLATZO,I., MENZEL,D. B., AND HAYMAKER, irradiated brain. Acta neuropath., 2, 482490. NAIR,V., SUGANO, H., AND ROTH,L. J., (1963); Recovery of central nervous system functions impaired by lethal head X-irradiation. Proc. SOC.exp. Biol. ( N . Y . ) , 112, 273-277. OKSCHE, A., (1961); Der histochemisch nachweisbare Glycogenaufbau und Abbau in den Astro-

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cyten und Ependymzellen als Beispiel einer Funktionsabhangigen Stoffwechselaktivitat der Neuroglia. Z. Zellforsch., 54, 307-361. PALAY,S. L., (1958); An electron-microscopical study of neuroglia. Biology of Neuroglia. W. F. Windle, Editor. Springfield, Thomas (pp. 24-38). PALLADIN, A. V., (1956); La biochimie du cerveau. Proc. 3rd Internat. Congress Biochem., Brussels. C . Liebkcq, Editor. New York, Academic Press (pp. 375400). POPE,A., (1958); Implication of histochemical studies for metabolism of the neuroglia. Biology of Neuroglia. W. F. Windle, Editor. Springfield, Thomas (pp. 21 1-233). PROCHOROVA, M. I. Cited by R. V. Coxon, (1963); Some biosynthetic activities of central nervous tissue. Int. Rev. Neurobiol., 5, 313-314. RAIMONDI, A. J., EVANS, J. P., AND MULLAN, S., (1962); Studies of cerebral edema. 111. Alterations in the white matter: an electron-microscopic study using ferritin as a labeling compound. Acta neuropath.,2, 177-197. SCHOLZ,W., SCHOLTE, W., AND HIRSCHBERGER, W., (1962); Morphological effect of repeated low dosage and single high dosage application of X-irradiation to the central nervous system. Response of the Nervous System to Ionizing Radiation. T. J. Haley and R. S. Snider, Editors. New York, Academic Press (pp. 211-232). SHIMIZU,N., AND HAMURO, Y., (1958); Deposition of glycogen and changes in some enzymes in brain wounds. Nature, 181, 781-782. SMART,I., AND LEBLOND,C. P., (1961); Evidence for division and transformations of neuroglia cells in the mouse brain, as derived from radioautography after injection of thymidine-H3).J. comp. Neurol., 116,349-367. SNEZHKO, A. D.: Cited by N. N. Livshits, (1960); Physiological effects of nuclear radiations on the central nervous system. Advances in Biological and Medical Physics. C . A. Tobias and J. H. Lawrence, Editors. New York, Academic Press (pp. 225-226). TSCHIRGI, R. D., (1958); The blood-brain barrier. Biology of Neuroglia. W. F . Windle, Editor. Springfield, Thomas (pp. 130-138). UNGAR,G., AND PSYCHOYOS, S., (1963); Diffusion across rat diaphragm. 11. Movement of sugars; effect of insulin and other agents. Biochim. biophys. Actu, 66, 118-122. VANDYKE,D. C., JANSSEN, P., AND TOBIAS, C. A., (1962); Fluorescein as a sensitive, semiquantitative indicator of injury following a-particle irradiation of the brain. Response of the Nervous System to Ionizing Radiation. T. J. Haley and R. S. Snider, Editors. New York, Academic Press (pp. 369-382). I., Kuss, B., BREURER, H., AND PARCHWITZ,H. K., (1962); Wirkung einer RontgenWESEMANN, bestrahlung auf den Glukose- und Fettsauretransport im Diinndarm der Ratte. Strahlentherapie, 119,425-434.

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DE ESTABLE: One of the virtues of this Symposium, whose organizers deserve the most sincere praise, consists in its fostering new research. I feel that I am expressing a unanimous judgement when I say that all the reports we have listened to are excellent. This statement does not mean that a unanimous agreement should exist on the many problems here debated. It is necessary that the complex unity of the concepts obtained by integration should prevail upon the simple and schematic unity obtained by elimination. The prevailing characteristic of the physiology of the nervous system was interpreted as an exclusive function of the neurons and it was even claimed by a great physiologist (Nobel laureate) that the axon stands in relation to Neurology as the atom stands in relation to Chemistry and the electron to Physics! It is true that, starting with the ideas of Cajal, Achticarro and del Rio Hortega, it was thought that the neuroglia would play an important role in the process of myelination, in the isolated propagation of the excitatory status, in the trophism of the neuron and it was even speculated that it could secrete some hormone (at present, Bremer is of the opinion that perhaps the neuroglia could elaborate pseudocholinesterase). On the basis of outstanding experiments with microelectrodes, microdissection techniques, ultramicrocytochemistry and microscopic and submicroscopic control, much progress has been made in the knowledge of the meaning of the neuroglia in some aspects of the physiology of the nervous system and its physiopathology (such as the importance of the neuroglia in the blood-brain barrier and in brain oedema). We have here among us some of the people who are to be credited for this advancement. Without naming them, it is well known who they are. One has the tendency to conceive the functions of the neuroglia and the neuron as intimately related. With a certain amount of exaggeration, the importance of the first has been magnified, lessening the importance that the classic investigators and the majority of the modern ones ascribe to the second. It would be unproper for the scientific mind to choose sides and become appassionated for any other thing but truth. Four factors should be kept in mind in considering the nervous system life (functionality). These are: (I) the neuron and its synaptic spectrum; (2) the glia cells, their contaets between them and with: (a) the neurons, (b) the vessels, (capillaries, arteries,

* This discussion refers to the papers of Prof. E. D. P. De Robertis, Dr. M. Polak, Dr. R. L. Friede, Dr. A. Lasansky, Dr. I. Klatzo et al., Dr. J. Miquel and Dr. W. Haymaker. References p. I23

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veins); (3) the blood vessels; (4) the molecules flowing in the narrow intracellular spaces (internal media) and the molecules which are not ordered in structures or substructures, and cannot be detected either with the electron or the light microscope. The metabolism of the neuron does not depend solely on the glia activity; when the synaptic spectrum is destroyed without damaging the neuroglia, the neuron undergoes atrophy and dies. On the other hand every glia cell (even astrocytes) is not in contact by endfeet with capillaries. Millions of them are only related with arteries and veins. It could be inferred, therefore, that the neuroglia has the substance flowing in the intracellular space as a nutrient medium. The nerve cell contacts although rarely with the capillary

Fig. 1. Ventricular ependyma of a rat irradiated in the head with an X-ray load of 20,000 roentgen. Osmic perfusion fixation 24 h after irradiation. PAS and hematoxylin staining following dimedone. The arrow shows the glycogen evenly distributed in an ependymary cell. Fig. 2. Cerebellar cortex of rat fixed by osmic perfusion 24 h after receiving an X-ray load of 20,000 roentgen. PAS and hematoxylin staining after dimedone. Notice the positive PAS material in the perikaryon of the Von Bergmann cells (BC) and their processes.

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and it should be emphasized that the cytoarchitecture is modelled in accordance with the angioarchitecture and not with the glia architecture. Undoubtedly, each glial type and particularly the astrocytes, perform multiple functions. That the astrocytes participate in the circulatory regulation of the nervous system mitigating the effect of the beating vessels on neurons is inferred from experiments made with vasodilators and vasoconstrictor drugs and with experimental oedema provoked by substitution of blood with perfusion liquids. The existence of physiological sphincters along the vessels related to the glia cells corroborates this hypothesis. It is not possible in this short lapse of time, to compare the innervation of the vascular system of the neuroaxis with the one existing along the vessels of other organs. It is surprising that in the nervous system the vascular innervation is poorer than in other systems. The glia cells may compensate this deficiency. Some confusion exists among some functional types of glia with neurons, some neurons are interpreted as glia. This mistake can be opposed to that made by investigators of the last century who described glia cells as being neurons. The differential diagnosis cannot be discussed here but the point seems clear to most researchers. ESTABLE-PUIG: We would like to show here some light and electronic photomicrographs of our irradiation material. We have studied this (De Estable et al., 1965) with a modification of Palay’s osmic perfusion fixation method (Palay et al., 1961) and we have attempted to establish if this technique could be useful for parallel optic and electronic microscopy studies in order to do what could be called ultrastructural histochemistry, particularly with glycogen (De Estable et al., 1963). Previous investigations have demonstrated the appearance of glycogen following ionizing radiation (Klatzo et al., 1961; Miquel et al., 1963; Kruger and Maxwell, 1963). This material corresponds to X-ray irradiated rat brains perfused with an osmic solution. From

Fig. 3. Similar to the previous Fig., with greater enlargement. Notice that the glycogen is restricted to the glial cells and is not seen in the cytoplasm of the Purkinje cells. Many grains with pyknotic nuclei can be observed. BC = Von Bergmann cell. References p . 123

3

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those brains blocks for electronic-microscopylwere imbedded in Epon and the remaining material was imbedded in paraffin for light microscopy. Following removal of paraffin the sections were treated with dimedonemfor the practically; specific differential block of the nonglycogenic aldehydic groups, following Bulmer’s technique (Bulmer, 1959). With this technique the PAS staining shows the glycogen without the disturbing background staining which results from osmic fixation of the nervous tissue. The specificity of such method was demonstrated by amylatic digestion,

Fig. 4. Cerebellar cortex of rat fixed by osmic perfusion 24 h after administration of an X-ray load of 20,000 roentgen. Topographic photomicrography of the limiting zone between the myelinic axis of the cerebellar lamellae and the granular layer. Notice that the tissue appears very compact, without the usual artifacts of fixation. Numerous myelinic fibers (FM) sectioned at different angles and glial fibers can be seen. Epon embedding. Lead staining following Karnovky method. x 17,000.

Our observations have indicated that this is an excellent method to preserve and demonstrate different cellular elements and particularly glycogen. The latter appears evenly distributed in the cytoplasm without the heavy artifactual precipitates that follow picroalcoholic fixation. The first figure corresponds to the periventricular cerebral tissue. Blood vessels are

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dilated and empty on account of the perfusion. Some ependymary cells contain glycogen evenly distributed in the cytoplasm. Fig. 2 shows the cerebellar cortex of a rat which received 20,000 roentgens 24 h before being dispatched. Hardening and staining was similar to that of the previous

Fig. 5. Cerebellar cortex of rat with osmic perfusion fixation 24 h after an X-ray load of 20,000 roentgen in the head. Electronic photomicrography of the granular layer in which morphologically preserved cells can be seen. The arrows show the modified grains. Epon embedding. Lead staining following Karnovky method. x 13,000.

case. We believe that this suggests that the PAS-positive staining of the molecular layer corresponds with the outline of the Von Bergmann cells. Glycogen is mainly located in these cells but not in the Purkinje cells. A small amount of glycogen can be found in some astrocytes of the granular layer close to the Purkinje cells. Many pyknotic cells can be seen in the granular layer. This is a well-known fact to those studying the radiobiology of the nervous system, since granular cells are the neuronal elements most sensitive to ionizing radiation injury. We do not know yet References p . 123

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which is the relation of these neurons, which are known to have large branches ramified in the molecular layer, with the glycogen accumulation in the glial cells. Fig. 3 shows with greater enlargement one area of the previous field. Pyknotic granular cells are better seen and the glycogen accumulation restricted to the glial cells can also be clearly observed.

Fig. 6. Similar to the previous figure showing a pyknotic granule and an astrocyticprocess filled with glycogen. Osmic perfusion fixation. Epon embedding. Lead staining following Karnovky method. x 60,000. G1= glycogen; PA = astrocytic process.

Fig. 4 is a topographic electronic photomicrography showing the general appearance of the limiting zone between the myelinic axis of the cerebellar lamellae and the granular layer as seen when the osmic perfusion fixation method is employed, the tissue appearing quite compact. The endothelium of the empty vessels dilated by the perfusion can be observed in the lower right angle of the figure. Many myelinic fibers sectioned at different angles can be seen exhibiting neurofilaments very clearly. Glial fibers are seen across the field in the upper part of the Figure.

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Fig. 5 is an electronic photomicrography of the granular layer where the morphology of some normal grains can be observed with the large nucleus and its nucleole and the scarce cytoplasm withmitochondria and RNA particles, which are the functional equivalent of Nissl substance in other neurons. Some granular cells are abnormal

Fig. 7. Rat cerebellar cortex with osmic perfusion fixation 24 h after receiving an X-ray load of 20,000 roentgen. Electronic photomicrography of an area of the molecular layer showing a Von Bergmann cell process filled with glycogen granules. Epon embedding. Lead staining following Karnovky method. x 60,000. PA = astrocytic process.

showing a reduction of their diameter as a consequence of the retraction of the cytoplasm and particularly of a retraction and densification of the nucleus, where lacunar spaces with granular content can be seen. With greater enlargement Fig. 6 shows that the glial processes adjacent to the pyknotic cells are completely filled with grains stained with Karnovky’s technique. These grains are interpreted as glycogen inclusions on account of their size, the lead affinity and the correlation with observations performed with light microscopy. References p. 123

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The Von Bergmann cell processes in the molecular layer are also filled with glycogen granules which are also found in the astrocytic processes of the neuropil but not in the nervous fibers, nor in the axonal ends of those dendrites making synaptic contact there (Fig. 7). The astrocytic nature of the space where the glycogen granules are found is certified by the fact that they are found coexisting with glial fibers (Fig. 8).

Fig. 8. Similar to the previous Fig. showing the coexistence of glycogen grains and glial fibers (FG) on the left upper angle. Lead staining. Epon embedding. GI = glycogen; PA = astrocytic process.

At present we are performing a serial study with the purpose of making certain the glycogen formation mechanism and its possibilities of relation with different cellular organs.

Luco: According to the law of Langley and Anderson, it seemed impossible to re-innervate with cholinergic fibers the smooth muscles that are normally innervated by adreneIgic fibers. In a paper reported by Vera, Vial and Luco, functional inner-

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vation of the nictitating membrane of cats was obtained by hypoglossal nerve. This heterogeneous innervation was demonstrated by the contraction of the smooth muscles during the stimulation of the hypoglossal nerve and by the normalization of the sensitivity to adrenaline, noradrenaline and acetylcholine. The myelinated fibers of the hypoglossal nerve grew through neuron tubes of non-myelinated fibers of the postganglionic sympathetic nerve. The observation of Simpson and Young was confirmed i.e. the growing fibers appear at the periphery as myelinated fibers. But the terminal portion of the fibers in our experiments was different from those observed in the hypoglossal nerve that innervate the tongue muscles. The terminals in our experimental situation were very much like the normal terminals of the sympathetic system in the nictitating membrane. It was suggested that the Schwann cells are not able to wrap the nerve terminals because a peripheral factor from the smooth muscles is blocking this reaction. Furthermore, it can be thought that in order to activate the fibers of the smooth muscles the presence is necessary of a long nude terminal to permit the release of a relatively large amount of chemical mediator, that may reach all the muscle fibers. These histological observations under the optical microscope suggested a problem on the relation between the axon and the reaction of the Schwann cells in wrapping theaxon. In other words, is this reaction induced by the axon or are there specific Schwann cells that result in myelinated or non-myelinated fibers? The electron microscopic observations recently made by Dr. Vial in the preparation of hypoglossal nerve-nictitating membrane confirmed the previous observations and added new data. At the periphery, the myelin is formed by ‘tongue’-like lamellae at the outer layers of the myelin, a situation not seen at a normal myelinated motor nerve. REFERENCES BULMER, D., (1959); Dimedone as an aldehyde blocking reagent to facilitate the histochemical demonstration of Glycogen. Stain Technol., 34, 95-98. DE ESTABLE, R, F., ESTABLE-PUIG, J. F., AND HAYMAKER, W., (1965); Electron microscopy of cerebellar cortex following ionizing radiation injury. Znt. J. Neurol. (Montevideo), in press. DE ESTABLE, R. F., ESTABLE-PUIG, J. F., AND MIQUEL,J., (1963); Fixation and identification of glycogen in the nervous system for light and electron microscopy studies. 39th Meeting of the American Association of Neuropathologists. KLATZO,I., MIQUEL,J., TOBIAS, C., AND HAYMAKER, W., (1961); Effects of alpha particle radiation on the rat brain including vascular permeability and glucogen studies. J. Neuropath. exp. Neurol., 20,459483.

KRUGER,L., AND MAXWELL, D., (1963); Electron microscopy of radiation induced laminar lesions in the cerebral cortex of the rat. The Second International Symposium on the Response of the Nervous System fo Ionizing Radiation. University of California, Los Angeles, August 29-31. MIQUEL,J., KLATZO, I., MENZEL, D. B., AND HAYMAKER, W., (1963); Glycogen changes in X-irradiated brain. Acta neuropath. (Bed.), 2, 482490. PALAY,S. L.; MCGEERUSSELL, S. M., GORDON, S., AND GRILLO,M. A., (1961); Fixation of neural tissue for electron microscopy by perfusion with solutions of osmium tetroxide. J. Cell Biol., 12, 385.

124

The Extracellular Space of the Brain H. DAVSON

AND

M. B R A D B U R Y

Medical Research Council, University College London, London (Great Britain)

In discussing the problem of the extracellular space of central nervous tissue we enter a field in which considerable uncertainty as to the nature of this entity, and of its physical magnitude, prevails. In the present paper, the anatomical concept, as revealed by modern electron-microscopicalstudies, will be only briefly touched on, and it will be sufficient to refer the reader to papers of Wyckoff and Young (1956); Luse (1956); Schultz et al., (1957); Fernhndez-Morhn (1957); Farquhar and Hartmannl(l957); Horstmann and Meves (1959) among others. The consensus of opinion, based on these experimental studies, is that the traditional concept of neurones with lightmicroscopically resolvable spaces between them must be abandoned. Instead, we must envisage a tightly packed mass of neurones and glia with spaces no greater than some 200 A wide between individual cells. An estimate of the extracellular space on this basis leads to some 5 % of the volume of the tissue, small by comparison with, say, the skeletal muscles of rat with values ranging between 13 and 20 %, or with that of heart muscle with a value of some 25 % (Fisher and Young, 1962). In the present paper we shall discuss the magnitude of a space in the tissue that conventionally would have been called, by virtue of the methods employed for its measurement, the extracellular space; it may, because of the differing physiology of the nervous tissue from that of other parts of the body, not correspond to an anatomically measurable entity such as the summated spaces between cells, but until this has been unequivocally proved, it is proposed to call it the extracellular space. To the physiologist and pathologist the most pressing problem is, besides the magnitude of the space, the nature of the fluid filling it and the possibilities of control over both its composition and volume; this involves us, too, in the question as to whether there is a consistent circulation of the fluid comparable in nature, if not in magnitude, with that of the cerebrospinal fluid. Magnitude of the extracellular space. With most tissues it is not too difficult to measure the extracellular space ; the animal may be injected with an ‘extracellular tag’, i.e. with a molecule or ion that is thought not to penetrate into cells. If a steady concentration of this is maintained in the blood for some minutes, and the tissue and blood are sampled simultaneously, it is a simple matter to compute the extracellular space :

% space =

Concn. in tissue x 100 Concn. in plasma dialysate

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Thus the ‘inulin-space’ is considered to represent the extracellular space of muscle reasonably accurately, although, in view of the relatively slow rate of diffusion of this molecule through a tissue containing large amounts of collagenous connective tissue, the estimate may well be a low one, i.e. sufficient time may not have been allowed for complete equilibration between plasma and the whole of the extracellular space. Other spaces that have been recorded are those employing sucrose and sulphate as the extracellular tags, sulphate being valuable because it may be used labelled with 35s. In the study of the brain and spinal cord, however, such a method is inapplicable because of the phenomenon of the blood-brain barrier, i.e. the great slowness with which an extracellular tag such as sucrose may escape from the vascular system. Some idea of this slowness is given by Fig. 1, which compares

Durotion of infusion (min)

Fig. 1. Penetration of sucrose ( O ) , and lz1I (0) into brain and diaphragm muscle of rabbits infused with the test substance for 6 or 120 min. The ordinate refers to the volume of tissue water (per 100 g tissue) that contains the same concentration of the test substance as in a plasma dialysate. Each point represents the average of determinations in 4-10 animals; s.e. shown as vertical lines. (From Davson and Spaziani, 1959).

the sucrose-, and 1311-spaces of rabbit brain with those of skeletal muscle in the same animal. These results suggest, either that there is virtually no space for these tags to enter, or that the barrier to their passage out of the blood stream is so high that it would take very many hours before equilibrium with the extracellular space were achieved. To carry out an experiment of this duration is not impracticable, however, since we could nephrectomize an animal and leave it for some 24 h after intravenous injection of a tag such as labelled sulphate. However, the resulting estimate of extracellular space will not necessarily be a correct one, because of the ‘sink-action’ of the cerebrospinal fluid (Davson, 1963). Thus, the extracellular tag is found to penetrate into the cerebrospinal fluid very slowly indeed - there is a blood-cerebrospinal fluid barrier to this molecule comparable with the blood-brain barrier to it. Although the molecule has penetrated into the cerebrospinal fluid with great difficulty, the passage outwards through the arachnoid villi is unrestricted, References p . 1331134

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consequently, on simple kinetic grounds it is easy to show that the steady-state concentration of the tag in the cerebrospinal fluid will be only a small fraction of that in the plasma. An example is given by a protein such as serum albumin; because the plasma concentration is virtually constant we may regard the concentration in the cerebrospinal fluid as an equilibrium, or steady-state, value; and, because of the high barrier to passage of protein from the choroid plexuses into the ventricles, and the relatively unrestricted passage out through the arachnoid villi (Welch and Friedman, 1960), the concentration in the cerebrospinal fluid is only some 1.3% of that in the plasma. Consequently, when our extracellular tag, such as inulin or sulphate, has been in the blood long enough to establish a steady-state concentration in the cerebrospinal fluid, and in the extracellular space of the surrounding nervous tissue, the concentration in the extracellular space will be less than that in the plasma because of the steady diffusion of the tag into the adjacent cerebrospinal fluid, a steady diffusion that is possible because of the absence of significant restraint on the passage of even large molecules like inulin across the ependymal lining of the ventricles and, presumably, the mesothelial lining of the subarachnoid spaces. Schematically we may represent this ‘sink-action’of the cerebrospinal fluid by Fig. 2, where it is assumed

Fig. 2. Illustrating the manner in which the cerebrospinal fluid may impose a low concentration on an extracellular tag.

that the concentration of tag in the plasma is 100 and that in the cerebrospinal fluid is 2 ; the concentration in the extracellular fluid achieves a steady-state level between these two values, its magnitude depending on the blood-brain barrier to this molecule, the diffusion coefficient of the latter through the nervous tissue, the rate of flow of cerebrospinal fluid, and so on. In the figure, the value has been put at 30, but it could well be less; if we accept this value, then the extracellular space computed will be only a third of the actual. This may be illustrated by the work of Barlow et al. (1961) who injected 35S04 into ureter-ligated animals and after 6 h analysed brain and blood; they computed a space of only 2-4 %. Since the concentration of 35S04 in the cerebrospinal fluid was only 9 % of that in the plasma, it is clear that there was a very considerable ‘sink-action’, so that the true extracellular space lies between 2-4 % at one extreme and 18-36 % at the other, when it is assumed that the extracellular space and cerebrospinal fluid have the same concentrations. Again, we have injected creatinine into nephrectomized rabbits (Bradbury and

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Davson, 1964) and determined its concentration in brain water and cerebrospinal fluid after 24 h. The concentrations relative to plasma are 33 % for brain water and 17 % for cerebrospinal fluid. Here the ‘sink-action’of the cerebrospinal fluid is affecting the distribution of a substance which normally attains equality of concentration in cell water and plasma water in the steady state (Schloerb, 1960). Chloride and sodium spaces. Classical measurements of extracellular space were based on the concentration of certain ‘endogenous tags’ in the tissue, notably chloride and sodium, i.e. the extracellular space was equated with the chloride space or sodium space, the assumption being made that these ions were extracellular. If these ions were, indeed, confined to the extracellular compartment of a tissue, their ‘spaces’ would be a fairly accurate measure of this compartment because there is reason to believe that the concentrations in cerebrospinal fluid and extracellular fluid are not greatly different*. It is now recognized that even in muscle, where the chloride space is not greatly different from the sucrose or sulphate space, some of this ion is intracellular, and the same applies to the sodium ion. Thus, estimates based on chloride or sodium space are too high, but in the case of skeletal muscle the discrepancy is not serious. In brain the chloride space is 31.4 % whilst the sodium space is even higher, namely 34.6% (Manery and Hastings, 1939; Davson, 1955) so that it seems very likely that a large proportion of these ions is intracellular unless, of course, the extracellular space is very high. As we shall see, however, although measurements of the space with extracellular tags do indicate a space larger than that conceded by the electron microscopists, the computed spaces are nevertheless considerably smaller than the chloride space. Circumventing the barrier. From the foregoing it becomes clear that the application of conventional techniques for estimating the magnitude of the extracellular space of brain or cord must necessarily give misleading results : (a) because of the presence of the blood-brain and bloodxerebrospinal fluid barriers, and (b) because such endogenous tags as Na+ and C1- are to a considerable extent intracellular. In applying our extracellular tag to the tissue we must, in consequence, circumvent the blood-brain barrier, and this may be carried out by studies in vitro on thin pieces of cerebral hemisphere, or by studies in vivo in which the tag is presented to the nervous tissue by way of the cerebrospinal fluid instead of by way of the blood. For the studies in vitro the use of very thin slices must be avoided since very large numbers of cells will be cut; if, on the other hand, the pieces are thick, the effects of anoxia become more significant. The cells of an anoxic tissue, in general, tend to swell at the expense of the surrounding extracellular fluid, so that an estimate based on a tissue that is becoming anoxic will probably be too low. In Fig. 3 we see the increase in the sucrose and PAH spaces with time of incubation of pieces of rabbit cerebral hemisphere; these were some 2 mm thick and obtained

* Actually the concentrations of these ions are higher than would be expected of a dialysate of plasma, so that the estimate of extracellularspace on the basis of these endogenous tags will depend on whether we assume that the extracellular fluid is a plasma dialysate or has the same composition as that of the cerebrospinalfluid, or has a composition between these limits. References p . 1331134

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h

m

w 80 __

' 0

20

40 60 Incubation time (min) ~~

Fig. 3. Penetration of PAH and sucrose in pieces of cerebral hemisphere incubated at 40";ordinate as in Fig. 1. Each point represents the average of 4-9 incubations; s.e. shown as vertical lines.

Incubation time (min)

Fig. 4. Penetration of 24Naand l3II, and changes in chloride space, in pieces of cerebral hemisphere incubated at 40".Ordinate as in Fig. 1. Each point represents the average of 6 1 0 incubations; s.e. shown as vertical lines. (From Davson and Spaziani, 1959).

with the minimum of cutting. The sucrose space levelled off at about 15 % whilst the PAH space rose continuously, indicating that there was some intracellular penetration. The penetration of into the slices is of special interest; when presented to the tissue by way of the blood, i.e. by intravenous infusion, we have seen (Fig. 1) that there is remarkably little penetration into the brain, as with PAH and sucrose; by contrast, the penetration into the isolated tissue is rapid (Fig. 4) and kinetic analysis indicates that there is no barrier to diffusion. The tag continues to penetrate the tissue and approaches the chloride space. This study effectually answers the claim that the concept of the blood-brain barrier is invalid. Thus, it has been argued that the failure of such tags as sucrose, iodide, PAH, etc. to appear rapidly in the nervous tissue is not because of a barrier to escape from the vascular system, i.e. not to the presence of a blood-brain barrier, but because of the absence of a space for them to enter. If there were indeed 'nowhere to go' for these tags, then it should not matter whether

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the tissue were presented with them by way of the blood or by direct contact, as in the studies in vitro. Thus, the concept of the blood-brain barrier is validated by these studies, as indeed by a number of others, notably of Van Harreveld et al. (1961), Bakay (1960); Davson and Pollay (1963) and Bradbury and Davson (1964). The alternative method of presentation of the extracellular tag, namely by way of the cerebrospinal fluid, profits by the circumstance that the barrier to escape from this fluid into the adjacent tissue - the cerebrospinal fluid-brain barrier - is not high, in fact recent studies in Rall’s laboratory (Rall et al., 1962) suggest that the passage of even large molecules like inulin is unrestrained. Davson et al. (1961) replaced the cerebrospinal fluid of the rabbit repeatedly with a Ringer’s solution containing sucrose and PAH as extracellular tags; by this technique the spinal cord (but not the cerebral hemispheres) was exposed to the tag at a known concentration ; analysis of the cord after 2 to 3 h of this exposure gave values for the space in the region of 12%. By perfusing the ventricular space continuously over long periods of time, and cutting out sections of brain at known distances from the ventricle, Rall et al. (1962) studied the diffusion of such extracellular tags as inulin into the tissue and, from the steady-state values, computed an extracellular space in the region of 7-14%. It is difficult to escape the conclusion from these measurements that at least 10% of the brain tissue is readily accessible by way of the extravascular pathway to substances such as sucrose and inulin, substances that in the past have been regarded as being exclusively extracellular. These studies, moreover, by demonstrating the existence of ‘spaces’ of at least 10 % for these substances, unequivocally demonstrate at the same time that there is, indeed, a barrier to their escape from the blood. Control of extracellular volume. An extracellular fluid separated from the blood capillaries by a highly selective membrane such as that denoted by the concept of the blood-brain barrier, may well be different from the simple filtrate from plasma that we assume to constitute the extracellular fluid of other tissues, such as skeletal muscle. Thus, such molecules as sucrose and inulin do not only cross this barrier with great difficulty, but also the far more mobile ions, Na+, C1- and CNS- cross the barrier with difficulty; their low permeability would thus constitute a severe viscous drag on the formation of a filtrate from plasma. This would apply not only to Na+ and C1but also to such non-electrolytes as urea and creatinine which also cross the bloodbrain barrier with difficulty (Davson, 1955; Bradbury and Coxon, 1962; Reed and Woodbury, 1962; Kleeman et al., 1962). Without entering into any calculations it becomes quite clear that the small hydrostatic pressure available for filtration would be quite inadequate to permit filtration on a scale comparable with that which occurs in skeletal muscle. We may conclude, therefore, that a significant filtration and reabsorption of fluid in the tissue is unlikely, and this may account for the absence of a lymphatic system in this tissue; the absence of a significant normal flow, together with the barrier that prevents any significant escape of protein, making an overflow mechanism unnecessary. We have reached this conclusion, however, solely on the assumption that the fluid filling the extracellular compartment of brain is formed by the mechanical process of filtration across the blood-brain barrier, the available References p . 1331134

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energy for this process being the hydrostatic difference of pressure. If we admit the possibility that the fluid is formed with the aid of chemical energy, i.e. if it is secreted by certain cells, then this limit to the rate of formation is lifted, and we may envisage the elaboration of a fluid in a quantity that might well contribute to the total turnover of cerebrospinal fluid - supposing that it drains away into this system -and a fluid that also has a specific chemical constitution different from that of a simple filtrate as determined by the Gibbs-Donnan equilibrium. This concept of a secreted extracellular fluid is not new and was actually proposed as a source of cerebrospinal fluid by Hassin (1948) and Tschirgi et al. (1954), although the arguments proposed by these authors are not necessarily acceptable. Let us assume, therefore, that the glial cells, covering with their footprocesses the capillaries of the brain and spinal cord, have secretory powers, so that they may abstract from the capillaries the water, ions and non-electrolytes that constitute a secreted extracellular fluid. Because there is a space between the endothelial lining of the capillary and the glial coating, we must now define two extracellular spaces of brain. Space I, the space between capillary endothelial and glial cells, and Space 11,the space between the cells of the tissue, i.e. between neurones and other glial cells (Fig. 5). Space I might well be

-

{

-

7 BLOOD

Blood-brain barrier

s1

C.S.F.

Fig. 5. Two hypothetical extracellularspaces. S1 is assumed to be filled by a plasma filtrate whilst SZ is assumed to be filled by a secretion elaboratedfrom this filtrate by the glial cells lining the capillary. S2 is, of course, grossly exaggerated.

occupied by a simple capillary filtrate, if we assumed that the site of the blood-brain barrier was the layer of glial processes covering the capillary. Space I1 would be a secretion, with a composition that was specifically different from that of the plasma filtrate, and might well be similar to that of the cerebrospinal fluid. In fact, it is the evidence for a similarity of the two fluids that is the compelling reason to adopt such a view of the nature of the extracellular fluid in space I1 as that we are presenting now. In an earlier discussion (Davson, 1958) essentially devoted to this aspect of the cerebrospinal fluid, namely its relationship with the extracellular fluid of the adjacent nervous tissue, it was pointed out that the concentrations of such ions as Na+, C1-, K+, and Mg++ were considerably different from those in the plasma, or a filtrate of plasma; and this necessarily raised the question as to how these differences of concentration were maintained throughout the sojourn of the fluid in the ventricles and subarachnoid spaces, since experiments had shown that exchanges between the

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cerebrospinal fluid and the adjacent nervous tissue were relatively unimpeded. It was argued that if, indeed, the fluid as secreted were different in composition from the extracellular fluid of the adjacent tissues, then we should expect the fluid to approach the composition of the latter on its progress through the ventricles and subarachnoid spaces. Thus there should be large differences between ventricular and lumbar fluid in man, and in small animals like the rabbit, where analysis of lumbar fluid is difficult, we might expect to find differences in composition of successive small samples taken from the cisterna magna as fluid was drained from parts that were more and more remote from the ventricles. In fact, inhomogeneities of this kind could not be detected so far as glucose and chloride were concerned (Davson, 1958), and later the attempt to find differences in urea concentration in successive samples was attended with the same failure (Kleeman et al., 1962). If, on the other hand, the extracellular fluid in space I1 were secreted with the same composition as that of the cerebrospinal fluid, then the difficulty would disappear. Some recent experiments with K+, carried out in our laboratory, lend support to this view of the extracellular fluid as a secretion. The concentration of this ion in cerebrospinal fluid is approximately one half that in the plasma; when the concentration of K+ in the plasma is raised artificially, that in the cerebrospinalfluidremainsunchanged(Bekaert andDemeester71951a,b,1952;Bradbury and Davson, unpublished) so that it appears that the cerebrospinal fluid is secreted at a concentration that is virtually independent of the plasma concentration. When the concentration in the blood is raised it is also found that the amount of K+ in the nervous tissue is virtually unchanged, although if the extracellular fluid concentration had risen we should have expected to find increases not only in this but also in the amount in the intracellular compartment in accordance with the Gibbs-Donnan equilibrium (Boyle and Conway, 1941). Suppose, now, that we perfuse the ventriculocisternal system with an artificial cerebrospinal fluid, the perfusate entering through a lateral ventricle and being drained away at the cisterna magna. It is found that, if the concentration of K+ in the artificial fluid is made higher than normal, it falls rapidly during the perfusion because of escape into the surrounding tissue, i.e. into

6'0c

.-. 5.0

- O . '00

20

40

60

80

100

120

Time h i n )

Fig. 6. Effect of ventriculo-cisternal perfusion with Ringer's solution containing different concentrations of K+. Ordinate: Concentration of K+ in outflowing fluid. Abscissa: Time from beginning of perfusion. The different concentrations of Kf in the perfusion fluids (CI,) are indicated at the right of each curve. References p . 1331134

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the extracellular fluid (Fig. 6). If, on the other hand, the concentration in the artificial fluid is made less than that in normal cerebrospinal fluid, the concentration rises rapidly, owing this time to diffusioninto the fluid from the surrounding tissue (Fig. 6). Finally, if the concentration in the artificial fluid is made equal to that in the normal cerebrospinal fluid, then the concentration remains virtually unchanged (Fig. 6), These experiments make it very probable indeed that the extracellular fluid in space 11 has a composition, at any rate so far as K+ is concerned, similar to that of the cerebrospinal fluid. Sinceit is only possible to account for the concentration of this ion in cerebrospinal fluid on the assumption that active transport, i.e. secretory, mechanisms are involved in the elaboration of the fluid, so we must concludethat similar mechanisms are operativein controlling the composition of the extracellular fluid of the nervous tissue. The picture that has emerged, then, is that of two processes, namely secretion of cerebrospinal fluid by the choroid plexuses and of an extracellular fluid by the glial processes surrounding the capillaries. By adjusting the compositions of these fluids so that they are not greatly dissimilar in respect to important constituents such as Na+, C1- and K+, the organism has effected an economy of effort. Thus, if the cerebrospinal fluid were secreted with a high concentration of K+, diffusion from here into the adjacent brain would undo the effect of the secretory process in this tissue, since the latter is attempting to maintain a low concentration of K+ surrounding the neurons. It may be argued, however, that only the extracellular fluid is secreted with a specifically different composition from that of a dialysate of plasma, and that this fluid, by virtue of its greater bulk, imposes its composition on the cerebrospinal fluid as soon as it is formed*. The complete solution to this problem must await further experiments; it must be appreciated that the cerebrospinal fluid is, indeed, a secretion, formed at least in part if not exclusively by the choroid plexuses, so that the postulate of a secreted cerebrospinal fluid of specific composition is consistent with numerous recent studies. Consequently, the main problem is to determine to what extent there is interaction between the two secreted fluids, rather than to consider the one as a passive filtrate and the other as a specific secretion. The finding of Rougemont et al. (1960) that freshly secreted cerebrospinal fluid, collected under oil from the exposed choroid plexus, had a higher concentration of K+ than that taken from the cisterna magna suggests that the fluids are not perfectly matched, and that the brain tends to impose its concentration on that of the cerebrospinal fluid; similarly, the C1concentration in the freshly secreted fluid was less than that in the cisterna; since the concentration of C1- in cerebrospinal fluid is higher than that in a plasma filtrate, it appears once again that the freshly secreted fluid is nearer to a plasma filtrate than that which has been in the system some time, i.e. that the nervous tissue once again has tended to impose its own concentration on that of the cerebrospinal fluid. In each of these two instances the concentration imposed has been that of a specific * The bulk of the extracellular fluid need not be greater than that of the cerebrospinal fluid if both are, say, 10% of the brain volume. From the point of view of K+, however, the intracellular K+ is in equilibrium with the extracellular K+ so that changes in the extracellular concentration will be buffered by relatively rapid transmembrane adjustments along the lines discussed by Boyle and Conway (1941). The same consideration might well apply to urea since the estimates of Kleeman et al. (1962) indicatethat its intracellularconcentration is probably equal to that in the extracellularfluid.

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secretion, since the cerebrospinal fluid has been made less like a plasma filtrate than it was when primarily secreted. In the case of urea, it would seem from the recent study of Stubbs and Bradbury (1963) that the change is in the reverse sense. Thus the concentration of urea in cerebrospinal fluid is considerably less than that in plasma or a plasma dialysate; when ventricular and lumbar fluids are compared in man, it is found that the concentration has risen on passing from ventricle to lumbar region; in this case, then, the nervous tissue is imposing a concentration on the cerebrospinal fluid that is closer to that of the plasma. Hence, it appears that the extracellular fluid of the nervous tissue, which is presumably imposing this concentration on the cerebrospinal fluid, has the higher concentration of urea, and is closer therefore to a dialysate of plasma. Recent studies of Bradbury and Davson (1964) suggest that the urea concentrations in both compartments are determined by simple physical forces, the low concentration in the cerebrospinal fluid being a consequence of molecular sieving in the choroid plexuses, resulting from the flow of fluid and restricted diffusion of urea into this fluid. The higher concentration of urea in the extracellular fluid of the nervous tissue, revealed by direct analysis of the tissue (Kleeman et al., 1962) and suggested by this ventriculo-lumbar difference in concentrations, indicates that the extracellular fluid does not flow in a manner comparable to the cerebrospinal fluid. Since the total turnover of cerebrospinal fluid is of comparable magnitude with the rate of secretion in the ventricles, this view of a relatively stagnant extracellular fluid is necessary, since it is difficult to see where else the secreted extracellular fluid would escape if not into the subarachnoid spaces by way of spaces surrounding the blood vessels, i.e. by the somewhat discredited Virchow-Robin spaces. SUMMARY

Some 10-15 % of the volume of the brain behaves as though it were extracellular, in the sense that under appropriate conditions an extracellular ‘tag’, such as sucrose, will come into equilibrium with it. The appropriate condition is that the sucrose should be presented by a non-vascular route, so that the blood-brain barrier has been circumvented. Quantitative aspects of this space have been discussed and the problem as to why it is inaccessible to extracellular tags when given by the vascular route has been considered. It is suggested that there are two extracellular spaces: a very small one between the blood vessels and their glial covering, and a larger one representing the spaces between the tissue cells generally; it is this latter space that is inaccessible to intravascularly administered extracellular tags, by virtue of the low permeability of the glial coating of the capillaries. It seems very likely from recent studies of the exchange of ions between cerebrospinal fluid and brain that the composition of this latter space is controlled by secretory processes. REFERENCES BAKAY,L., (1960); Studies in sodium exchange. Neurology (Minneap.), 10, 564-571. BARLOW, C. F., DOMEK, N. S., GOLDBERG, M. A., AND ROTH,L. J., (1961); Extracellular brain space measured by 35S sulphate. Arch. Neurol. (Chicago), 5 , 102-110.

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BEKAERT, J., AND DEMEESTER, G., (1951a); The influence of glucose and insulin upon the potassium concentration of serum and cerebrospinal fluid. Arch. int. Physiol., 59, 262-264. BEKAERT, J., AND DEMEESTER, G., (1951b); The influence of the infusion of potassium chloride on the cerebrospinal fluid concentration of potassium. Arch. int. Physiol., 59, 393-394. BEKAERT, J., AND DEMEESTER, G., (1952); The influence of dinitro-a-naphthol upon the potassium concentration of serum and cerebrospinal fluid. Arch. int. Physiol., 60, 172-175. BOYLE, P. J., AND CONWAY, E. J., (1941); Potassium accumulation in muscle and associated changes. J. Physiol., 100, 1-63.

BRADBURY, M. W. B., AND COXON, R. V., (1962); The penetration of urea into the central nervous system at high blood levels. J. Physiol., 164, 423435. BRADBURY, M., AND DAVSON,H., (1964); The transport of urea, creatinine, and certain monosaccharides between blood and fluid perfusing the cerebral ventricular system of rabbits. J. Physiol., 170, 195-21 1.

DAVSON, H., (1955); A comparativestudy of the aqueous humour and cerebrospinalfluid in therabbit. J.Physio1. (Lond.), 129, 111-113. DAVSON, H., (1958); Some aspects of the relationship between the cerebrospinal fluid and the central nervous system. Ciba Symp. The Cerebrospinal Fluid. London, Churchill (pp. 189-208). DAVSON, H., (1963); The cerebrospinal fluid. Ergebn. Physiol., 52, 21-73. DAVSON, H., KLEEMAN, C. R., AND LEVIN,E., (1961); Blood-brain barrier and extracellular space. J. Physiol. (Lond.), 159, 67-68. DAVSON, H., AND POLLAY, M., (1963); The turnover of 24Nain the cerebrospinal fluid and its bearing on the blood-brain barrier. J. Physiol., 167,247-255. DAVSON, H., AND SPAZIANI, E., (1959); The blood-brain barrier and the extracellular space of brain. J. Physiol. (Lond.), 149,135-143. FARQUHAR, M. G., AND HARTMANN, J. F., (1957); Neuroglial structure and relationships as revealed by electron microscopy. J . Neuropath. exp. Neurol., 16, 18-39. FERNANDEZ-MORAN, H., (1957); Electron microscopy of nervous tissue. Metabolism of the Nervous System. D. Richter, Editor. London, Pergamon Press (pp. 1-34). FISHER, R., B. AND YOUNG,D. A. B., (1962); Direct determination of extracellular fluid in the rat heart. J. Physiol., 158, 50-58. HASSIN, 0.B., (1948); The morphology of the pial blood vessels and its bearing on the formation and absorption of the cerebrospinal fluid. J . Neuropathol., 7,432-438. HORSTMANN, E., AND MEVES,H., (1959); Die Feinstruktur des molekularen Rindengraues und ihre physiologische Bedeutung. Z. Zellforsch., 49, 569-604. KLEEMAN, C. R., DAVON, H., AND LEVIN,E., (1962); Urea transport in the central nervous system. Amer. J . Physiol., 203, 739-747. LUSE,S., (1956); Electron microscopic observations of the nervous system. J. biophys. biochem. Cytol., 2, 531-542.

MANERY, J. F., AND HASTINGS, A. B., (1939); The distribution of electrolytes in mammalian tissues. J . bioL Chem., 127,657-676. RALL,D. P., OPPELT,W. W., AND PATLAK, C. S., (1962); Extracellular space of brain as determined by diffusion of inulin from the ventricular system. Life Sci., 2, 4348. REED,D. J., AND WOODBURY, D. M., (1962); Effect of hypertonic urea on cerebrospinalfluid pressure and brain volume. J . Physiol., 164,252-264. ROUGEMONT, J., AMES, A., NESBITT, F. B., AND HOFMANN, H. F., (1960); Fluid formed by choroid plexus. J . Neurophysiol., 23,484-495. SCHLOERB, P. R., (1960); Total body water distribution of creatinine and urea in nephrectomised dogs. Amer. J . Physiol., 199,661-670. SCHULTZ, R. L., MAYNARD, E. A., AND PEASE, D. C., (1957); Electron microscopy of neurons and neuroglia of cerebral cortex and corpus callosum. Amer. J . Anat., 100, 369-407. STUBBS, J. D., AND BRADBURY, N. W. B., (1963); Regional differences in urea concentration in human cerebrospinal fluid. J . Physiol., In the press. R. D., FROST, R. W., AND TAYLOR, J. L., (1954); Inhibition of cerebrospinal fluid forTSCHIRGI, mation by a carbonic anhydrase inhibitor (Diamox). Proc. SOC.exp. Biol. ( N . Y . ) ,87, 373-376. VAN HARREVELD, A., HOOPER, N. K., AND CUSICK, J. T., (1961); Brain electrolytes and cortical impedance. Amer. J . Physiol., 201, 139-143. WELCH, H., AND FRIEDMAN, V., (1960); The cerebrospinal fluid values. Brain, 83,454469. WYCKOFF, R. W. G., AND YOUNG, J. 7., (1956); The motor neuron surface. Proc. roy. SOC.B, 144, 440-450.

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The Distribution of Water in Brain Tissues Swollen in v h o and in vivo H. M. PAPPIUS The Donner Laboratory of Experimental Neurochemistry, Montreal Neurological Institute and the Department of Neurology and Neurosurgery, McGill University, Montreal (Canada)

INTRODUCTION

Brain tissues have a marked tendency to take up fluid under a variety of conditions. In vitro the swelling occurs when cerebral cortex slices are incubated in isotonic salt solutions under optimal metabolic conditions and it cannot be adequately controlled even in hypertonic solutions (Elliott, 1946). In vivo cerebral edema develops in response to various types of trauma and despite numerous studies of this problem (e.g. Clasen et al., 1957; Gerschenfeld et al., 1959; Ishii et al., 1959; Klatzo et al., 1958; Raimondi et al., 1962) the mechanisms responsible for its production remain to be elucidated. Thus a clarification of the processes involved in the uptake of fluid by, and its distribution within, cerebral tissues is not only of neurochemical interest, but also of considerable practical importance. STUDIES

in vitro

For some years now we have made detailed studies of the distribution of the swelling fluid in cerebral cortex tissue (and in some cases in subcortical white matter and nerve) under a variety of experimental conditions in vitro. We have incorporated into the incubating medium substances which were believed to equilibrate only with the water in the extracellular spaces (Pappius and Elliott, 1956a, b; Pappius et al., 1962). The results of these studies, which I wish to summarize here, show clearly that three distinct fluid compartments exist in incubated cerebral cortex slices and three types of swelling can occur. Experimental procedures and presentation of results

Rat or cat cerebral cortex slices, about 0.5 mm thick, were cut with a Stadie-Riggs microtome, without moistening, in a humid chamber (Elliott, 1955). Representative samples of the slices were weighed and incubated in glucose-containing Ringerbicarbonate medium closely resembling cerebral spinal fluid in its electrolyte composition. After the incubation, the slices were drained, reweighed and then suitably References p. 1531154

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extracted for analysis of the ‘tracer’ substance under study. The substances incorporated into the incubating medium as ‘tracers’ were inulin, sucrose, thiocyanate and two types of protein labelled with a fluorescent dye i.e. rat serum protein labelled with lissamine rhodamine B 200 (LLP) and fluorescein-labelled bovine albumin (FLA). The concentrations used were as low as was compatible with subsequent analysis. Usually the final concentration of protein and thiocyanate was 0.1 %, of sucrose 0.3 % and of inulin 0.5 %. The concentration of the ‘tracer’ in the medium and in the tissue was then determined and the ‘tracer’ space in each case was taken as equal to that volume of the medium which would contain the same amount of the ‘tracer’ as was found in the slice. It must be pointed out that the time required for the equilibration of the ‘tracer’ with all the tissue water available to it varied with the substance used. Thus thiocyanate rapidly entered the tissue space available to it and within 15 min equilibrium was established. Sucrose, inulin and protein penetrated into the tissue much more slowly and at least 60 min incubation with the ‘tracer’ was required to obtain steady state conditions. Details of the composition of the media used, of the conditions of incubation and of the chemical methods for the determination of the ‘tracer’ substances have been published (Pappius and Elliott, 1956a, b; Pappius et al., 1962). All our results are expressed on the basis of 100 mg of fresh weight of tissue. Assuming the densities of the tissue and fluids to be 1, a 100 mg slice has a volume of 100 pl. In an unincubated cerebral cortex slice, prepared in the humid chamber, 80 pl of the initial volume is the ‘original tissue water’, the rest being dry weight residue. After incubation the difference between the total weight of the swollen slice and its dry weight is the ‘total fluid volume of the swollen tissue’. This would amount to 121 pl in a 100 mg slice which on incubation had taken up 40 mg (or pl) of fluid and lost 1 mg of dry weight. The ‘tracer’space would then be that fraction of the swollen tissue water which is in equilibrium with the tracer substance in the medium. The difference between the total fluid volume and the ‘tracer’ space would be the tissue water not penetrated by the ‘tracer’ substance and we refer to it as the ‘non-tracer’ space. In the case of the example cited above, if the thiocyanate space was found by analysis to be 85 pl, then the non-thiocyanate space obtained by difference would be 36 pl (i.e. 121-85 pl). Fluid compartments in incubated cerebral tissue

Results obtained with 4 tracer substances in incubated cerebral cortex slices are summarized in Fig. 1. For comparison a representation of an assumed unincubated slice is also included, based on the electron-microscope evidence that no extensive extracellular space exists in such tissue (see e. g . Schultz et al., 1957). In Fig. 1 the extracellular space of the unincubated slice is taken as 7 % of the total tissue volume. Varon and McIlwain (1961) obtained essentially the same results with inulin as we did. During the incubation at 38” under our standard aerobic conditions the swelling of the cerebral cortex slices was 40% of the initial tissue weight or 40 pl of fluid in

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a 100 mg slice. Inulin was found to equilibrate with the fluid of swelling and also with a little of the initial tissue water, so that the inulin space amounted to approximately 50 p1 of water. When labelled protein was incorporated into the medium its distribution was quantitatively the same as that of inulin. We assume, therefore, that these two tracer substances penetrate the same fluid compartment. (In liver slices inulin and 14C-polyglucose, a much larger molecule, were found to penetrate the same fraction of the tissue water (Parsons and Van Rossum, 1962a).) In contrast to inulin and protein, sucrose and thiocyanate equilibrated not only with the fluid taken up on incubation but also with a considerable fraction of the initial tissue water. The sucrose and thiocyanate spaces amounted to approximately 85 p1 of fluid. About 35 pl of the swollen tissue water was not permeable by any of the tracer substances used in this study. This is the non-thiocyanate and/or non-sucrose space. Thus three distinct fluid compartments exist in incubated cerebral cortex slices : (1) the non-thiocyanate and non-sucrose space, (2) the inulin and protein-space, and (3) the compartment which is penetrated by sucrose and thiocyanate but not by inulin and protein. In Fig. 2 results of similar studies with subcortical white matter slices of the cat and sciatic nerve of the rat are presented. In both these tissues after 60 min of aerobic incubation the swelling was somewhat smaller than in cerebral cortex. In nerve as References p. l53lI54

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in cerebral cortex, inulin penetrated into little more than the swelling fluid. However, in the white matter a greater portion of the original tissue water was equilibrated with inulin. Sucrose occupied a space in the white matter similar to that occupied in grey matter. In both the subcortical white matter and nerve thiocyanate slowly equilibrated with most of the tissue water. We were able to definitely identify one of the 3 fluid compartments in incubated cerebral tissue slices. The use of protein labelled with a fluorescent dye enabled us to visualize microscopically the tissue water compartment equilibrated with this tracer. Photomicrographs of cat cerebral cortex and subcortical white matter slices incubated aerobically in fluorescein-labelled albumin solution (FLA) were prepared by Dr. Klatzo and some representative examples are shown in Fig. 3. Freeze-dried preparations revealed a striking green fluorescence of the margins in sections from both white matter (Fig. 3,l) and cerebral cortex (Fig. 3,2). In both tissues, areas underlying the peripheral zones were devoid of FLA, except for occasional blood vessels, which showed intense green fluorescence. In Figs. 3,3 and 3,4 the frozen section was prepared from the peripheral zone of slices fixed in formaldehyde-saline. The less intense green fluorescence, due to elution of about 40% of the FLA from the slice during fixation in formaldehyde-saline, permitted better localization of the remaining FLA in particular cell components. Swollen nerve fibers can be identified in the white matter (Fig. 3,3) and neurons and glia in the cortex (Fig. 3,4). It is clear that the protein ‘space’ of incubated brain tissue slices is mainly limited to a zone near the edge which consists of tissue damaged during preparation of the slice. Thus protein and, by inference, inulin equilibrate with the fluid in damaged regions of the slice and their distribution in vitro is not a measure of a true extra-

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Fig. 3. Fluorescence photomicrographs of cat brain tissue slices incubated aerobically in medium containing 0.3 % fluorescein-labelled bovine albumin (FLA). (1) White matter, showing edge (e) of slice. Slices processed by freeze-drying. Intense green fluorescence of the marginal zone and of the blood vessels (bv). White globules and granules scattered in the section are due to the presence of autofluorescent pigments, x 230. (2) Cerebral cortex, showing the edge of two slices (e). Section processed by freeze-drying. Intense green fluorescence of the leptomeninges and of the blood vessel (bv) penetrating into the cortex. Superficial areas of the cortex showed a diffuse green fluorescence. The granular material in the cortex represents autofluorescent cellular pigment, x 230. (3) White matter. Formaldehyde-saline fixed frozen selion. Area next to edge of slice. Green fluorescent nerve fibers (f) show distorted swollen outlines, X 450. (4) Cerebral cortex. Formaldehyde-saline fixed frozen section. Area next to edge of slice. Neurons (n), glial cells (g), and vessels (bv) are green fluorescent. The occasional bright, small granules in the cells represent orange autofluorescent pigment, x 450.

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cellular space or of a specific intracellular compartment. On the other hand, the tissue water not penetrated by inulin or protein (the non-protein, non-inulin space) can be regarded as the intracellular space of undamaged cells. This interpretation of our results with inulin and labelled protein is compatible with the electron-microscopic evidence that in brain tissue ‘essentially all of the space is filled with living cell processes of one sort or another’ (Schultz et al., 1957). We interpret the equilibration of thiocyanate and sucrose with a larger fraction of the tissue water as evidence of their entrance into an intracellular compartment. One possibility, namely that thiocyanate and sucrose penetrate into some subcellular structures of all cells, seems unlikely. We have shown (Pappius and Elliott, 1956a) that under our experimental conditions thiocyanate can penetrate intracellular space in liver and kidney cortex slices and occupies all the tissue water of these tissues. Thusthere is no subcellular structure common to all cell types which is impermeable to thiocyanate. It is more probable that the thiocyanate or sucrose space in brain tissue corresponds to the intracellular compartment of a particular group of cells. We suggest that these may be glial cells. This view is consistent with the hypothesis of Gerschenfeld et al. (1959) that astrogha functions as a water-ion compartment replacing extracellularspace of other tissues. In the virtual absence of an extracellular space of brain tissue the sodium, chloride and other ions and molecules normally present must be largely intracellular, though not necessarily homogenously distributed throughout all cells. Experimental data of Katzman (1961), mostly from a study of glial cell turnours, provide suggestive evidence that some, if not all glial cells may be high sodium cells. A similar conclusion may be drawn from the electron-microscopic studies of Birks (1962). He observed definite structural changes in the mitochondria of neurones when rat sympathetic ganglia were perfused with digoxin which were shown to be related to the drug’s inhibitory action on sodium-extrusion mechanisms. It is strikingthat in thesamepreparationsthemitochondriaofnon-neuronalcellswerecompletely unaffected.This lack of effect of digoxin on mitochondria of non-neuronal cells may indicate that these organellesnormally function in an environment high in sodium. The third fluid compartment in incubated cerebral cortex tissue, the non-thiocyanate or non-sucrose space, may correspond to the neuronal space. Slow equilibration of thiocyanate with all the tissue water in the white matter and nerve is compatible with this interpretation because in the preparations all nerve processes are cut. Coombs et al. (1955) have evidence that just as chloride, thiocyanate crosses neuronal membranes readily but is excluded from intracellular spaces for electrochemical reasons. It is of interest that our estimate of non-thiocyanate space agrees with that of the non-chloride space calculated from the figures reported by Leaf (1956). Membrane permeability and not cell membrane potential must govern the distribution of the electrically neutral molecule, sucrose. Distribution of swelling fluid within cerebral cortex tissue under various experimental conditions in vitro

Three types of swelling can be distinguished in the incubated cerebral cortex slices.

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Uptake offluid by damaged tissue elements. As already mentioned, marked uptake of water occurs in the inulin space, which represents the damaged regions of the slice. The fact that the non-inulin space does not change, while swelling and inulin space do increase during incubation (Pappius et al., 1962), suggests that the increase in the ‘damaged space’ is not a spread of the area of damage but rather uptake of fluid by the damaged elements. A further indication that this swelling is related to the extent of damage caused to the tissue during preparation of the slices is the finding that in thick slices, containing a smaller proportion of damaged cells, the swelling was significantly smaller than that found in the slices of the usual thickness (Pappius and Elliott, 1956a).

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Fig. 4. The inulin space in cerebral cortex slices incubated under various experimental conditions. For explanation see Fig. I . RBG = Ringer-bicarbonate.-glucose. * Inulin could not be determined in the presenceof high concentration of sucrose (10%sucrose in the medium). Inulin space was assumed to be the same as in isotonic medium since the larger sucrose space was unchanged in hypertonic medium (see Fig. 5).

It will be seen from Fig. 4 that the size of the inulin space is quite constant under a variety of experimental conditions, namely, after aerobic and anaerobic incubation, under certain aerobic conditions which result in an increase of non-inulin space (e.g. when glutamate is present in the incubating medium) and in hypotonic (e.g. half isotonic saline in the medium) and hypertonic (e.g. 10% sucrose in standard medium) solutions. Thus neither metabolic conditions nor osmotic forces are apparently involved in this swelling. Our studies on the distribution of electrolytes indicate that the fluid taken up has about the same osmolarity as the medium (Pappius and Elliott, 1956b). The incorporation of polyvinyl pyrollidone (PVP) into the incubating medium was the only condition which appeared to control the uptake of fluid into the inulin space, but only at concentrations of PVP equivalent to at least twice the colloid osmotic pressure of the plasma. The mechanism of the fluid uptake into the damaged regions of cerebral cortex References p . 1531154

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slices is still not understood. Possibly damage to the tissue decreases certain natural cohesive forces between and within the cells opening small channels in the affected regions and allowing the tissue to act like a sponge. In liver slices Parsons and Van Rossum (1962b) observed limited swelling of the extracellular compartment (using inulin as tracer), which was not reversible (osmotically) and which they attributed to changes in the arrangement and mechanical properties of extracellular supporting tissue. In brain tissue hydration of hydrophilic molecules, normally not in contact with aqueous fluids, may also be involved (Elliott, 1961). Osmotically induced swelling under aerobic conditions. Increased swelling of cerebral cortex slices occurs under aerobic conditions in the presence of glutamate or when the potassium concentration in the medium is increased. As will be seen from Fig. 5, Slicer incubated aerobically for 60 min

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this additional swelling is accounted for by an increase in the non-thiocyanate, nonsucrose space. Under these conditions, however, potassium is taken up by the tissue against a concentration gradient (Pappius and Elliott, 1956b), and the increase in the amount of water in the slice can be correlated with the increase in the potassium content of the tissue. Thus, in the presence of glutamate or high potassium in the medium, the increase in that part of the intracellular water which is not equilibrated with either thiocyanate or sucrose, can be explained as a consequence of the osmotic

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gradient generated by an active uptake of solute from the surrounding medium. We have further evidence that the size of this fluid compartment can be modified osmotically, in contrast to the lack of osmotic control which we have demonstrated in the case of inulin space (see Fig. 4). It is clear from Fig. 5 that the non-thiocyanate, non-sucrose space decreased after incubation in hypertonic solution and increased in hypotonic medium. These changes in an intracellular water compartment are in keeping with the general phenomenon of net exchange of water between cells and their immediate surroundings to maintain osmotic equilibrium (Robinson, 1960). Such movement of water can occur in response to changes in the tonicity of the environment (e.g. during incubation in hyper- or hypotonic media), or when the osmotic gradient is brought about by an active uptake of solute into the cells (e.g. swelling of cerebral cortex slices in the presence of glutamate). Swelling under conditions of impaired metabolism. Cerebral cortex slices, like a variety of isolated tissues (Robinson, 1960), swell appreciably when their metabolism is impaired. Under anaerobic conditions the additional swelling fluid is taken up in the non-sucrose (see Fig. 5 ) and non-inulin spaces (see Fig. 4), thus into the intracellular compartments of the tissue. (The thiocyanate space cannot be estimated under anaerobic conditions as thiocyanate slowly penetrates all of the tissue water when metabolism is inhibited (Pappius and Elliott, 1956a).) The intracellular swelling when metabolism is impaired can be correlated with the entry of extracellular solutes. In our experiments with cerebral cortex slices the increase in their sodium content was greater than the loss of potassium. At the same time an increase in the chloride content would be expected (Leaf, 1959). The observed swelling can be compared to swelling in hypotonic solution, the formerly isotonic medium becoming hypotonic with respect to the tissue because a solute, sodium ion, which is in effect non-penetrating under aerobic conditions, becomes penetrating and without osmotic activity under anaerobic conditions. We now know that even under our standard aerobic conditions some intracellular swelling occurs which may be attributed to a transient impairment of metabolic processes. If brain slices are placed in ice-cold medium and kept ice-cold, instead of at room temperature, during the gassing and setting up period (approximately 10 min) preceding the incubation, the total swelling observed after aerobic incubation at 38" is considerably decreased. It appears that carrying out the preliminary manipulations in the cold eliminates or reduces a period of hypoxia with its concomitant additional swelling, which cannot be reversed by subsequent restoration of optimal metabolic conditions. It will be seen from Fig. 6 that both the non-thiocyanate and the inulin spaces were the same whether the tissue slices were prepared in the cold or at room temperature *. The pre-cooling resulted, however, in a significant decrease of the water compartment penetrated by thiocyanate but not by inulin. In a discussion of

* It is also clear from Fig. 6 that in slices set up in the cold inulin penetrates a greater fraction of the original tissue fluid than was initially realized. This, however, does not in any way invalidate the conclusion that most of this fluid is in the damaged tissue elements. References p . 1531154

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Tissue water not equilibrated with either inulin or thiocyanate

Tissue water equilibrated with both inulin qnd thiocyonot.

Fig. 6. The effect of initial cooling on the fluid compartments in cerebral cortex slices incubated aerobically for 60 min. For general explanation see Fig. 1. P <0.01 for the difference in swelling and in thiocyanate space under the two conditions.

neuroglia it is of special interest that following a period of partial hypoxia the increase of the swelling can be totally accounted for by an increase in that water compartment which we think is the glial space of cerebral tissue. We suggest that this is the glial swelling observed by Gerschenfeld et al. (1959) in their electron-microscope studies of incubated cerebral cortex slices. We do not believe that glial swelling accounts for the total fluid uptake by brain tissue incubated in vitro. Furthermore, as a result of our studies of the effect of several fixative solutions on the water content of brain cortex slices (Pappius et al., 1962), we are confident that suitably directed electronmicroscope studies of appropriately fixed preparations would demonstrate the swelling in the damaged regions. Summary. Our evidence indicates that the damaged tissue elements take up the major portion of the swelling fluid in cerebral cortex slices in vitro. The mechanism of this swelling remains to be elucidated. STUDIES

in vivo

Our studies of brain tissue swelling have been extended now to the in vivo conditions. We have investigated the distribution of water in cerebral tissues in traumatically induced edema (Pappius and Gulati, 1963) and in swelling arising from an osmotic gradient established experimentally between the cerebral tissues and the plasma (Pappius et al., 1963). Traumatic edema Experimental procedures. Cerebral edema was induced in cats by local freezing of cortex or by partial removal of the occipital lobe. The results provide a comparison between the two types of traumatic lesion. Freezing lesions were produced by a modified apparatus originally described by Klatzo et al. (1958). It consisted of a

145

WATER D I S T R I B U T I O N I N SWOLLEN B R A I N

brass cup with a copper rod extending beyond the base of the cup to form a circular end plate 80mmzarea. The whole instrumentwascooled to 50" and the end plate was then applied for 20 sec to the surface of the exposed cortex. The general procedure was as follows: Cats were anaesthetized with nembutal and the bone over the right hemisphere was removed. For freezing lesions the dura was left intact; for occipital lobectomy the dura was laid open and the right occipital lobe was removed. The animals so treated were killed at intervals of 1 to 10 days after the lesion was made. Replicate determinations of dry weight, sodium and potassium were carried out on samples of white and of grey matter from both hemispheres. Since all the experimental lesions were made on the right side, the left hemisphere in each animal was the control. The method of sampling the experimental hemisphere is critical. Following the production of freezing lesions samples of white matter were taken from areas grossly edematous or from the region immediately underlying the lesion when edema was not visibly obvious. In sampling the cortex the area of cell necrosis where the freezing lesion was produced was not analyzed. Samples were taken from the tissue surrounding the lesion area and, in cases where the white matter was grossly edematous, overlying the affected white matter. In animals from which the occipital lobe had been removed samples of both white and grey matter were taken at the edge of the lesion. TABLE I THE D R Y W E I G H T A N D THE S O D I U M A N D POTASSIUM C O N T E N T OF BRAIN TISSUES O F T H E CAT 24 H A F T E R T H E APPLICATION O F A F R E E Z I N G LESION

Freezing lesion: 80 nun2;20 sec. Dry weight: mg per 100 mg fresh weight of tissue. Na and K : mmoles per kg fresh weight of tissue. Number of animals in brackets. White

Normal (6) Following freezing lesion (4) Control hemisphere Experimental hemisphere

Grey

Dry weight

Na

K

NaIK Dryweight Na

K

Na/K

32.0

58

82

0.74

18.2

68

90

0.76

31.2 22.1*

59 86*

81 61*

0.74 1.40*

18.9 18.5

68 78*

96 93

0.70 0.85*

* Significantly different from the control hemispheres (P
146

H. M. P A P P I U S

from 68 to 78 %. This represents 45 % swelling of the tissue*. At the same time there was a rise in sodium and a fall in potassium content of the white matter resulting in a sharp rise of Na/K ratio. In striking contrast to the changes observed in the white matter, the dry weight of the cerebral cortex adjacent to the lesion or overlying swollen white matter was within the normal limits and there was no change in its potassium content. A small increase in sodium produced a significant, but small increase in Na/K ratio. These results show that the accumulation of the fluid brought about by trauma due to freezing occurred almost exclusively in the white matter and that the fluid taken up by the tissue had a high sodium and low potassium content. A highly significant correlation in the edematous white matter between the decrease in dry weight on one hand and the decrease in potassium and increase in sodium content on the other (Pappius and Gulati, 1963), is consistent with our belief that the fluid taken up is closely related to a plasma liltrate. The time course of the changes in water and electrolyte content of white matter following freezing lesions is not pertinent to the present discussion. Suffice it to say that the fluid content of the affected white matter remained high for at least the first 3 days. Changes observed in cerebral tissues 3 days after production of a freezing lesion and after occipital lobectomy, are summarized in Table 11. It will be seen that surgical removal of the occipital lobe resulted in the same changes in the white matter as those following freezing lesions. In grey matter adjacent to the area of removal there was no measurable uptake of fluid after occipital lobectomy. Similar results have been reported in a study of localized cerebral edema associated with intracranial tumours TABLE I1 T H E D R Y WEIGHT A N D T H E NA/K RATIO O F B R A I N TISSUES OF T H E C A T 2 D A Y S AFTER DIFFERENT TYPES OF LESION

Freezing lesion :80 mm2;20 sec.Dry weight :mg per 100 mg fresh weight of tissue. Number of animals in brackets. White

Grey

Types of lesion

N o lesion (6) Experimental hemisphere Freezing lesion (4) Occipital lobectomy (8)

Dry weight

NaIK

Dry weight

NalK

32.0

0.72

18.2

0.76

23.5* 24.6*

1.36* 1.24*

19.4 18.5

0.70 0.86*

*

Significantly different from the control hemisphere (PtO.O1).

*

Per cent swelling can be calculatedfrom percentage dry weight by the formula: per cent swelling

P-P1

=

x 100 where P is the normal percentage dry weight, P I is the dry weight of the edematous P1-p * tissue and p is the percentage dry weight of the fluid taken up (Elliott and Jasper, 1949). Assuming the swelling fluid to be plasma filtrate, p = 1.

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WATER D I S T R I B U T I O N I N SWOLLEN B R A I N

(Stewart-Wallace, 1939). It appears that trauma causes an uptake of fluid in the white matter when the damage is to both the white and the grey matter as in occipital lobectomy, or when only the superficial layers of the cortex are directly damaged, as in our freezing lesions. Effect of hypertonic urea infusion on the water content of cerebral tissues in the presence of traumatic edema. Hypertonic urea solutions have been used clinically as a means of reducing intracranial pressure in the presence of a variety of brain lesions (e.g. Javid, 1958; Langfitt, 1961). We found that in normal animals the water content of both the white matter and the cerebral cortex was decreased after the administration of hypertonic (30%) urea solution (1.5 g/kg) (Pappius and Dayes, 1965). The effect was maximal between 60 and 120 min after the start of the infusion and was correlated with a concentration gradient for urea between the plasma and the cerebral tissues. Similar studies have been reported by Bradbury and Coxon (1962), Kleeman et al. (1962) and Reed and Woodbury (1962). To find out if the accumulation of fluid in the white matter associated with trauma could be osmotically controlled, Dr. Dayes and I infused hypertonic urea solution into a series of cats 24 h after making freezing lesions. The animals were killed 90 min after the start of the urea infusion and the water content of tissues in both hemispheres was determined. Results of these experiments, with suitable controls are summarized in Table 111. The infusion TABLE 111 THE EFFECT OF HYPERTONIC UREA I N F U S I O N O N THE D R Y WEIGHT OF BRAIN TISSUES OF T H E CAT

24

H AFTER A FREEZING LESION

Freezing lesion: 80 mm2;20 sec. Urea: 90 rnin after infusion of 30%urea in saline solution; 1.5 g/kg. Dry weight: mg per 100 mg fresh weight of tissue. Number of animals in brackets. Dry weight White

Normal (6) Normal urea (3) Freezing lesion urea: control hemisphere (4) experimental hemisphere (4) no urea: experimental hemisphere (5)

*

Grey

32.0 35.1:

18.2 21.2*

33.4" 21.3* 20.6:

20.3* 20.3: 18.3

Significantly different from normal: P < 0.01 ; ** P < 0.05.

of hypertonic urea solution under these conditions resulted in a significant increase

of the dry weight of the cerebral cortex of both hemispheres and of the white matter of the control hemisphere (i.e. the water content was decreased). But, hypertonic urea infusion had no effect on the dry weight (or water content) of the edematous white matter beneath the freezing lesion. These results clearly show that traumatically induced edema cannot be controlled osmotically. Clasen et al. (1957) came to the same conclusion when studying the effects of hypertonic glucose infusion on edema produced by massive freezing lesions. References p. 1531154

148

H. M. P A P P I U S

Distribution of edemaJZuid within cerebral tissues. We do not have direct evidence on the distribution of the edema fluid within the white matter. Because we have shown that the bulk of the swelling fluid accumulates in white matter, no conclusion as to the intracellular nature of cerebral edema can be drawn on the basis of electron micrographs of cerebral cortex tissue (Gerschenfeld et al., 1959; Luse and Harris, 1960). The suggestion that the edema fluid is closely related to a plasma-filtrate and our finding that traumatically induced edema is not osmotically controlled, are compatible with the electron-microscope evidence of Raimondi et al. (1962) that changes in the white matter due to edema were associated with a significant increase in the extracellular compartment. Glial elements in the cortex and in the white matter are undoubtedly affected in edema (Gerschenfeld et al., 1959; Raimondi et al., 1962; Klatzo et al., 1962) but quantitatively the fluid involved in this cellular swelling can represent only a negligible part of the total brain swelling. Thiocyanate space in vivo. At this point I would like to mention briefly some experiments designed to test in vivo our suggestion based on studies in vitro that thiocyanate can equilibrate with the water in an intracellular, possibly glial, compartment. The fairly rapid equilibration in vitro of a variety of substances with the fraction of cerebral tissue water available to them is in contrast to their relatively very slow penetration in vivo. Because of this the situation in vitro has been dismissed by some investigators as due to the damage artefact (Dobbing, 1961) and by others regarded as evidence for a barrier in vivo to diffusion into cerebral tissues (Davson TABLE IV T H E PENETRATION O F T H I O C Y A N A T E I N T O CEREBRAL CORTEX TISSUE in ViVO

Percent of tissue water equilibrated with plasma CNS 4 h after intravenous sodium thiocyanate 0.25 g/kg. Average final concentration of plasma thiocyanate 8.3 mmoles/l of water. Number of animals in brackets. Cerebral cortex Control side

No lesion (3) Lesion, 3 h (5)

*

21.5 23

Experimental side

22 35*

Significantly different from control P <0.01.

and Spaziani, 1959). If the latter explanation is in fact the correct one then in regions where the so-called ‘blood-brain barrier’ is no longer operative, as in the region around a freezing lesion (Klatzo et al., 1958), the uptake in vivo of thiocyanate should be increased. In preliminary experiments, sodium thiocyanate solution was infused intravenously in anaesthetized cats (0.25 g/kg) and a freezing lesion was made in the usual manner. Three hours after the lesion was made the animals were killed and the thiocyanate content of cerebral cortex adjacent to the lesion and from the control hemisphere

WATER DISTRIBUTION I N S W O L L E N B R A I N

149

was compared with that of the plasma. Animals killed 4 h after the thiocyanate infusion without a lesion being made served as additional controls. The results summarized in Table IV show that, with the relatively high concentration of thiocyanate in the plasma in our experiments, about 20% of the water in the control tissues was in equilibrium with the circulating thiocyanate. This is considerably higher than the currently accepted estimate of extracellular space in cerebral tissues and suggests intracellular distribution of thiocyanate in normal cerebral cortex in vivo. The fraction of the tissue water equilibrated with thiocyanate was significantly increased in tissue adjacent to a freezing lesion. Since the total water content of cerebral cortex in the experimental hemisphere was not elevated and since no evidence exists that an abnormal extracellular space is formed in cortical tissue under these conditions, it is reasonable to tentatively conclude that this increase represents increased penetration of thiocyanate into an intracellular compartment. Experiments are in progress to clarify this problem further. Osmotically induced swelling of brain tissues in vivo During an investigation into the causes of neurological complications associated occasionally with otherwise successful hemodialysis of uremic patients, we were able to accumulate data on what can be described as osmotically induced cerebral swelling (Pappius et al., 1965). The results are interesting because they provide a comparison with traumatically induced changes in the water and electrolyte content of cerebral tissues. The experimental procedure briefly was as follows : Mongrel dogs were made uremic by ligation of both ureters 48 h before the start of dialysis. The urea content of the plasma was further increased by a slow infusion of urea solution more than 24 h before dialysis. Hemodialysis was carried out on anaesthetized dogs with the Kloff twin-coil artificial kidney, usually for 60 to 90 min. Blood urea, electrolytes, total osmolarity and pH were measured throughout the experimental period. Bath water was similarly analyzed before and after dialysis. Brain biopsies were taken at appropriate times during the experiments.The dry weight, urea, sodium and potassium determinations were done on both the subcortical white matter and the cerebral cortex tissue. Changes in the plasma and tissue urea concentrations in a typical experiment are presented in Fig. 7. The urea content of the cerebral tissues in uremic dogs was found to be in equilibrium with that of the plasma. Rapid hemodialysis and the resultant sharp decrease in the concentration of plasma urea were accompanied by a considerable but slower decrease in the urea content of both the white and the grey cerebral tissue. As a result a significant but transient discrepancy developed between the concentration of urea in the plasma and in cerebral tissues during and immediately after dialysis. In 8 successfully dialyzed animals the brain biopsies were taken immediately on termination of the dialysis. In 6 of these, swelling of cerebral tissues was observed. Table V summarizes the results obtained with cerebral tissues before and immediately References p. 153IJS.l

150

H. M. P A P P I U S '0 r

a

BOO

-

700

-

Y

2

3

s

600

8 - 500 I n

& 400 I Ly

-

200

-

0

E

100

--- ____ 7.

-

300

3

-

..............

-

BLOOD

I .

0 0

...-...............

.........

--

CEREBRAL C O R T f X

WHIIE M A I T E R

0

DIALYSIS

0-

I

0

3

1

1

4

h Fig. 7. The effect of hemodialysis on the urea content of blood and cerebral tissues of uremic dog.

after dialysis. It will be seen that the dry weight of both the white matter and cerebral cortex was significantly decreased immediately after hemodialysis. This decrease (calculated by the formula of Elliott and Jasper, 1949) is equivalent to a swelling of 17% and 19 % respectively. In both tissues the Na/K ratio remained unchanged. Thus, in striking contrast to traumatic edema, osmotically induced swelling of cerebral tissues affects both the white and the grey matter and it does not involve uptake of fluid with electrolyte content similar to that of plasma. Under these conditions intracellular swelling may account for the bulk of the fluid accumulated in both cerebral tissues. Luse and Harris (1960) were unable to demonstrate any abnormal extracellular space in cerebral cortex tissue from rabbits in which edema was TABLE V THE EFFECT OF HEMODIALYSIS O N THE D R Y WEIGHT A N D THE SODIUM A N D POTASSIUM CONTENT OF BRAIN TISSUES O F UREMIC DOG

Dry weight: mg per 100 mg fresh weight of tissue. Na and K: mmoles per kgfreshweightof tissue. Number of animals in brackets. ~~

White Tissue biopsy

Uremic : Pre-dialysis (10) Post-dialysis (6 of 8) Normal : Post-dialysis (1)

*

~~~~~~

Grey

Dry weight

Na

K

NaIK

Dry weight

Na

K

Na]K

33.0 f 1.5 28.4 f0.9*

51 52

76 73

0.67 0.68

19.6 f 1.0 16.8 -+ 1.2*

52 58

90 87

0.58 0.68

33.6

62

75

0.83

19.3

61

103

0.59

Significantly different from pre-dialysis value (Pt0.01).

WATER D I S T R I B U T I O N I N S W O L L E N B R A I N

151

produced by osmotic means (injection of distilled water). It is interesting that only glial cells appeared swollen in their electron micrographs. It is clear that cerebral edema associated with trauma and osmotically induced swelling of cerebral tissues are basically different phenomena and the two methods of producing accumulation of fluid in cerebral tissues cannot be used interchangeably in experimental studies. CONCLUSION

Intracellular swelling which occurs as a result of osmotic action in brain tissue slices in vitro has a counterpart in vivo in osmotically induced cerebral swelling. Impaired metabolism in vitro also results in intracellular swelling which can be correlated with the entry of extracellular solutes. A similar mechanism may be envisaged as underlying the swelling observed in both white and grey matter in response to a variety of metabolic poisons (e.g. Hurst, 1940; Hicks, 1950; Lumsden, 1959). A question arises : What relevance has the swelling in vitro in the damaged regions of cerebral cortex and white matter to the accumulation in vivo of the edema fluid in response to trauma, the bulk of which is found in the white matter? Both processes involve the uptake of fluid into an abnormal space. The extracellular nature of these spaces explains why increased tonicity of the incubating medium or the circulating plasma has no effect on the swelling associated with damage. However, our results with freezing lesions indicate that in vivo the major effect is not on the cellular elements immediately involved in trauma, since the fluid accumulated in the white matter while only the superficial layers of the cortex sustained the damage. In vitro on the other hand, the abnormal space which appears consists of the tissue elements directly damaged in slicing. In vivo structural characteristics may be responsible for the exclusion of the edema fluid from the cerebral cortex, where the closely interwoven cellular processes might be expected to offer considerable resistance to the influx of extraneous fluid. A smaller degree of mechanical resistance may be expected in the white matter. It could be argued that in cerebral cortex slices mechanical resistance is decreased when a large proportion of the cellular processes are cut. These are, however, highly speculative considerations, and they leave unresolved the basic problem of the mechanisms involved both in vitro and in vivo in cerebral tissue swelling associated with trauma. In a discussion on neuroglia the role which these cells play in cerebral swelling is of special interest. Our results show clearly that glial swelling does not account for the bulk of the fluid uptake due to trauma either in vitro or in vivo. Nevertheless such swelling may reflect a vital function which has been postulated for glial cells in the maintenance of a normal water and electrolyte environment within cerebral tissue (e.g. Gerschenfeld et al., 1959). Further quantitative studies in vitro of factors which affect that fluid compartment in cerebral cortex which, we think is the glial space should be worthwhile, especially if a suitable tracer substance were found for microscopic visualization of this compartment. Rejkrances p. 1531154

152

H. M. P A P P I U S

SUMMARY

Cerebral cortex slices swell appreciably when incubated in vitro in a medium resembling cerebral spinal fluid in its electrolyte composition. The distribution o f water in swollen slices is studied by incorporating thiocyanate, sucrose, inulin or protein labelled with a fluorescent dye into the incubating medium. The fraction of the swollen tissue fluid which is permeable by inulin consists mostly of fluid taken up in swelling but also of a little of the initial tissue water. It is found to be quantitatively the same as that permeable by the labelled protein. This makes possible the microscopic visualization of the water compartment in question. The protein labelled with a fluorescent dye is found to be restricted to the periphery of the slice. This indicates that protein and, by inference, inulin equilibrates with the fluid in the damaged regions of the slice. Their distribution in cerebral cortex slices in vitro is not a measure of a definite extracellular or intracellular compartment, while the tissue water not penetrated by inulin or protein presumably represents the intracellular space of undamaged cells. Such a conclusion accords with the finding that the extent of the inulin space is related to the proportion of the tissue elements damaged in slicing but is not greatly affected by experimental conditions of incubation, including increased osmotic pressure of the medium. The mechanism of fluid uptake into the damaged regions of the tissue is not understood, but may be of interest in relation to the problem of cerebral edema. Under certain aerobic conditions and under anaerobic conditions fluid is taken up by cerebral cortex slices which is not penetrated by inulin and is therefore intracellular. This may be the glial swelling observed with the electron microscope. In contrast to protein and inulin, sucrose and thiocyanate equilibrate not only with the fluid taken up on incubation but also with a considerable fraction of the initial tissue water. The more extensive penetration of sucrose and thiocyanate is interpreted as due to their distribution in an intracellular compartment. The possibility that this compartment corresponds to the intracellular space of glial cells will be considered. Swelling of brain tissue in vivo, or cerebral edema, was produced in cats by discrete freezing lesions and by trauma involved in partial removal of the occipital lobe. Twenty four hours following both procedures there is a sharp decrease in the dry weight (or conversely an increase in the water content) of the white matter of the traumatized hemisphere. At the same time there is sharp rise in the Na/K ratio of the swollen white matter. In striking contrast to changes observed in the white matter, the water content of the cerebral cortex tissue in the traumatized hemisphere remains unchanged within the limits of the techniques used. It is clear from the results obtained that the accumulation of fluid, or edema, brought about by trauma occurs almost exclusively in the white matter and the electrolyte changes observed are in agreement with the assumption that the fluid concerned is derived from the circulating blood. The glial swelling in cerebral cortex observed with the electron microscope in studies of cerebral edema can thus represent only a negligible part of the total brain swelling. In contrast to the water content of normal cortex and white matter, which is decreased by intravenous infusion of hypertonic urea solution, edematous fluid in the white

WATER D I S T R I B U T I O N IN S W O L L E N B R A I N

153

matter is not osmotically controlled. These findings will be discussed in relation to the evidence that edema fluid accumulates in an abnormal not intracellular space. Of interest in connection with the suggestion that thiocyanate penetrates into the glial cells in vitro is the finding that increased uptake of thiocyanate can be demonstrated in vivo 3 h after freezing in cerebral cortex tissue surrounding the lesion area. This penetration of thiocyanate into brain tissue in vivo occurs at a time when the tissue water content is unchanged. Preliminary experiments in uremic dogs subjected to hemodialysis indicate that osmotically swelling of brain tissue occurs in both grey and white matter and is not associated with a change in the Na/K ratio. Thus the mechanism of such swelling is quite different from that of cerebral edema induced by trauma and may involve intracellular swelling. ACKNOWLEDGEMENTS

I wish to express my appreciation to Professor K. A. C. Elliott for his continued encouragement and interest and I am grateful to Dr. L. S . Wolfe and Dr. J. A. Lowden for helpful criticism of the manuscript. REFERENCES BIRKS,R. I., (1962); The effects of a cardiac glycoside on subcellular structures within nerve cells and their processes in sympathetic ganglia and skeletal muscle. Canad. J. Biochem., 40, 303-315. BRADBURY, M. W. B., AND COXON, R. V., (1962); The penetration of urea into the central nervous system at high blood levels. J. Physiol., 163, 423435. CLASEN, R. A., PROUTY, R. R., BINGHAM, W. G., MARTIN, F. A., AND HASS,G. M., (1957); Treatment of experimental cerebral edema with intravenous hypertonic glucose, albumin and dextran. Surg. Gynec. Obstet., 104, 591-606. COOMBS, J. S., ECCLES, J. C., AND FATT,P., (1955); The specific ionic conductances and the ionic movements across the motorneuronal membrane that produce the inhibitory postsynaptic potential. J. Physiol., 130, 326-373. DAVSON, H., AND SPAZIANI, E., (1959); The blood-brain barrier and the extracellular space of brain. J . Physiol., 149, 135-143. DOBBING, J., (1961); The blood-brain barrier. Physiol. Rev., 41, 130-188. ELLIOTT,K. A. C., (1946); Swelling of brain slices and the permeability of brain cells to glucose. Proc. SOC.exp. Biol. ( N . Y.), 63, 234236. ELLIOTT, K. A. C., (1955); Tissue Slice Technique. Methods in Enzymology. New York, Academic Press Inc. (pp. 3-9). ELLIOTT, K. A. C., (1961); Brain Swelling and Fluid and Electrolyte Distribution. Chemical Pathology ofthe Nervous System. New York, Pergamon Press. H., (1949); Measurement of experimentally induced brain swelling ELLIOTT,K. A. C., AND JASPER, and shrinkage. Amer. J. Physiol., 157, 122-129. GERSCHENFELD, H. M., WALD,F., ZADUNAISKY, J. A., AND DE ROBERTIS, E. D. P., (1959); Function of astroglia in the water-ion metabolism of the cerebral nervous system. Neurology (Minneap.), 9, 412425. HICKS,S. P., (1950); Brain metabolism in vivo. I. The distribution of lesions caused by cyanide poisoning, insulin hypoglycemia, asphyxia in nitrogen and fluoroacetate poisoning in rats. Arch. Pathol,, 49, 111-137. HURST,E. W., (1940); Experimental demyelination of the central nervous system. I. The encephalopathy produced by potassium cyanide. Aust. J. exp. Biol. med. Sci., 18,201-223. ISHII,S., HAYNER, R., KELLY,W. A., AND EVANS,J. P., (1959); Studies of cerebral swelling. 11. Experimental cerebral swelling produced by supratentorial extradural compression. J. Neurosurg., 16, 152-166. JAVID, M., (1958); Urea - New use of an old agent. Surg. Clin. N . Amer., 38, 907-928.

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KATZMAN, R., (1961); Electrolyte distribution in mammalian central nervous system. Are glia high sodium cells? Neurology (Minneup.), 11, 27-36. KLATZO, I., MIGUEL,J., AND OTENASEK, R., (1962); The application of fluorescein labeled serum proteins (FLSP) to the study of vascular permeability in the brain. Actu neuroputh., 2, 144-160. KLATZO, I., PIRAUX, A., AND LASKOWSKI, E. J., (1958); The relationship between edema, blood-brain barrier and tissue elements in a local brain injury. J. Neuropath. exp. Neurol., 17, 548-564. c.R.,DAVSON, H., AND LEVIN,E., (1962); Urea transport in the central nervous system. KLEEMAN, Amer. J. Physiol., 203, 739-747. LANGFITT, T. W., (1961); Possible mechanism of actions of hypertonia urea in reducing intracranial pressure. Neurology (Minneup.), 11, 196-209. LEAF,A., (1956); On the mechanism of fluid exchange in tissue in vitro. Biochem. J., 62, 241-248. LEAF,A., (1959); Maintenance of concentration gradients and regulation of cell volume. Ann. N . Y. Acad. Sci., 72, 396-404. C. E., (1950); Cyanide leucoencephalopathy in rats and observations on the vascular and LUMSDEN, ferment hypotheses of demyelinating diseases. J. Neurol. Neurosurg. Psychiut., 13, 1-1 5. LUSE,S. A., AND HARRIS,B., (1960); Electron microscopy of the brain in experimental edema. J. Neurosurg., 17, 4 3 9 4 6 . PAPPIUS, H. M., AND DAYES,L. A., (1965); Effect of hypertonic urea on the distribution of water and electrolytes in normal and edematous brain tissues. Arch. neurol., In the press. H. M., DAYES, L. A., AND DOSSETOR, J. B., (1965); Cerebral swelling associated with rapid PAPPIUS, hemodialysis of uremic dogs. J. Neuroputh. exp. Neurol., 24, 147. K. A. C., (1956a); Water distribution in incubated slices of brain and PAPPIUS, H. M., AND ELLIOTT, other tissues. Canad. J. Biochem. Physiol., 34, 1007-1022. PAPPIUS, H. M., AND ELLIOTT,K. A. C., (1956b); Factors affectingthe potassium content of incubated brain slices. Canud. J. Biochem. Physiol., 34, 1053-1067. PAPPIUS, H. M., AND GULATI,D. R., (1963); Water and electrolyte content of cerebral tissues in experimentally induced edema. Actu neuropath., 2, 451460. I., AND ELLIOIT,K. A. C.; (1962); Further studies on swelling of brain PAPPIUS, H. M., KLATZO, slices. Canud. J. Biochem. Physiol., 40, 885-898. PARSONS, D. S., AND VANROSSUM, G. D. V.,(1962a); On the determination of the extracellular water compartment in swollen slices of rat liver. Biochim. biophys. Actu, 57,495-508. G. D. V., (1962b); Observations on the size of fluid compartments PARSONS, D. S., AND VANROSSUM, of rat liver slices in vitro. J. Physiol., 164, 116-126. RAIYONDI,A. J., EVANS, J. P., AND MULLEN, S., (1962); Studies of cerebral edema. 111. Alterations in the white matter: An electron-microscopic study using ferritin as a labeling compound. Actu neuroputh., 2, 177-197. D. M., (1962); Effect of hypertonic urea on cerebral spinal fluid REED,D. J., AND WOODBURY, pressure and brain volume. J. Physiol., 164, 252-264. J. R., (1960); Metabolism of intracellular water. Physiol. Rev., 40, 112-149. ROBINSON, E. A., AND PEASE,D. C., (1957); Electron microscopy of neurons and SCHULTZ, R. L., MAYNARD, neuroglia of cerebral cortex and corpus callosum. Amer. J. Anal., 100, 369-407. A. M., (1939); A biochemical study of cerebral tissue and of the change in STEWART-WALLACE, cerebral edema. Bruin, 62, 426-438. VARON,S., AND MCILWAIN, H., (1961); Fluid content and compartments in isolated cerebral tissues. J. Neurochem., 8, 262-275.

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The Movement of Electrolytes and Albumin in Different Types of Cerebral Edema LOUIS B A K A Y The Division of Neurosurgery, State University of New York at Buffalo Medical School and the Neurosurgical Research Laboratory, Buffalo General Hospital, Buffalo, N . Y . (U.S.A.)

The study of the morphological and chemical changes of central nervous tissue rendered edematous by various conditions has a far reaching significance. Although an abnormal condition, cerebral edema is useful in casting light on some of the more puzzling aspects of the water and electrolyte household of the central nervous system. It serves as an experimental model where increased hydration of the tissue and changes in its chemical milieu can be studied hand in hand with the morphological changes, in vivo and without irreversible damage to the tissue or interference with its blood supply. Experimental edema is more suitable for this purpose than human pathological edema for many reasons; most importantly, experimental edema can be easily calibrated and controlled acd the brain can be removed for investigation after death without delay. Furthermore, the dynamic equilibrium of various constituents between circulating blood and edematous brain tissue can be studied with labeled compounds to an extent that would be impossible in humans. Observations on cerebral edema were an important aspect of the pioneering experiments of De Robertis and Gerschenfeld (1961) and Gerschenfeld et al. (1959) who called attention to the morphological absence of an extracellular space in the brain. Swelling of the brain was intracellular and was associated with an increase in volume of the astrocytes. From this observation and from other findings, they concluded that the astroglia serves as the main supplier of metabolites, at least of water and electrolytes, to the nerve cells. The importance of this theory is far reaching because it involves basic issues of the biology of the central nervous system. If proven correct in every detail, it would dispense entirely with the cerebral extracellular space, it would prove cerebral metabolism to be a completely cell-to-cell process and would delegate the function of the blood-brain barrier to the membranes of individual cells. Cerebral edema, being most instrumental in the establishment of this new theory, is not adequately known yet to serve this purpose without reservation. A great gap exists between the advanced knowledge of brain swelling by electron microscopy and the inadequacy of our understanding of its biochemistry. In his recent review of the cerebrospinal fluid, Davson (1963) pointed out that a great deal of careful experimentation on brain edema is required. Chemical and morphological changes will References p . 181-183

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have to be correlated and different types of edemas will have to be kept separate in discussing the results. This review aims to summarize briefly our present knowledge on the dynamic exchange of substances between plasma and edematous brain tissue under various experimental circumstances, with special regard to the author’s own experience. The term ‘cerebral edema’ was used although by definition edema is an increase of extracellular fluid, a term that is not applicable to brain tissue in its pure form. The lack of participation on the brain’s part in generalized edema of the body is wellknown both clinically and experimentally. Cerebral edema in this review connotes the increase of brain volume due to generalized or localized overhydration of the tissue. The results of experiments obtained in water intoxication, intravenous water infusions, etc. were not included because in the author’s experience they increase the water content of the brain only slightly and very transiently and without any definite change in its microscopic appearance. Objections raised against the validity of such ‘inflation edema’ can be found in the work of Elliott and Jasper (1949) and Gerschenfeld et al. (1959). I Cold induced edema

Clasen et al. (1953) demonstrated that local freezing over the surface of one cerebral hemisphere is followed by widespread edema; their technique was developed further by Klatzo et al. (1958). Bakay and Haque (1964) studied the exchange of various substances between plasma and cold induced edema in cats. The following description summarizes their findings. Cortical freezing was produced by the application of solid CO2 to a circumscribed area of the dura covered cortex, protected by an additional layer of plastic to avoid direct trauma to the brain. At the 24-h stage following cold application, a sharply circumscribed area of the cortex is hyperemic. This area corresponds to the area of freezing and has all the outward appearance of a hemorrhagic infarct. It involves only the upper layers of the cortex although on rare occasions it reaches as far as the subcortical white matter. The most striking edema is seen in the subcortical white matter which shows the spongy structure typical for edema (Fig. 1). The vascularity of the edematous white matter is not increased. There is moderate proliferation of glia cells within the distinctively edematous area (Fig. 2). The astrocytes are considerably enlarged as compared with those of normal white matter. In addition to their hypertrophy, the astrocytes in edematous tissue show some degenerative changes, such as irregular swelling of their processes. It was found that the edema is quite widespread and involves much of the affected hemisphere including regions that are quite remote from the original point of cold application (Fig. 3). The cortical gray matter was not seriously affected, except for those gyri that were frozen. The increased volume of the edematous hemisphere was, therefore, almost entirely caused by swelling of the white matter. The average water content of the normal cortex was 80.6 % and that of the white matter 72.4 %. The amount of water averaged 78.1 % in the edematous white matter. This represents a significant increase of 5.7% over the normal value.

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Fig. 1. Spongy appearance of white matter in cold induced edema. Loyez stain, x 400.

The increase in water content corresponds well with the increase in volume in edematous cat brain given by White et al. (1942). Vital staining. Trypan blue was injected intravenously at various intervals following freezing. There was intense blue coloration of the marginal zone of the cortex surrounding the necrosis. The staining of the edematous white matter was directly related to the time elapsed from the injection of trypan blue to the time of death. Although the white matter was enlarged and obviously edematous, it was not colored 1 h after trypan blue administration. Six hours later, the vital staining extended somewhat from the zone around the necrotic cortex into the adjacent white matter. However, even at this stage there was only a faint streak of blue extending toward the centrum semiovale along the fiber tracts and sparing the arcuate fibers (Fig. 4). At 24 h, the entire white matter involved in edema was intensely blue with no sparing of any portion. Radioactive serum albumin. There was a marked increase in radioactive albumin in both lesion and edema, edematous white matter being consistently more radioactive than the necrotic lesion of the cortex itself. The RISA content of edematous brain and lesion closely followed that of plasma. Radioautography revealed a striking change that occurred with the lapse of time from the injection (Fig. 5). One hour after injection, RISA concentrated only in that portion of the edematous white matter that was in closest vicinity to the lesion, although edema already involved much of the white matter of the ipsilateral hemisphere. None the less, radioactivity References p . 181-183

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Fig. 2. Proliferation of astrocytes in edematous white matter, 24 h after cold injury. Cajal's gold chloride stain, x 180.

of the remote areas of edema was only slightly higher than normal. The spread of RISA in the edematous brain was well seen at the 6- and 24-h stage, respectively. The similarity between these radioautographs and the pictures showing vital staining with trypan blue is very great indeed. Microscopic examination revealed that RISA under normal conditions did not penetrate the brain tissue to any significant degree, neither the cortex nor the white matter (Fig. 6 ) . However, the granules representing radioactive albumin could be seen in edematous white matter although never in such concentration that would equal or even approximate that of the plasma (Fig. 7). The distribution in tissue was very even without any predilection to any particular cell or intercellular space of any size. Electrolytes. Sodium and potassium values obtained in the severely traumatized frozen part of the cortex were practically identical with that of plasma (Na between 130 and 150 mM/kg of wet weight, K between 2 and 8 mM/kg). This is not surprising considering the appearance of the lesion which is one of a hemorrhagic infarct with little of the original tissue remaining. There was a considerable variation in the electrolyte content of various portions of the edematous white matter. However, the sodium concentration showed definite correlation to the distance from the lesion in form of a concentration gradient. The highest concentration, only slightly less than that of plasma, was found in the edematous tissue next to the frozen cortex. The

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Fig. 3. Coronal section of cat brain 24 h after cortical freezing. Arrow indicates cortical lesion. Swelling is limited to the white matter of the hemisphere, Rasmussen stain, x 4.

Fig. 4. Extension of trypan blue staining from the cortical lesion into the edematous white matter, 6 h after intravenous administration of the dye. References p . 181-183

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Fig. 5. Radioautographs of coronal sections of brains, 1 h (a), 6 h (b), and 24 h (c), after intravenous injection of lSII-labeledserum albumin. Arrows indicate site of freezing that was performed 24 to 48 h prior to death. (From Bakay and Haque, 1964).

sodium decreased in more remote edema to 70-80 mM/kg. It started to approach the values for normal brain tissue (45-50 mM/kg) in the peripheries of edema. The difference in sodium content between normal cortex and white matter is due to the different water content. It disappears when sodium is calculated to water content

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Fig. 6. Microscopic radioautograph of normal white matter 1 h after intravenous injection of 'SlI-labeled serum albumin. Significantconcentration of albumin is limited to the blood vessel, X 800. (From Bakay and Haque, 1964).

Fig. 7. Microscopic radioautograph of edematous white matter 1 h after intravenous injection of 131I-labe!ed serum albumin. Even distribution of albumin in the edematous tissue, x 1OOO. (From Bakay and Haque, 1964). References p . 181-183

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Fig. 8. Radioautographs of coronal sections of brains 24 h after cold injury (arrow) and 1 h (a) and 4 h (b), respectively, after intravenous injection of 24Na.The radioactivity in the center of the sections represents the choroid plexuses. (From Bakay and Haque, 1964). TABLE I SPECIFIC ACTIVITY O F 2 4 N A A N D

42K IN

B R A I N TISSUE

Values are expressed in counts/g of wet tissue/min per mM of sodium and potassium/kg of tissue. Potassium

Sodium

Plasma Cerebrospinal fluid Cortical lesion Edematous white matter next to lesion intermediate zone far from lesion Cortex, normal White matter, normal

f h

4h

24 h

l h

4h

48 19 46

35 31 32

27 27 22

1000 17 1200

510 640

320 130 400

38 35 38 21 8

30 28 37 26 16

21 25 30 34 30

120 74

160 98 42 44 18

100 71 50 47 23

25 30 12

133

24 h

rather than wet weight. Similar correlation between sodium and water was observed in other parts of the normal central nervous system (Aprison et al., 1960). The increased sodium of edematous white matter, on the other hand, exceeded any increment that could be calculated from overhydration. Potassium remained uniformly

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distributed throughout the edematous white matter. Its concentration, when related to wet weight, was about one-half of normal brain potassium. This is undoubtedly caused by the increase of the water and plasma content of the tissue. Studies with isotopes. The frozen cortical lesion was more radioactive than normal brain tissue and the z4Na and 42K content of the lesion more or less paralleled that of the blood plasma. The decline in radioactivity of the lesion indicates a fast exchange of sodium and potassium with that in the plasma and contrasts with the slow exchange between plasma and normal brain tissue which manifests itself in a slowly increasing radioactivity of the latter. The 24Na,42K and 32Pcontent of edematous white matter followed a course that was halfway between that of the frank lesion and normal brain. However, just as in experiments with RISA, radioautographs and individual tissue analysis revealed a wide variety of radioactive sodium, potassium and phosphate concentration within the edematous white matter itself. During the first hour after injection, truly high isotope concentration could be found only in that portion of the edema that was immediately adjacent to the cortical infarct (Fig. 8). With the lapse of time, the wave of increased isotope concentration, be that 24Na,42Kor 32P, reached deeper and deeper portions of the edematous tissue. Table I shows the result of 24Na and 42Kexperiments expressed in terms of specific activity. Equilibrium of 24Na between plasma and the cortical lesion is practically instantaneous, followed by the equilibrium between plasma and edematous white matter. Although sodium in the edematous brain does not equilibrate with plasma immediately, its exchange with the sodium pool of the blood is reasonably fast and includes all parts of the edematous white matter. Potassium behaves quite differently. The exchange of potassium between blood and cortical infarct is very rapid just as it was for sodium. This is obviously due to the fact that the cold induced infarct is saturated with plasma and is much closer chemically to plasma than to normal cerebral cortex. The specific activity for potassium in the edematous white matter shows a gradual decrease with distance from the lesion. The discrepancy between specific activity in various parts of the edema does not change significantly with the lapse of time and equilibrium with 42K of plasma is not reached in 24 h. Discussion. These experiments show that various substances have a definite port of entry to thekdematous white matter. This place is the marginal zone to the cortical injury itself. The spread is gradual and rather slow not only for larger molecules, such as vital dye particles and labeled albumin, but also for 32P and 4%. At the same time the stage of the edema was the same in every experiment and there was no morphological difference in the edematous white matter and in its vasculature between those portions that were adjacent to the cortical infarction and those which are far remote from it. The findings with trypan blue are very similar to those of Klatzo et al. (1958) who used Na fluoresceinas indicator. They also noticed a delay in the staining of edematous white matter although the lesion itself stained promptly. Clasen and his associates (1962) raised the question whether this vital staining is not a part of the protein exudation that takes part in edematous brain because of the known protein binding of acid dyes. This view was corroborated by the latest experiments of Klatzo et al. References p. 181-183

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(1962) who described vital staining of edematous white matter by fluorescein-labeled serum proteins. It seems that these extravasated proteins are taken up by the glia cells through pinocytosis. The temporal and spatial aspects of the exchange of trypan blue and RISA between plasma and edematous brain tissue are identical in the experiments of Bakay and Haque (1964). This speaks strongly in favor of all visible dye being protein bound. The primary source of this vital staining is the necrotic lesion itself rather than the cooling of the entire hemisphere. Lourie et al. (1960) pointed out that the blood-brain barrier for trypan blue does not become affected in general hypothermia unless the temperature of the brain drops below 9", a temperature that is considerably lower than that of the white matter after freezing of the cortex (Klatzo et al., 1958), because of the low thermal conductivity of brain tissue (Rowbotham et al., 1959). The data of Bakay and Haque (1964) shed some light on the mechanism of vital staining of edematous brain at least as far as trypan blue is concerned. In their original paper, Klatzo et al. (1958) pointed out that there is no evidence of gross capillary changes in cold induced edema. The astrocytes, on the other hand, did reveal marked pathological alterations. It was, therefore, logical to assume that the astrocytic membranes might determine the permeability of the blood-brain barrier. However, in that case the increase in barrier permeability for vital dyes should occur simultaneously in the entire edematous white matter characterized by abnormal astroglia. This is obviously not the case because the experiments of Bakay and Haque (1964) show that vital staining advances from its focal point, the point of vascular and general tissue injury into the adjacent edematous white matter through a gradual and slow process. The electrophoretic studies of Klatzo et al. (1958) performed at the time when freezing edema reached its maximum intensity, indicated an appreciable rise in total proteins with a striking (17 fold) increase in the albumin fraction of edematous white matter. Similar results were obtained by Hauser et al. (1961) in cerebral edema produced by the implantation of psyllium seeds. The greatest concentration of albumin was found nearest to the psyllium mass ; it decreased progressively with increasing distance from the lesion. Kaps (1954) reported marked increase in albumin content in the edematous white matter of man in the vicinity of tumor, with occasional increase in p-globulins. There is a considerable difference between various types of cerebral edemas with special regard to the protein content of the edema fluid. Kiyota (1959) described two types of edema in man. The 'simple' type was found mostly in non-neoplastic brains. The soluble proteins revealed a normal electrophoretic pattern. Edema of the 'complicated' type was frequently limited to the white matter in the vicinity of tumors or subdural hematomas. Here, there was an increase of albumin or tx-globulins. The former was explained by the migration of serum albumin into the tissue and the latter by denaturation of tissue proteins. The increase of albumin in the edematous brain is not surprising since it represents the smallest and fastest migrating component of proteins. Leduc (1955) studied the passage of albumins and globulins of various molecular weights from plasma into the choroid plexuses and area postrema of normal animals. Although these regions take up substances from the blood relatively freely, not being protected by the blood-brain barrier, globulin

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concentration in them reached its peak only 8 to 24 h after administration as compared with 10 min for the smallest sized albumin. The findings of Bakay and Haque (1964) with l311-labeled serum albumin are in good agreement with the changes in albumin content observed by other techniques. It is easy to trace RISA because it does not penetrate the blood-brain barrier under normal circumstances. Normally, it is pooled in the lumen of capillaries to such an extent that it can be used to measure the regional blood content of brain (Barlow et al., 1958; Everett and Simmons, 1958). Lee and Olszewski (1959) made observations similar to those of Bakay and Haque (1964) in heat-induced edima. RISA exuded from the injured blood vessels, particularly from the vicinity of the necrotic cortex and involved the adjacent edematous white matter. In their opinion, the extravasated radioactive serum spread along the nerve fibers and reached remote areas gradually rather than by an increase in capillary permeability of the entire edematous brain. The results of Bakay and Haque (1964) seem to prove this point quite clearly. Recently, Klatzo et al. (1962) studied the movement of fluorescein-labeled protein under the microscope in cold induced edema. They came to the conclusion that this substance extravasates from the injured blood vessels and diffuses throughout the extracellular space of the edematous white matter because the form of their distribution under the microscope did not appear to be spatially related to any cellular structures. Nevertheless, the distribution of serum proteins in an extracellular compartment of the brain remains a somewhat problematic matter. Penetration of proteins in swollen brain tissue slices in vitro occurred only in the injured marginal regions of the tissue (Pappius et al., 1962). Cumings (1961) suggested on basis of his enzyme studies that both water and albumin were extracellular in edematous brain. When cerebral edema takes place around a tumor or abscess, albumin exudes through the capillary walls, which might be damaged, into the extracellular space. Here, according to Cumings (1961), some of the albumin might be absorbed onto the astrocytes with resulting damage to them or their enzyme systems thereby causing rupture of some of these cells. The relative distribution of electrolytes in the cells and in the interstitial fluid of the central nervous system is not known. This question is not settled even under normal circumstances let alone such pathological changes as edema. Apart from the controversy raging about the presence or absence of a cerebral extracellular space, exact determination of the intracellular electrolyte concentration might not solve the problem either, because it is altogether possible that nerve cells and glia cells are quite differently composed. The electrical properties of neurons suggest that these cells contain high concentration of potassium and are surrounded by a medium containing mostly sodium. A certain amount of interchange has been demonstrated since cerebral function is accompanied by a shift of electrolytes in and out of the nerve cells. The electrical impedance of the cerebral cortex is quite low. Swelling of the neurons results in a shift of electrolytes (mostly sodium chloride) into the cells, accompanied by water transfer. This increases the electrical impedance markedly. From their experiments, Van Harreveld and SchadC (1960) concluded that a good deal of electrolytes must either be in a relatively large extracellular space or in glial cells with easily permeable References p . 181-183

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membranes. In fact, Katzman (1961) advanced the theory that glia are high sodium cells. It is interesting in this respect that experimental neuronal degeneration of the lateral geniculate body in the cat with the ensuing preponderance of glia cells results in an increase of sodium and chloride while potassium remains unchanged (Koch et al., 1962). The tendency to identify glial plasma with the extracellular space of the brain is shared by a number of electron microscopists. However, there is still no definite proof that an intercellular space, albeit small, does not exist. Even the permeability of glia cells is not boundless. Recently, Ranck (1963) analyzed the cortical impedance in rabbit. He came to the conclusion that only one-third of the volume of neuroglia could be considered as an effective interstitial space. It is known that in normal brain and in normal white matter in particular, the clearance rate of tissue for a given electrolyte, that '{to say, the ratelof disappearance of an isotope injected directly into the brain substance is generally slower than the clearance rate of the same isotope would be-when injected, for instance, into 3 muscle. This has been observed even for rapidly migrating ions, such as sodium (Boatman et al., 1950; Lawrence and Hellman, 1954; and Knapp et al., 1955). The rate of disappearance from tissue has been generally attributed to the degree of blood'circulation, but in view of the possibility of a high concentration of electrolytes in brain cells, this explanation might not be sufficient. It is, therefore, of particular interest to recall the investigations of Austin et al. (1956) who found that not only was 24Na present in a higher than normal concentration in cerebral edema, but also that the clearance rate of this isotope from edematous white matter was significantly decreased. This observation indicates that sodium (and for this matter water) is bound to a certain degree in edematous white matter. The protein and electrolyte concentration in cold induced edema suggests that the edema fluid closely resembles plasma. Its high albumin content would classify it as 'edema exudate' because it exceeds the limit set for interstitial fluid and commonly seen in transudates. Its source is leakage of fluid from injured capillaries. The raised protein content of the tissue fluid is indicative of increased capillary permeability. The isotopic investigations show that this originates from the area rendered necrotic by direct freezing. Additional escape of water and sodium might occur in areas of edema more remote from the primary lesion, but such secondary factors are probably of not much significance. When related in terms of nomenclature used in other parts of the body, this is an inflammatory edema brought up by a physical trauma. Several factors in addition to the rupture of minute blood vessels can be held responsible for the ensuing edema, such as the effect of chemical substances liberated by the inflammatory reaction (histamine, leucotaxine), as well as the increased hydrostatic pressure in the engorged capillaries. It is hard to say at the present time whether this gradual penetration into the uniformly edematous white matter is the sequence of diffusion in a relatively small extracellular space or active cell transport. At any event, it shows that direct tissue and vascular damage with a breakdown in the blood-brain barrier plays an extremely important role in this phenomenon and that the small blood vessels of the edematous tissue itself are not, or at least not particularly, permeable when compared with

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normal white matter. The lack of uniform appearance in the edematous white matter of labeled albumin and electrolytes following cold induced edema is almost certainly identical or at least similar to the situation that exists around another type of focal lesion such as trauma, infarct, tumor or abscess. They all share in common a well localized breakdown in vascular continuity or permeability which acts as a port of entry for substances into the surrounding edema. This was mentioned already by Stewart-Wallace (1939) who found that in human brain edema the increase in water, sodium, chloride and potassium is in the same relative proportion as would be the case if the added fluid were a serum filtrate. This suggested that there is an increase in interstitial fluid, derived from the circulating blood. Finally, the question should be raised whether the extracellular space of the white matter is identical with that of the cortex or not. Most of the electron microscopic studies that indicate a very small extracellular space were limited to observations in the cortex. The predisposition of edema in the white matter could be explained by the assumption that the extracellular space is very small or absent in gray matter, but present in a larger form in the white matter. The original electron microscopic pictures of Torack et al. (1959) in brain swelling secondary to cold injury suggest that much of the excess fluid is intracellular, but recent reports by Raimondi et al. (1962) and by Gonatas et al. (1963) indicate that the extracellular compartment of the white matter does enlarge in traumatic and inflammatory edemas. Further investigations in different types of edemas are needed before this question is settled. Inflammatory edema Inflammatory edema can be produced in animals by intracerebral injection of a dry mixture of PPD (purified protein derivative) of tubercle bacilli and graphite. When implanted into the brain tissue, this pellet forms an inflammatory reaction within 10 to 12 h that is noted almost exclusively in the white matter of the corresponding hemisphere. Under the light microscope, this reaction consists of an exudate of neutrophil leucocytes. The white matter shows a spongy appearance indicative of edema. Observations by Levine et al. (1963) showed that the exudate had a remarkable selectivity for white matter. They believe that the neutrophil exudate is superimposed on what otherwise might be simple edema. This process is probably due to the chemotactic properties of PPD. It is possible that the structure of the white matter supplies special means for the spread of substances, a special mean that is not present in gray matter. The affinity of cerebral edema for white matter was explained by them through this hypothesis, although they were not able to identify the supposed mediator or to clarify the pathway of propagation. In subsequent experiments, Gonatas et al. (1963) studied the ultrastructure of this inflammatory edema in rat brain. They found that the edema fluid was mostly extracellular. The widening of the interstitial space occurred only in the white matter. The normal, small extracellular space of the neuropil of cortex and basal ganglia was not enlarged. They concluded that edema of this type is based on enlargement of the extracellular space in the white matter without destruction of tissue. In addition, tbey References p. 161-183

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found the astrocytes showing signs of pinocytosis suggestive of fluid flow from the extracellular space into the cell and phagocytosis of myelin by macrophages. The edematous white matter stained vitally with Geigy blue. The breakdown of the blood-brain barrier to this vital dye occurred locally in the gray matter, but spread extensively throughout the white matter. Recently, Katzman et al. (1963b) found that the 24Na uptake was altered only to a small extent by the edema as compared to [35S]04 whose uptake was markedly increased. In acute experiments, the inorganic sulfate space was found to be as high as 18 % in blue staining white matter, 12% in blue staining gray matter and 7 % for the rest of the brain as compared to a normal value of 2-4 %. The increased space in the white matter corresponded well with the true extracellular space as seen under the electron microscope. However, such explanation cannot be applied to the gray matter. Edema caused by cerebral hypoxia Clinical experience as well as experimental work showed that under certain circumstances hypoxia or anoxia is followed by brain swelling. Anoxia in man and experimental animals is followed by an increase in water content of the brain (Richardson et al., 1959; Gunn el al., 1962; Hills and Spector, 1963). The changes in brain volume and the alteration of blood and CSF content of the intracranial cavity was well described by White et al. (1942). The use of different vital dyes by Broman (1949), Grontoft (1954), as well as Becker and Quadbeck (1952) seems to indicate that the blood-brain barrier becomes permeable for vital dyes only when the anoxia is severe and the vital dye applied is an easily penetrating one such as astraviolet or fluorescein. Some of these experiments involve the production of asphyxia rather than controlled hypoxia and consequently the state of edema and the permeability of various substances from plasma into edematous tissue were hard to evaluate. Agonal or postmortem changes cannot be ruled out and the various factors involved in asphyxia are usually impossible to separate. Shmidt (1960) reported that asphyxia increased the cerebral uptake of 32P. Asphyxia combined with considerable COz accumulation produced a stronger effect than asphyxia with a lesser degree of hypercapnia. The passage of 32P from blood to brain was enhanced in animals rendered hypoxic in simulated high altitude chamber when the atmosphere contained only 7 4 % of oxygen (Smirnov and Chetverikov, 1953). Factors other than hypoxic edema itself might be responsible for some of the transgression of substances from blood to brain. The inhalation of COZalone, when severe enough, might increase the cerebral concentration of various compounds. Goldberg et al. (1961) showed that exposure to 25 % of COZincreased the cerebral concentration of phenobarbital, salicylic acid, acetazolamide and urea in cats. To separate the various factors involved in asphyxia, Bakay and Bendixen (1963) performed experiments in cats by separating the various physiological components. The conditions studied were hypoxia with COa retention, hypoxia with normal COZ elimination, hypercapnia with normal oxygenation, and increased venous pressure.

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Fig. 9. Pressure recordings at the onset of severe hypoxia with hypercapnia. (From Bakay and Bendixen, 1963, p. 66).

Fig. 10. Recording of normal initial pressures (left) and pressures obtained after 30 min of hypoxia with normal C02 elimination (right). (From Bakay and Bendixen, 1963, p. 66).

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Fig. 11 A. For legend see next page.

Significant cerebral edema could be produced only by a combination of hypoxia and hypercapnia (Fig. 9). When the arterial pH dropped below 7.10 and the oxygen saturation became lower than 50 %, the CSF pressure increased to 350-400 rnm HzO. On removal, brains were congested and edematous. Even by gross examination, much of the change seemed to have been limited to the gray matter, both nuclear and cortical. Edema did not occur with pure hypoxia (Fig. 10) or following inhalation of 10% of C02. Increased venous pressure by abdominal tourniquets caused some venous engorgement of the brain and considerable, although temporary, increase in the

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Fig. 11. Autoradiographs of coronal sections of the cerebrum, cerebellum (lower left and center) and midbrain (lower right), 60 min after intravenous injection of szP. A = pure hypoxia; B = hypercapnic hypoxia. (From Bakay and Bendixen, 1963, p. 73-74).

CSF pressure. Within the range of the hypoxia applied in these experiments, no pathological changes developed in the brains except for edema. Microscopic examination showed no evidence of capillary rupture, petechial bleedings, or any other gross change that would have interfered with the interpretation of the findings. The blood-brain barrier remained impermeable for trypan blue. The combination of severe hypoxia and COz accumulation increased the cerebral uptake of sodium as well as of phosphate; neither hypoxia nor hypercapnia alone produced a similar References p . 181-183

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effect. The increase in 24Na and 32P concentration involved the cortical and nuclear gray matter more than the white matter, and this pathological increase was clearly correlated to the degree of cerebral edema (Fig. 11). It was most pronounced in the basal ganglia, but it was also noticed in the deeper layers of the cerebral and cerebellar cortex and in the midbrain. RISA given intravenously did not penetrate the brain tissue when increased venous pressure was applied alone or COZ inhalation was given. RISA did not penetrate the capillary wall in hypoxia with normal COz elimination, nor did it in hypoxia with hypercapnia until the arterial pH fell below 6.75 and the oxygen saturation became less than 20 to 25 %. Above these values the only excess RISA found in the brains was stored in the dilated and engorged vessels. This seemingly increased uptake of albumin was also seen in the experiments with increased venous pressure and in the vasodilation caused by CO:! inhalation; although in all of these conditions the tracer could be easily removed from the brain tissue by vascular perfusion. However, in severe hypercapnic hypoxia that led to pronounced cerebral edema, with the oxygen saturation and the arterial pH below the levelsjust mentioned, true transgressioh of albumin through the capillary wall into the edematous brain tissue occurred. The radioactive albumin under such circumstances could not be removed from the brain by vascular perfusion. It deposited almost exclusively in the gray matter where the brain swelling was also most pronounced. This deposition of radioactive albumin in the gray matter, when studied by radioautography, represents a striking contrast with the deposition of the same protein studied under identical circumstances in traumatic edema, when it is almost exclusively seen in the white matter (Fig. 12). Perfusion experiments and calculations on the vascular isotope

Fig. 12. Autoradiographic demonstration of the deposition of l3lI-labeled albumin in the white matter in cold induced edema (a) and in the gray matter in edema following severe hypercapnic hypoxia (b).

concentration showed that the increase in 24Na, 32P and RISA in hypoxic edema was caused by a true absorption of these tracers by the nervous tissue rather than by a mere increase in cerebral blood content. These observations are in good correlation with the microscopic findings. Severe histological changes in anoxia are limited to the gray matter. Hills and Spector (1963) found neuronal degeneration in the cerebral cortex and hippocampus of the hemisphere rendered experimentally anoxic, with nerve cell loss especially in the hippo-

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campus. Proliferation of microglia and astroglia was also seen. Anoxia was a cardinal factor in the development of these cellular changes because degeneration and loss of nerve cells did not follow cerebral edema produced by water administration alone. TriethyI tin produced edema Profound edema of the white matter can be produced in experimental animals by administration of triethyl tin compounds. Intraperitoneal injection of a massive dose is followed by acute edema. However, for experimental purposes chronic experiments are to be preferred by adding triethyl tin hydroxide or sulfide to the animal's diet and drinking water. Neurological signs develop within 1 or 2 weeks, depending upon the concentration of triethyl tin in food. The symptoms usually start with paralysis of the hind legs which is then followed by diarrhea, apathy, protrusion of the eyeballs, etc. The advantage of this type of experimental edema is that it is easy to reproduce and standardize. In some ways, triethyl tin produced edema can be considered as the prototype of toxic edema in human pathology. However, considerable differences exist, both clinically and pathologically between various types of toxic edemas. Lead encephalopathy, perhaps the best known of toxic edemas in man, seems to be quite

Fig. 13A. For legend see next page. References p . 181-183

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different from the experimental edemas produced by alkyl tin or alkyl lead compounds. Although the white matter is severely involved in lead encephalopathy, the cortex is not spared. The vascular changes and the protein-rich exudate in the edema (Popoff et al., 1963) show that in lead encephalopathy we are dealing with an entirely different type of toxic edema. Strangely enough, the clinical signs and the pathological findings vary considerably between poisonings by different organic tin or lead compounds. Cremer (1962) fmnd that although both triethyl tin and triethyl lead acted similarly in vitro by inhibiting the oxygen consumption of brain slices, the clinical symptoms and the degree of cerebral edema was quite different following administration of the two compounds which again differed from the tetraethyl compounds (Vardanis and Quastel, 1961). One of the most unusual features of alkyl tin produced edema is the prompt reversibility of clinical symptoms. Unless in a terminal stage, animals survive and recover from the neurological signs within a week or two after feeding with triethyl tin ceases, although considerable edema of the myelinated structures can still be seen after recovery. On gross examination, the brains of rats rendered edematous are swollen and pale. The microscopic changes are generally localized to the white matter with remarkable sparing of both cortical and nuclear gray. The white matter is enlarged and spongy in

Fig. 13. Edematous white matter of the fornix (A) ofrat in chronictriethyl tin poisoning as compared with normal fornix (B). Loyez stain, x 160.

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Fig. 14A.

Fig. 14. Cellular changes in the lateral geniculate body of rat in triethyl tin poisoning (A), compared with normal tissue (B). Rasmussen stain, X 600. References p. 181-183

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appearance (Fig. 13). There is a remarkable lack of cellular reaction in the edematous white matter even in chronic experiments. Although edema is limited essentially to the white matter, recent experiments by Bakay (1964) showed that some parts of the gray substance are not entirely normal. Distinct changes of vacuolar degeneration in the cytoplasm of some nerve cells were present also, particularly in the basal ganglia. The lateral portions of the thalamus and the nearby geniculate body showed unusually huge vacuoles conspicuously distorting the shape of the nerve cells (Fig. 14). In addition, there was a distinct loss of the small nerve cells in the striatum. It appears that these changes are probably caused by increased fluid within the myelinated fibers and that this process is extending within the substance of the 5th and 6th cortical layers and within the gray nuclei of the brain stem and basal ganglia along the medial borders of the internal capsule. Most impressive was the lack of glial and mesenchymal reaction about the severely involved nerve cells. These findings are similar to those described by Kalsbeck and Cumings (1963) in rat and cat brains. The astrocytes were slightly, if at all, increased in number, but many appeared swollen. Swollen oligodendroglia were numerous in a few sections. They found that nerve cells, as a whole, were unaffected. The exact distribution of water in the edematous white matter is still somewhat a matter of speculation. Originally, this water was thought to be located between the myelin fibers (Magee et al., 1957). However, Kalsbeck and Cumings (1963) suspect that many of the vacuolated spaces are within the myelin sheath. The electron microscopic observations of Aleu et al. (1963) indicate that there might be a ‘split’ in the myelin lamellas which leads to the formation of large, clear spaces between the layers of myelin. This split occurs in the interperiod line, and the space thus created is analogous to the extracellular space seen in this location in the developing animal. As far as the relationship of glia cells and edema fluid is concerned, Torack et al. (1960) revealed severe swelling of the clear glial cell under the electron microscope without an increase in prominence of the minute intercellular space. None the less, there is slight uncertainty as far as the location of fluid is concerned because of the large number of ruptured glial cell membranes that attest to the tremendous amount of edema. Such ruptured cells are unusual in other types of edema. However, Torack et al. (1960) points out that even in the immediate vicinity of these broken membranes there was no significant dissection of fluid into the intercellular space. Adjacent cell membranes maintained their normal spacing. The water content of the brains increases in triethyl tin poisoning. In the experiments of Magee et al. (1957) the increase was in order of 80% water versus 78% normal. The values were 78.5 versus 74% in the experiments of Bakay (1964). However, it has to be pointed out that the true increase in the water content of the white matter is probably greater than these figures suggest. It is almost impossible to perform correct determinations of the water content in the white matter of such small animals as the rat. A higher degree of hydration was recently reported in triethyl tin poisoned rabbits by Katzman et al. (1963a). Mageeetal. (1957) showed a marked increase of sodium in the edematous brain without change in the potassium

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level when calculated to dry weight of tissue. However, the sodium content of the white matter is significantly elevated even in terms of wet weight (Katzman et al., 196313; Bakay, 1964). Triethyl tin intoxication produces an edema that is unique with its relation to the blood-brain barrier. Seemingly, in edema of this type permeability of the barrier is very little affected except for increase of the water content in the edematous tissue which, of course, is not under the control of the barrier. The edematous white matter does not stain vitally with trypan blue (Magee et al., 1957; Torack et al., 1960; Bakay, 1964). There was never any histological evidence of abnormal protein accumulation and the recent investigations by Katzman et al. (1963b) and Bakay (1964) showed that RISA does not penetrate this edematous tissue from the blood stream. No change in the permeability of the blood-brain barrier for amino acids could be demonstrated. Bakay (1964) was able to demonstrate on microscopic radioautography a perfectly normal distribution of 14C-labeled glycine in the edematous white matter after its injection into the blood stream. Few data are available on the concentrations of labeled electrolytes in alkyl tin produced edema. Magee et al. (1957) studied the distribution of 32P.Although the specific activity of the total acid soluble phosphate of the brain in the treated animals was greater than in the controls, its ratio to the specific activity of the inorganic phosphate of the plasma was lower than in the controls. The difference in the ratios are thought to be mainly due to the higher specific activity of the plasma inorganic phosphate after tin poisoning, and, therefore, the results on the permeability of the blood-brain barrier are difficult to interpret. On the whole, Magee et al. (1957) thought that this alkyl tin compound does not greatly alter the permeability of the barrier for 32P. Bakay (1964) confirmed this finding to the extent that gross difference in 32P uptake by the brain of poisoned and normal rats could not be demonstrated. Katzman et al. (1963b) found that 24Nauptake was depressed in the edematous white matter, while, on the other hand, it remained normal in the cortex. In the edematous brain 43 h was required for half the brain sodium to exchange as compared to 23 h in controls. A similar lag in 24Naexchange between plasma and edematous white matter was found by Bakay (1964) in triethyl tin poisoned rats. The slowness of 24Nauptake was most marked within the first hours after injection of the isotope. The difference in the 24Na uptake between normal and edematous white matter became manifest only when expressed in terms of specific activity. Without correction for the increased sodium content of the edematous tissue, the radioactivity in the normal and affected white matter in terms of wet weight was about equal. Due to this fact, no difference could be demonstrated by radioautography. Streicher (1962) found that cerebral edema produced by triethyl tin is not accompanied by a change in the extracellular space as measured by the volume of distribution of the thiocyanate ion. However, Kalsbeck and Cumings (1963) believe that the fluid accumulation is not largely intracellular. Evidence presented by these authors suggests that at least some of the fluid increase is extracellular. Kalsbeck and Cumings (1963) found some serum albumin by electrophoresis in the edematous brain. From the presence of serum albumin and alkaline phosphatase from the blood, without an increase in intracellular enzymes, they concluded that the fluid increase must be References p. 181-183

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extracellular. Recently, Katzman et al. (1963a) determined the inorganic sulfate space with 35Sin triethyl tin edema. They found it to be 2.6 %, a normal value. From their chemical and electron microscopic studies, they concluded that in triethyl tin edema the excess fluid, although similar or identical with an extracellular fluid, is loculated within the intramyelinic vacuoles and is not free in communication with the fluid of the extracellular space. M E C H A N I S M OF C E R E B R A L E D E M A

The various data presented here and particularly the results of recent electron microscopic and chemical investigations demonstrate clearly that ‘cerebral edema’ is not a uniform concept, but varies dependingon its pathogenesis. Fluid accumulationbrought up by physical or thermal trauma or produced by tumors or abscesses of the brain are essentially serum exudates characterized by enlargement of the extracellular space, increased permeability of the blood-brain barrier for large molecules and protein-rich edema fluid. In contradistinction to this, the excess fluid in triethyl tin produced edema is probably a serum ultrafiltrate. The sparse cellular response and the absence of excess protein excludes an inflammatory process. Already, Magee et al. (1957) demonstrated a close agreement between the chemical composition of the tissue and the theoretical calculations in which they assumed the fluid being an ultrafiltrate of plasma. This view is further supported by the constancy of lipid and nucleic acid constituents (Magee et al., 1957), and by the electrolyte andIprotein concentration (Gonatas et al., 1963; Kalsbeck and Cumings, 1963; Katzman et al., 1963b). In addition, the permeability of the blood-brain barrier seems to be largely unaffected in triethyl tin edema and the cerebral extracellular space, at least in terms of thiocyanate and inorganic sulfate space remains normal. However, the molecular mechanism by which triethyl tin and possibly other chemical compounds produce edema remains unknown. An inhibition of the sodium pump by enzyme action leading to reduced efflux of sodium and accumulation of edema fluid was considered by Katzman et al. (1963b) but could not be proven. These recent informations carried us a long way toward the understanding of cerebral edema, but some of the characteristics of this condition still defies interpretation. Among others, it is still unexplained why the great majority of different types of edema are limited to the white matter. Scheinker (1947) thought that the selective vulnerability of the white matter is due to its characteristic pattern:of blood supply. He thought that the relative paucity of capillaries of the white matter and their peculiar arrangement and size is much more prone to develop congestion, stasis, and retarded circulation than the richly supplied and arborized vascular network of the gray substance. He thought the absence of extensive anastomoses in the white matter would be particularly vulnerable. In his view then, cerebral edema would be secondary to local or generalized circulatory changes. However, the same discrepancy in vascularity between the white and the gray matter could be used to arrive at a completely different explanation. The possibility of edema being primarily a vascular lesion is made unlikely by the fact that the gray matter is more vascular than the white. It is justly pointed out that in most edemas, except for those that are secondary to gross

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traumatic changes within the white matter itself, the capillaries are well preserved and show no gross structural changes. It can also be pointed out that in the edema that is unmistakably associated with circulatory changes, namely in edema that follows hypercapnic anoxia, the pathological alterations are more or less restricted to the gray matter. The experiments of Bakay and Bendixen (1963) show that edema in hypoxia is almost entirelylimited to the cortical and nuclear gray matter while on the other hand the white matter is remarkably spared. Widespread edema of the white matter with sparing of the cortex was also found following ultrasonic radiation of the brain (Bakay et al., 1956),under circumstances that strongly indicate that molecular changes of the brain tissue were an important factor in the damage. Assuming then that the selectivity of the white matter for edema is not due to vascular factors primarily, we are left with two alternate explanations. One would be that the white matter is more ‘edema-prone’ for structural-molecular reasons. The parallel course of the myelinated fibers might be more conducive to fluid accumulation and propagation between them. The particular structure of myelin itself with its cation binding capacity might retain electrolytes and water under abnormal circumstances to a greater extent than gray matter. The other explanation, albeit an entirely theoretical one at the present time, might implicate a difference in the extracellular space. It could be assumed that there is a sizable extracellular space in the white matter which in turn would be more prone to fluid accumulation than the gray matter which would have no extracellular space or only a very small one. It is possible that this extracellular space is a functional rather than anatomical space and includes some glia cells as well as the space between adjacent cells. This does not mean that the anatomical disruption of capillaries in trauma or the increased vascular permeability of neoplastic blood vessels is not the primary factor in exudative edema. They trigger off a chain of events by introducing serum into the brain tissue. However, the secondary edematous involvement of the white matter cannot be explained by widespread increase of capillary permeability of the entire region. SUMMARY

Investigations on cerebral edema in the past hardly extended beyond the determination of brain volume and water content and the reduction of increased pressure in clinical medicine. Valuable as these data are, they do not cast light on the pathophysiological factors that produce edema. The dynamic exchange of various constituents between plasma and edematous brain tissue is as yet poorly understood. Yet this aspect of the blood-brain barrier is of great importance not only for the basic understanding of cerebral edema but also because of the present controversy about the role of glia in the water and electrolytes support of nerve cells. It has been suggested that in a physiological sense glial tissue partly or completely constitutes the cerebral extracellular space. An important contributing factor to this assumption was the behavior of glia cells in cerebral edema. Electron microscopic investigations seem to show that experimental and clinical edema is caused by a swelling of the glial cells rather than by the accumulation of intercellular fluid. References p. 181-183

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The experiments presented by the author were performed to furnish data on the exchange of labeled proteins and electrolytes between circulating blood and edematous brain tissue as well as to compare the behavior of brain tissue rendered edematous by different means. Various types of cerebral edemas were produced experimentally. The tissues were examined uniformly by conventional histological methods and by analyzing their water and electrolyte content. The dynamic exchange of constituents between blood and brain tissue was studied with trypan blue, radioactive iodinated serum albumin, 14C-labeled amino acids, 24Na, 42K and 32P.The temporal and spatial aspects of the exchange of labeled substances were determined and correlated to the chemical composition of the tissue including its regional alterations. The experimentalprototype of focal and traumatic edema was produced by freezing the dura protected cortex. The resulting swelling was essentially localized to the white matter and was accompanied by hypertrophic changes of the astrocytes. The experiments reveal that this swelling is caused by a protein-rich serum exudate that originates in the cortical lesion. The process starts by an increased permeability of the involved cortical capillaries and continues by a gradual spread of the exudate into the underlying white matter. Microscopic radioautography revealed even distribution of albumin without any predilection to any particular cell. There is no evidence of primarily increased permeability in the edematous white matter itself. Experimental edema was also produced by triethyl tin poisoning. This was considered as a prototype of toxic edema. The swelling was limited to the white matter, although pronounced edematous changes in adjacent nerve cells were observed, particularly in the lower layers of the cortex and in the basal ganglia. Glial reaction was noticeably absent in this edema. The exchange of some of the electrolytes was increased in the edematous white matter in proportion to the increase in water content. However, there was no increase in the permeability of the blood-brain barrier for proteins or amino acids. Anoxic type of cerebral edema was produced by hypoxia with hypercapnia since hypoxia with normal C02 elimination does not produce cerebral swelling. Hypercapnic hypoxia below a certain level of pH and oxygen saturation in the arterial blood results in brain swelling. This, however, is localized almost entirely to the gray matter, both cortical and nuclear. The resulting increase in exchange of constituents is limited to water and electrolytes until the arterial oxygen saturation and pH drop below a certain level. At that point, the capillaries become permeable for albumin. As a conclusion, it can be stated that cerebral edema is not a uniform reaction of brain tissue. The role of glia in its development is still not clearly defined. The increase of proteins, and to a certain extent of electrolytes, in edematous brain tissue is the direct result of injury severe enough to be responsible for gross capillary damage. ACKNOWLEDGEMENT

This work was supported by Research Grant NB 03754-02 from the National Institute of Neurological Diseases and Blindness.

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REFERENCES R., AND TERRY, R. D., (1963); Fine structure and electrolyte analyses of cerebral ALEU,F., KATZMAN, edema induced by alkyl tin intoxication. J. Neuropath. exp. Neurol., 22, 4 0 3 4 1 3 . APRISON, M. H., LUKENBILL, A., AND SEGAR, W. E., (1960); Sodium, potassium, chloride and water content of six discrete parts of the mammalian brain. J. Neurochem., 5, 150-155. AUSTIN,0. M., CORSON, R., LINDER,J., CHAMBERLAIN, R., AND GRANT,F. C., (1956); Cerebral edema: Studies on its location and mode of action with radioactive sodium. Arch. Neurol. Psychiat., 75, 4 4 7 4 8 . BAKAY, L., (1964); Morphological and chemical studies in cerebral edema. 11. Triethyl tin induced edema. J. Neurol. Sci., To be published. BAKAY, L., AND BENDIXEN, H. H., (1963); Central nervous system vulnerability in hypoxic states: Isotope uptake studies. Selective Vulnerability of the Central Nervous System in Hypoxaemia. J. P. Schadb and W. H. McMenemey, Editors. Oxford, Blackwell Scientific Publications (pp. 63-78). BAKAY, L., AND UL HAQUE, I., (1964); Morphological and chemical studies in cerebral edema. I. Cold induced edema. J . Neuropath. exp. Neurol. 23, 393418, BAKAY,L., HUETER,T. F., BALLANTINE, H. T., AND SOSA,D., (1956); Ultrasonically produced changes in the blood-brain barrier. Arch. Neurol. Psychiat., 76, 457467. BARLOW, C. F., SCHOOLAR, J. C., AND ROTH,L. J., (1958); An autoradiographic demonstration of the relative vascularity of the central nervous system of the cat with iodine 131-labeled serum albumin. J. Neuropath. exp. Neurol., 17, 191-198. BECKER, H., AND QUADBECK, G., (1952); Untersuchungen iiber Funktionsstorungen der BlutHirnschranke bei Sauerstoffmangelund Kohlenoxydvergiftungmit dem neuen Schrankenindikator Astraviolett FF. Z. Naturforsch., 7b, 498-500. BOATMAN, J. B., KENDRICK, T. R., FRANKE, F. R., AND MOSES,C., (1950); The use of radioactive iodine, radioactive phosphorus and radioactive sodium in the determination of cerebral and muscle clearance. J . Lab. cfin. Med., 36, 456459. BROMAN, T., (1949) ; The Permeability of Cerebrospinal Vessels in Normal and Pathological Conditions. Copenhagen, Einar Munksgaard (pp. 33-35). CLASEN, R. A., BROWN, D. V. L., LEAVITT, S., AND HASS,G. M., (1953); The production by liquid nitrogen of acute closed cerebral lesions. Surg. Gynec. Obstet., 96,605-616. CLASEN, R. A., COOKE, P. M., PANDOLFI, S., BOYD,D., AND RAIMONDI, A. J., (1962); Experimental cerebral edema produced by focal freezing. I. An anatomic study utilizing vital dye techniques. J. Neuropath. exp. Neurol., 21, 579-596. CREMER, J. E., (1962); The action of triethyl tin, triethyl lead, ethyl mercury and other inhibitors on the metabolism of brain and kidney slices in vitro using substrates labeled with 14C. J. Neurochem., 9,289-298. CUMINGS, J. N., (1961); Soluble cerebral proteins in normal and edematous brain. J. clin. Path., 14,289-294. DAVSON, H., (1963); The cerebrospinal fluid. Ergebn. Physiol., 52, 20-73. DE ROBERTIS, E. D. P., AND GERSCHENFELD, H. M., (1961); Submicroscopicmorphology and function of glia cells. Int. Rev. Neurobiol., 3, 1-65. ELLIOTT, K. A. C., AND JASPER, H., (1949); Measurement of experimentally induced brain swelling and shrinkage. Amer. J. Physiol., 157, 122-129. EVERETT,N. E., AND SIMMONS, B., (1958); Measurement and radioautographic localization of albumin in rat tissues after intravenous administration. Circular. Res., 6, 307-3 14. GERSCHENFELD, H. M., WALD,F., ZADUNAISKY, J. A., AND DE ROBERTIS, E. D. P., (1959); Function of astroglia in the water-ion metabolism of the central nervous system. An electron microscope study. Neurology, 9, 412425. GOLDBERG, M. A., BARLOW, C. F., AND ROTH,L. J., (1961); The effect of carbon dioxide on the entry and accumulation of drugs in the central nervous system. J . Pharmacol. exp. Ther., 131, 308-3 18. GONATAS, N. K., ZIMMERMAN, H. M., AND LEVINE, S., (1963); Ultrastructure of inflammation with edema in the rat brain. Amer. J. Path., 42, 455469. ORONTOFT, O., (1954); Intracranial Hemorrhage and Blood-Brain Barrier Problems in the Newborn. Copenhagen, Einar Munksgaard (pp. 62-74). GUNN,C. G., WILLIAMS, G. R., AND PARKER, I. T., (1962); Edema of the brain following circulatory arrest. J . surg. Res., 2, 141-143.

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HAUSER, H. M., SVIEN,H. J., GOLDSTEIN, N. P., AND MCKENZIE, B. F., (1961); A study of cerebral protein and polysaccharide in the dog. 111. Albumin changes in experimental cerebral edema. Excerpta med., 36, 83-84. Transactions of the Second International Congress of Neurological Surgery, Washington. R. G., (1963); Anoxia and cerebral water content in the adult rat. Nature, H~LLS, C. P., AND SPECTOR, 199, 393. -BECK, J. E., AND CUMINGS,J. N., (1963); Experimental edema in the rat and cat brains. J. Neuropath. exp. Neurol., 22,237-247. QPS, G., (1954); uber electrophoretische Untersuchungen an Hirngewebe, insbesondere aus der Umgebung von Tumoren, zugleichein Beitrag zur Pathogenese von Hirnschwellungund Hirnodem. Arch. Psychiat. Nervenkr., 192, 115-129. KATZMAN, R., (1961); Electrolyte distribution in mammalian central nervous tissue. Neurology, 11, 27-36. KATZMAN, R., ALEU,F., AND WILSON,C., (1963a); Further observations on triethyl tin edema. Arch. Neurol., 9, 178-187. KATZMAN, R., GONATAS, N. K., AND LEVINE,S., (1963b); Na24 and S 3 5 0 4uptake in PPD induced inflammatory edema. Paper read on the Fifteenth Ann. Meet. Amer. Acad. Neurol., Minneapolis. KIYOTA, K., (1959); Electrophoretic protein fractions and the hydrophilic property of brain tissue. 11. The proteins of brains with edema. J . Neurochem., 4,209-216. KLATZO, I., MIQUEL,J., AND OTENASEK, R., (1962); The application of fluorescein labeled serum proteins (FLSP) to the study of vascular permeability in the brain. Acta neuropath., 2, 144-160. KLATZO, I., PrrCAux, A., AND LASKOWSKI, E. J., (1958); The relationship between edema, blood-brain barrier and tissue elements in a local brain injury. J. Neuropath. exp. Neurol., 2, 144-160. KNAPP,F. M., HYMAN, C., AND BERCEL,N. A., (1955); A method for the estimation of regional cerebral blood flow. Yale J. Biol. Med., 28, 363-371. KOCH,A., RANCK,J. B., JR., AND NEWMAN, B. L., (1962); Ionic content of the neuroglia. Exp. Neurol., 6, 186-200. LAWRENCE, W., JR., AND HELLMAN, L., (1954); Disappearance rates of Na24,K42,P32and 1131from cerebral cortex and muscle of dog. Surg. Forum, 593-598. LEDUC, E. H., (1955); Localization of injected proteins in structures comprising the hematoencephalic barrier in the rat. Anat. Rec., 121, 328. LEE,J. C., AND OLSZEWSKI, J., (1959); Permeability of cerebral blood vessels in healing of brain wounds. Neurology, 9, 7-14. LEVINE,S., ZIMMERMAN, H. M., WENK,E. J., AND GONATAS, N. K., (1963); Experimental leukoencephalopathies due to implantation of foreign substances. Amer. J. Path., 42, 97-1 17. Lourn, H., WEINSTEIN, W. J., AND O’LEARY, J. L., (1960);Effect of hypothermiaupon vital staining of the brain. A study of the blood-brain barrier. J. nerv. ment. Dis., 130, 1-5. MAGEE, P. N., STONER, H. B., AND BARNES, J. M., (1957); The experimental production of oedema of the central nervous system of the rat by triethyl tin compounds. J.Path. Bact., 73, 107-124. PAPPIUS, H. M., KLATZO, I., AND ELLIOIT,K. A. C., (1962); Further studies on swelling of brain slices. Canad. J. Biochem. Physiol., 40, 885-898. POPOFF, N., WEINBERG, S., AND FEIGIN, I., (1963); Pathologic observations in lead encephalopathy, with special reference to the vascular changes. Neurology, 13, 101-1 12. ~ O N D I A. , J., EVANS, J. P., AND MULLAN, S., (1962); Studies of cerebral edema. 111. Alterations in the white matter. An electron microscopic study using ferritin as a labeling compound. Acta neuropath., 2, 177-197. RANCK, J. B., (1963); Analysis of specificimpedanceof rabbit cerebralcortex. Exp. Neurol., 7,153-174. RICHARDSON, J. C., CHAMBERS, R. A., AND HEYWOOD, P. M., (1959); Encephalopathies of anoxia and hypoglycemia. Arch. Neurol., 1, 178-190. ROWBOTHAM, G. F., HAIGH,A. L., AND LESLIE,W. G., (1959); Cooling cannula for use in the treatment of cerebral neoplasms. Lancet, i, 12-14. SCHEINKER, M., (1947); Cerebral swelling. Histopathology, classification and clinical significance of brain edema. J. Neurosurg., 4, 255-274. SHMIDT,Z. M., (1960); Mechanism of change of permeability of biological barriers with lowering of atmospheric pressure. Dokl. Akad. Nauk SSSR, Otd. Biol., 132, 1216-1218. SMIRNOV, A. A., AND CHETVERIKOV, D. A., (1953); Phosphorus exchange in the brain under hypoxia with the aid of radioactive phosphorus. Dokl. Akad. Nauk SSSR, Otd. Biol., 90, 843-845. STEWART-WALLACE, A. M., (1939); A biochemical study of cerebral tissue and of the changes in cerebral edema. Brain, 62,426438.

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STREICHER, E., (1962); The thiocyanate space of rat brain in experimental cerebral edema. J. Neuropath. exp. Neurol., 21, 4 3 7 4 1 . TORACK, R. M., TERRY,R. D., AND ZIMMERMAN, H. M., (1959); The fine structure of cerebral fluid accumulation. I. Swelling secondary to cold injury. Amer. J . Path., 35, 1135-1147. TORACK, R. M., TERRY,R. D., AND ZIMMERMAN, H. M., (1960); The fine structure of cerebral fluid accumulation. 11. Swelling produced by triethyl tin poisoning and its comparison with that in the human brain. Amer. J. Path., 36, 273-287. VANHARREVELD, A., AND SCHADB,J. P., (1960); On the distribution and movements of water and electrolytes in the cerebral cortex. Structure and Function ofthe Cerebral Cortex. D. B. Tower and J. P. SchadC, Editors. Transactions of the Second International Meeting of Neurobiology, Amsterdam, Elsevier (pp. 253-256). VARDANIS, A., AND QUASTEL, J. H., (1961); The effects of lead and tin organometallic compounds on the metabolism of rat brain cortex slices. Canad. J. Biochem. Physiol., 39, 1811-1837. WHITE,J. C., VERLOT,M., SELVERSTONE, B., AND BEECHER, H. K., (1942); Changes in brain volume during anesthesia. The effects of anoxia and hypercapnia. Arch. Surg., 44, 1-21.

184

Changes in the Size of Astrocytes and Oligodendrocytes during Anoxia, Hypothermia and Spreading Depression* H. COLLEWIJN

AND

J. P. S C H A D E

Netherlands Central Institute for Brain Research, Amsterdam (The NetherIands)

INTRODUCTION

Various authors have demonstrated that the content of electrolytes and water in neurons may change according to the physiological conditions of the tissue. Swelling of apical dendrites of pyramidal cells during anoxia and spreading depression was observed by Van Harreveld (1957, 1958). That this change in size was accompanied by an accumulation of sodium chloride in the neuronal elements was made most probable by a number of techniques, such as impedance measurements (Van Harreveld and Ochs, 1956) and the histochemical demonstration of a chloride shift (Van Harreveld and Schadt, 1959a, b). In a later study it was shown that this chloride uptake was accompanied by a decrease in concentration of potassium ions (Collewijn and SchadC, 1962; Collewijn, 1963), which may be caused by a loss of potassium or dilution by water uptake. Such experiments have given ample evidence that under various conditions considerable transfer of electrolytes and water occurs between the neuronal and extraneuronal compartment in the central nervous system. These changes are most evident during anoxia caused by circulatory arrest, that is, in the absence of circulation. It seems highly probable that in this condition the water and electrolyte transport is completely intracortical. Since the advent of the electron microscope a controversy has arisen as to the definition, size and contents of the extraneuronal space. Previous evidence, based on electrical and chemical measurements, did not indicate that the extracellular space of nervous tissue would be fundamentally different from that in other organs. Electronmicroscopists, however, were unable to find a substantial non-cellular compartment in the brain. Upon these findings several hypotheses have been founded, some of them claiming that in the central nervous system glial cells fulfil the role of the extracellular space in other tissues. Specifically, they might be the source of the water taken up by the neurons in asphyxia and other conditions.

1 * This research was supported in part by grants from the National Institute of Neurological Diseases and Blindness (NB-3048) and the National Institute of Mental Health (MH-6825).

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The experiments which we present here, part of which have been communicated before (Collewijn, 1963), were directly inspired by this controversy. Recent improvements in fixation techniques for electron microscopy (Van Harreveld and Crowell, 1964) have brought new evidence (Van Harreveld et al., 1965) suggesting that the paucity of extracellular space is really a fixation artifact. For a more extensive discussion of this subject we refer to a monograph by Van Harreveld (1965). Though these recent findings strongly favor a more conservative image of the organization of the brain, quantitative analysis of volume changes of neuroglial elements in different experimental conditions remains of interest for an understanding of the contribution of glia to the functional state of the brain. MATERIAL A N D METHODS

Experiments were performed on adult rabbits (Alaska F 1 bastards) weighing about 2.5 kg. They were anesthetized with nembutal(30 mg/kg intravenously) and paralyzed with succinylcholine. Artificial respiration was applied. The skull was opened widely and the right and left cortex was exposed by removing the dura mater. The skin edges of the wound exposing the brain were sewn to a steel ring, forming a cup. At the desired moment, this cup was filled with isopentane, cooled to its fusion point (- 160") in liquid nitrogen. In this way the cortex is frozen instantaneously, so that the existing distribution of water and ions is preserved. The cortex was frozen in the following conditions: (a) during normal oxygenation and normal ECoG at a body temperature of 37" (control series); (b) after cooling to 28"; (c) after cooling to 20"; (d) 10 min after cardiac arrest at 37"; (e) during the progression of a spreading depression of the electrocorticogram. For conservation of the existing water distribution during fixation, freeze substitution at a temperature below the eutectic point of sodium chloride solutions (-22") was considered essential. After freezing of the cortex in situ with cold isopentane the head was severed from the body, rewarmed in isopentane to -50", and kept in absolute ethanol at -30' for one week. In that period, the ice is slowly dissolved by the alcohol, which moves in from the pial surface and penetrates gradually into the deeper layers of the cortex. After one week the alcohol front has penetrated to a depth of about 1.5 nun. At the moment that the tissue is reached by the alcohol it is fixed, while the pre-existing water and ion distribution is still present. We attempted to stain the glial cells in the material thus obtained by classical staining methods. Our starting points were the procedures described by Cajal (gold sublimate method) and Del Rio Hortega (silver carbonate method), modified by Penfield (Conn et al., 1960). As these methods are successful only on tissue prepared by special fixatives such as formol ammonium bromide (FAB), we tried to adapt them for alcohol fixation. The essential feature of fixation for glial stainings may be a lowering of the isoelectric point of tissue proteins to a suitable level (Lascano, 1958). This may be accomplished by fixing fresh tissue in FAB during a period of several days. With tissue that has been fixed for a long period in normal formalin, the same result is probably obtained by the method of Globus (1927), which consists References p . 195

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in treating frozen sections of old formalin fixed material successively in ammonia and hydrobromic acid. A new approach was worked out by Lascano (1958), who tried to lower the isoelectric point of tissue protein by a fixative, consisting of formalin, hydrochloric acid and glycine. On tissue fixed in this way, classical staining methods yielded excellent results. After considerable experimentation we found that, after freeze substitution with alcohol, astrocytes were made stainable by treatment first with FAB, and then by Globus' method. For the oligodendrocytes, mere treatment with Lascano's fixative gave the best results. The methods were actually carried out as follows. Astrocytes (1) A piece of cortex was excised from the freeze-substituted brain. To avoid cytoarchitectonical differences, specimens were always taken from the same area (visual cortex). (2) This tissue was treated in FAB for 5-7 days. The FAB was composed in the following way: formalin 33% (Merck) 15 ml; distilled water 85 ml; ammonium bromide 2 g; pH, 1.5. (3) Frozen sections of 15 p were cut. They were kept in ammonia 2.5 % for 24 h. (4) Sections were rinsed twice in distilled water and brought into hydrobromic acid 4 % in which they remained tor 3 h. (5) Sections were rinsed twice in distilled water, to which 0.5 vol. % of ammonia 2.5 % was added. (6) Sections were stained in the gold sublimate mixture for 4 h at a temperature of 24-29' in the dark. The gold sublimate solution was prepared as follows. Stock solution A, 1 % gold chloride (yellow) in distilled water; stock solution B, 5 % sublimate (HgC12) in distilled water. Shortly before use the following mixture was prepared : 5 ml sol. A 5 ml sol. B 40 ml distilled water. (7) The purple red sections were rinsed twice in distilled water, and fixed during 5 min in sodium thiosulphate 5 %. (8) They were rinsed three times in distilled water. (9) Dehydrating, clearing and mounting was done in the usual way.

+

+

Oligodendrocytes (1) A piece of cortex was excised as for the astrocytes and treated for 1 day in Lascano's fixative, which is composed as follows: glycine 1.05 g ; 1 N hydrochloric acid 14.8 ml; formalin 33 % (Merck) 15.0 ml; distilled water to make 100 ml; pH 1.5. (2) Frozen sections of 15 p were cut and rinsed three times in distilled water. (3) Sections were kept in sodium carbonate 5 % for 1 h. (4) Sections were stained during 5 min at room temperature in 'weak' silver carbonate solution which was freshly prepared as follows. To 5 d silver nitrate 10% was added 20 ml sodium carbonate 5 %. Concentrated ammonia was added drop by drop until the precipitate dissolved. Then several drops of the silver nitrate solution were added until the solution became slightly turbid.

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( 5 ) Sections were reduced in formalin 0. 33 % (Merck) for 1 min. (6) They were then rinsed three times in distilled water. (7) Dehydrating, clearing and mounting were done in the usual way. Measurements of glial cell bodies were made in the following way. Glial cells were observed with an oil immersion objective (100 x) which was focused on their plane of maximal cross section. With the aid of a drawing prism, the outline of the cell body was drawn on paper. Only those cells were considered of which the cell body was lying completely within the section. The surface area of the drawn figures was determined with a planimeter. The values obtained in this way were expressed in arbitrary units. After determination of the total enlargement of the system these values were recalculated in p3. Five animals were studied for each experimental condition. For every animal 100 cells were drawn. In this way, 500 cells were measured in each of the conditions studied. To prevent any bias, the slides were coded before observation. The effect of spreading depression was analyzed as follows: 14 mm long sagittal strips were cut from the cortex, containing the whole piece of cortex, in which the process of a spreading depression occurred, and from the heterolateral control cortex. The slides were divided into 7 areas with a length of 2 mm. In each of these areas 50 astrocytes and 50 oligodendrocytes were measured. R E S U L TS

The staining as described for the astrocytes demonstrates these cells clearly. They are stained dark blue or black, whereas nerve cells and background stain red and pink. The pyramidal cells with their apical dendrites are usually visible, and are distinguished from the glial cells by their characteristic shape. The glial cells, however, have not exactly the same appearance as in normally stained tissue, the branches being poorly stained. In most instances only the cell body and the origin of the branches are demonstrated. If a branch is visible, it can often be followed up to a blood vessel or nerve cell. The protoplasm showed in general a granular appearance. Usually the cells are seen throughout the whole depth of the cortex; many of them occur in connection with the pial membrane. In the deeper layers of the cortex they are often stained in larger numbers. The same type of cells is seen in the subcortical white matter; here they are neither present in large numbers, nor arranged in long rows, which latter situation is thought to be characteristic for oligodendrocytes. Although the cells occur often in connection with nerve cells, they have not the distribution of satellite cells (oligodendrocytes). Differentiation between the several kinds of glial cells by glial staining methods is not very specific. Although the original gold sublimate method is regarded as rather specific for astrocytes, it could not be ruled out that by our modification oligodendrocytes are also stained. The spatial distribution of the cells stained, however, was more characteristic for astroglia. As in the cortex mainly protoplasmic astrocytes occur (Glees, 1955) we suppose that the greater part of the cells stained in our sections belongs to this category. Referonces p . I95

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The correctness of this identification was confirmed by the results of the method for oligodendroglia. The cells stained by this technique have a quite different appearance. Their shape is more spherical, in general they are much smaller, and they have the typical spatial distribution of oligodendrocytes: they are grouped as satellites around the neuronal cell bodies. The branches, also here, are only occasionallv stained. In the white matter the stained cells are numerous, and arranged in long rows, E I n view of these results we feel justified in identifying the stained cells as astrocytes and oligodendrocytes respectively. The fact that the branches are so poorly visible may be ascribed to the freezing technique used. As a result of this, ice crystals are always formed, especially in the deeper layers of the cortex. These crystals,'the size of which is about 1 p, give the background a foamlike appearance, which will probably interfere with the visibility of fine structures such as glial fibers. Changes in size of glial cells

r i ( a ) Anoxia at 37". The appearance of astrocytes in sections, taken from a well oxygenated cortex at 37" is illustrated in Fig. 1, A. In Fig. 1, B the same cells are

Fig. 1. Microphotographs of rabbit cortices, stained by the modified method for astroglial cells described in the text. A section of cortex was frozen (A) at 37" while the circulation was intact; @) 10 min after circulatory arrest at 37"; (C) at 28" while the circulation was intact.

shown in a cortex frozen 10 min after cardiac arrest at 37". The decrease in size of the cell bodies is evident. Apart from this, the cells have the same appearance as in the control cortex. The difference in size is so obvious that on microscopical inspection slides from anoxic cortex are easily distinguished from those of normal cortex. The results of size measurements are shown in Fig. 2 and Table I. The difference between the histograms of well oxygenated and anoxic cortex is evident. The mean cross section of an astrocyte cell body in series Control 37' amounts to 226 arbitrary units (S.E. f 5). In series Anoxia 37" this value amounts to 78 (S.E. f 3). The difference is significant (P < 0.001).

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Artroglio YMHAExp. A37'

-E x p S 3 7 '

Fig. 2. Histograms of cross sections of 2 x 500 astrocyte cell bodies. Normal and anoxic cerebral cortex at 31".

Volumes can be calculated from cross sections if the shape of the cell body is known. In fact, this shape is very different in individual cells. For an approximation the volume was considered spherical. The calculation then gives a value of 358 p3 in series Control 37" and a value of 83 p3 in series Anoxia 37". The latter value is only 23% of the former. For the oligodendrocytes,the results are presented in Fig. 3. The mean cross section of an oligodendrocyte in control slides was 100 arbitrary units (S.E. f 2). In anoxic cortex a value of 123 (S.E. f 2) was found. Consideringthe cells as sphere, the volumes were calculated as 105 p3 in normal cortex, and 140 p3 in anoxic cortex. Thus in the TABLE I SURFACE AREA A N D VOLUME OF NEUROGLIAL CELLS

Astrocytes

Oligodendrocytes

References p. I95

Experiment

Surface area (arbitrary units f S.E.)

(p3)

Control 37" Control 28" Control 20" Anoxia 37" Control 37" Control 20" Anoxia 37"

226 kt 5 ) 245 (4= 6) 162 Ct 4) 78 (=t3) 100 (+ 2) 115 (f2) 123 (rt 2)

358 410 228 83 105 124 140

VOI.

VOl. (relative)

100 115 64 23

100 118 133

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latter state they are swollen by 33 %. The difference is significant (P < 0.001). Summarizing these results, we may state that 10 min after cardiac arrest astrocyte cell bodies have shrunk to 2 3 % of their normal volume, while on the other hand oligodendrocytes have swollen to 133 % of their original size.

Oligodendrog!!a

Fig. 3. Histograms of cross sections of 2 x 500 oligodendrocyte cell bodies. Normal and anoxic cerebral cortex at 37".

(b) Hypothermia. In Fig. 4 the sizes of astrocytes at 37" and 28", both during normal oxygenation, are compared. A size difference is not very obvious from this graph. By calculation, however, we found a mean cross section of 245 arbitrary units (S.E. & 6) at 28 ",which is larger than at 37 ".The probability of exceeding this difference (P) was, however, only 0.02. The computed mean volume of the astrocyte cell body at 28 is 410 p3. This is 1 15 of the normal volume at 37 '. At 20°, on the other hand, astrocytes are of a smaller size than at 37", as shown in Fig. 5. Their cross section amounts at 20" to 162 arbitrary units (S.E. f 4). This difference is highly significant (P < 0.001). Expressed as mean volume, the size is 228 p3, or 64 % of the normal value at 37 '. O

Artroglia

-Exp. c 37'

Fig. 4. Histograms of cross sections of 2 x 500 astrocyte cell bodies. Normal cortex at 37" and 28".

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Surface area (arbitrary units)

Fig. 5. Histograms of cross sections of 2 x 500 astrocyte cell bodies. Normal cortex at 37" and 20"

Measurements of the oligodendrocytes were only made at 20" (Fig. 6 ) . They proved to be swollen in comparison with their size at 37". Their cross section amounted to 115 arbitrary units (S.E. f 2). The difference is significant (P< 0.001) and corresponds to a volume increase of 18 %.

7

Oligodendroglia

Surface area farbitmry units)

Fig. 6. Histograms of cross sections of 2 x 500 oligodendrocyte cell bodies. Normal cortex at 37" and 20".

( c ) Spreading depression. In Fig. 7 cross sections are presented of both types of glial cells in a cortex, frozen during the progression of a spreading depression. Each point in the graph represents 50 cells. The spreading depression was elicited by application of a crystal of glutamic acid on the left frontal cortex (point 0 at the abscissa of Fig. 7). Eight mm caudally from this point (arrow in Fig. 7) electrodes were placed for ECoG registration and impedance measurements. At the moment that the ECoG in the left cortex was severely depressed, and the impedance had increased by 11 %, the cortex was frozen. The right cortex, in which the ECoG was normal, was used as control. It follows from Fig. 7 that in the part of the cortex in which the spreading depression was situated or had just passed (the rostra1 part of the cortex) the astroReferences p . 195

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20 1 "

4 Add

I

o

SCHADB

2

i

k

8

io

i2

bmm

Fig. 7. Changes in the size of glial cell bodies during a spreading depression, elicited by application of glutamic acid rostrally on the left cortex. Surface areas of astrocyte and oligodendrocyte cell bodies in left (experimental) and right (control) cortex are presented (ordinate), as measured at various distances in caudal direction from the point of origin (zero) of the spreading depression. The arrow indicates the cortical zone where the depression was maximal, as judged from ECoG extinction and impedance rise. (A) Astroglia: A = control side, A = exp. side. (B) Oligodendroglia: 0 = con= exp. side. trol side,

cytes are considerably smaller than in the symmetrical part of the control cortex. Caudally in the cortex, where the spreading depression had not yet arrived, there is no size difference. The shrinkage of the astrocytes during spreading depression can be estimated to be about half as great as during anoxia. Exact calculations are omitted in view of the small number of cells measured in the zone of maximal depression. For the oligodendrocytes no clear difference in size is obvious from the graph. Apparently a change in size of oligodendrocytes during spreading depression is either absent or too small to be detected in this experiment. DISCUSSION

The absence of space in the central nervous system claimed by electron microscopists has led to many speculations about the role that glia might play as a functional extracellular space. Koch et al. (1962) and Katzmann (1961) found support for a high chloride content of glial tissue in their chemical analysis of parts of nervous tissue with a high glia content. Glial swelling in cerebral edema was found by Gerschenfeld et al. (1959), Luse (1959), and Luse and Harris (1960), although these authors differ in opinion about the identification of the swollen cells as astro- or oligodendroglia. Hild and Tasaki (1962) found a low electrical membrane resistance in glial cells cultured in vitro which might explain the relatively high conductivity of the brain in vivo, in the absence of an extracellular space. In view of the recent findings of Van Harreveld et al., mentioned in the introduction it would seem that one should be cautious in assuming such revolutionary properties

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for glial cells. Van Harreveld et al., using rapid freezing on a silver disc and substitution at low temperatures, find an extracellular space of about 23 vol. % in electronmicrographs of the molecular layer of the cerebellum. Such a value would be in agreement with chemical and electrical estimates made in the past. Fixation in the usual way does not prevent the uptake of the extracellular fluid into the cells, owing to Donnan forces. As the extracellular fluid contains virtually no protein, in asphyxia extracellular fluid will move into the cells. To reach a Donnan equilibrium, a considerable hydrostatic pressure in the cells would be required. As the cell membranes do not possess the mechanical strength to sustain such a pressure, cell swelling will occur, a Donnan equilibrium will never be reached, and therefore no space will be left. Cell swelling can be expected in any tissue after the arrest of active membrane transport (see Tosteson, 1964), though in the brain this shift is very rapid. Results of Kuffler and collaborators on the leech central nervous system too are at variance with an extracellular space function of glia. The electrical resistance of the glial cell membrane as determined by Kuffler and Potter (1964) is considerably higher than that found by Hild and Tasaki. Nicholls and Kuffler (1964) elegantly demonstrated that substances such as sodium ions, sucrose and choline reach the neurons by diffusion through the extracellular channels, and not through the glial cytoplasm. All these recent findings indicate that at present it is not justified to regard neuronal and glial volumes as complementary, as we did before (Collewijn, 1963). Our results seem to indicate that in general neuronal swelling is accompanied by swelling of satellite cells and shrinkage of astrocyte cell bodies. The swelling of oligodendrocytes may be interpreted as just another example of the general phenomenon of cell swelling in asphyxia. More surprising is the decrease in volume of astrocytes. Not only would the latter not participate in the general cell swelling, but they would even lose water. However, before drawing such a conclusion we have to emphasize that our staining method does not demonstrate the fibers of the astrocytes which may occupy a considerable part of the total volume of these cells. Van Harreveld (1961) observed asphyxial swelling of Bergmann fibers in the cerebellar cortex, in preparations stained with a method for chloride ions. These fibers, generally considered to be astrocytes, would thus take up water and chloride in anoxia, like the apical dendrites of pyramidal cells in cerebral cortex. This chloride uptake was not observed in the cell bodies from which the Bergmann fibers originate. The characteristic orientation of Bergmann fibers facilitates their identification. In cerebral cortex it has never been possible to recognize any asphyxial chloride uptake in glial cells or fibers. Furthermore, efforts to stain Bergmann glia in frozen cerebellar tissue with similar methods as presented here for the cerebral cortex remained unsuccessful (Collewijn, unpublished observations). Therefore, no conclusion about glial fibers in cerebral cortex is possible. The observed shrinkage of astrocyte cell bodies might be due to a change in shape of astrocytes, in the sense of a transfer of cell body material into the fibers. It might also be due to a loss of water to the extracellular space, and from there to the neurons. The eventual distribution of water and salts in the anoxic cortex will be determined by the large molecules in solution, molecules that cannot cross the membranes. The References p . 195

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extracellular space is almost devoid of such particles and will therefore disappear almost entirely (Van Harreveld, 1965). All the tissue water will then be contained by cellular elements. The proportions will be determined by the relative content of proteins and other large particles, and the relative size of these and their capability to pass through anoxic membranes. The number and relative distribution of big molecules in the anoxic tissue may also change owing to anoxic breakdown - or lack of synthesis - of many components, for example ATP. I n the normal, oxygenated cortex cell volumes are, on the contrary, regulated by active membrane transport. This implies that observations on cell volumes in asphyxiated or traditionally fixed tissue do not really inform us on any function that these cells might have in vivo in ion and water transport. Neither is it possible to elucidate the cellular localization of brain edema in this way. Swelling of apical dendrites during spreading depression has been described by Van Harreveld (1958). We found that in this condition astrocyte cell bodies were smaller, while satellite cells remained of the same size. Again, it is not possible at present to know whether this is due to a direct water transport from glia to neurons, or whether glial cells participate in spreading depression with changes in shape. The slight volume increase of astrocyte cell bodies at 28 ' body temperature indicates that in this condition cell volumes do not change very much, which is in agreement with our earlier observation that at this temperature the diameters of apical dendrites are the same as at 37" (Collewijn, 1963; Collewijn and Schadt, 1964). At 20" there is shrinkage of astrocytes and swelling of satellite cells, combined with an increased diameter of apical dendrites (Collewijn, 1963; Collewijn and Schadt, 1964). All these phenomena resemble the changes in asphyxia, but they are smaller. Possibly, active membrane transport is inhibited at this temperature. Obviously, as these temperatures are easily survived, cell swelling is not an indication of cell death. The independence of these two phenomena was demonstrated by Van Harreveld and Tachibana (1962) who demonstrated that chloride and water uptake in apical dendrites was reversible even after periods of asphyxia as long as 60 min, although all these cells will die eventually. Recently, this reversibility of swelling was also found in Bergmann glia in cerebellum (Van Harreveld and Collewijn, unpublished observations). SUMMARY

Sizes of neuroglial cells stained with a modified Cajal gold sublimate method after freeze substitution with alcohol were determined in different conditions. (a) Ten minutes after cardiac arrest, astrocyte cell bodies had shrunk to 23 % of their normal value, while on the other hand oligodendrocytes were swollen to 133% of their original size. (6) At 20" astrocytes were of a smaller size than at 37". The oligodendrocytes appeared to be swollen in comparison with their size at 37". (c) During spreading depression the shrinkage of the astrocytes was estimated to be about halt as great as during anoxia. The oligodendrocytes did not show a clear difference in size. (d) The results have been discussed in view of recent findings by Van Harreveld and coworkers of measurements of the extracellular space in the brain.

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REFERENCES COLLEWIJN, H., (1963); Ionic Movements in the Cerebral Cortex. Thesis, University of Amsterdam. H., AND SCHADB,J. P., (1962); Potassium movements in the cerebral cortex during COLLEWIJN, asphyxia. Acta morph. need.-scand., 5 , 11-20. COLLEWIJN, H., AND SCHADE,J. P., (1964); Chloride, potassium and water content of apical dendrites and their changes after circulatory arrest, at body temperatures from 37°C to 20°C. Arch. int. Physiol., 72, 194-210. CONN,H. J., DARROW,M. A., AND EMMEL, V. M., Editors, (1960); Staining Procedures, used by the Biological Stain Commission. Baltimore, The Williams and Wilkins Co. H. M., WALD,F., ZADUNAISKY, J. A., AND DE ROBERTIS, E. D., (1959); Function GERSCHENFELD, of astroglia in the water-ion metabolism of the central nervous sytem. Neurology, 9, 412425. GLEES,P., (1955); Neuroglia. Morphology and Function. Springfield, Ill., Charles C. Thomas. GLOBUS, J. H., (1927); The Cajal and Hortega glia staining methods. Arch. Neurol. Psychiat. (Chic.), 18, 263-271. HILD,W., AND TASAKI, I., (1962); Morphological and physiological properties of neurons and dial cells in tissue culture. J. Neurophysiol., 25, 277-304. KATZMANN, R., (1961); Electrolyte distribution in mammalian central nervous system. Are glia high sodium cells? Neurology, 11, 27-36. KOCH,A., RANCK JR., J. B., AND NEWMAN, B. L., (1962); Ionic content of neuroglia. Exp. Neurol., 6,186-200. KUFFLER, S. W., AND POITER,D. D., (1964); Glia in the leech central nervous system: physiological properties and neuron-glia relationship. J. Neurophysiol., 27, 290-320. LASCANO, E. F., (1958); A glycine-HC1-formalin fixative for improved staining of neuroglia. Stain Technol., 33, 9-14. LUSE,S. A., (1959); Electronmicroscopic observations of the central nervous system. Inhibition in the nervous System and Gamma-aminobutyricAcid. E. Roberts, Editor. New York, Pergamon Press (p. 29). LUSE,S. A., A ~ D -IS, B., (1960); Electronmicroscopy of the brain in experimental edema. J. Neurosurg., 17, 439-446. NICHOLLS, J. G., AND KUFFLER, S. W., (1964); Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech; ionic composition of glial cells and neurons. J. Neurophysiol., 27, 645-671. TOSTESON, D. C., (1964); Regulation of cell volume by sodium and potassium transport. The Cellular Functions of Membrane Transport. J. F. Hoffman, Editor. Englewood-Cliffs, N.J., Prentice-Hall Inc. (p. 110). VANHARREVELD, A., (1957); Changes in volume of cortical neuronal elements during asphyxiation. Amer. J. Physiol., 191, 233-242. VANHARREVELD, A., (1958); Changes in the diameter of apical dendrites during spreadingdepression. Amer. J. Physiol. 192, 457-463. VAN HARREVELD, A., (1961); Asphyxia1 changes in the cerebellar cortex. J. cell. comp. Physiol., 57, 101-110. VAN HARREVELD, A., (1965); Butterworth Scientific Publications. In the press. VAN HARREVELD, A., AND CROWELL, J., (1964); Electronmicroscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec., 149, 381-385. VANHARREVELD, A., CROWELL, J., AND MALHOTRA, S. K., (1965); A study of extracellular space in central nervous system by freeze substitution. J. cell. Biol., 25, 117-137. VANHARREVELD, A., AND OCHS,S., (1956); Cerebral impedance changes after circulatory arrest. Amer. J. Physiol., 187, 180-192. VAN HARREVELD, A., AND SCHADE,J. P., (1959a); Chloride movements in cerebral cortex after circulatory arrest and during spreading depression. J. cell. comp. Physiol., 54, 65-84. VANHARREVELD, A., AND SCHADB,J. P., (195913); On the distribution and movements of water and electrolytes in the cerebral cortex. Structure and Function of the Cerebral Cortex. D. B. Tower and J. P. Schadk, Editors. Amsterdam, Elsevier (pp. 239-256). VAN HARREVELD, A., AND TACHIBANA, S., (1962); Recovery of cerebral cortex from asphyxiation. Amer. J. Physiol., 202, 59-65.

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Osmotic Behaviour and Glial Changes in Isolated Frog Brains J. A. Z A D U N A I S K Y *, F. W A L D

AND

E. D. P. DE ROBERTIS

Department of Biophysics and Institute of Anatomy and Embryology, University of Buenos Aires, Buenos Aires (Argentlna)

Immersion of mammalian tissues in buffered isotonic solutions results in swelling due to the development of intracellular hypertonicity. This increased osmotic pressure of the cytoplasm was shown to be due to retention of salts and to the breakdown of large molecules into smaller metabolites, which causes gain of water by theIcells (Conway et al., 1955; Leaf, 1956). Mammalian brain slices are not an exception to this behaviour and their degree of swelling when immersed in appropriate solutions is greater than that of other tissues. Addition of metabolic poisons exaggerateslthis phenomenon, due probably to interference with the extrusion of sodium and accumulation of intermediate products inside the cells. In general, it is assumed that the water is accumulated in all the different types of cells of a tissue in similar amounts. In the case of mammalian brain slices, however, electron microscopy showed thatathe swelling was mainly produced in glial cells, whereas the rest of the structure, especially the neurons, did not show the watery cytoplasm or the increase in size which was observed especially in the astrocytes (Gerschenfeld et al., 1959). Previous observations showed that in the central nervous system the different cellular elements were separated only by spaces of about 200 A, suggesting that probably only a very small morphological extracellular space was present (De Robertis, 1955; Dempsey and Wislocki, 1955; Farquhar and Hartmann, 1957; Horstmann, 1957; Wyckoff and Young, 1956). The hypothesis was advanced then that glial cells could participate in the water and ion metabolism of the brain. This hypothesis was strenghtened by the fact that in hypotonic solutions, where water should move into all cells of the brain slice, only the glial cells identified as astrocytes were greatly swollen (Gerschenfeld et al., 1959). Though the functional existence of an extracellular space in the brain has been demonstrated (Davson and Spaziani, 1959;Zadunaisky and Curran, 1963) the relationships between glia and neurons as regards the movements of salts and water show peculiarities which are of physiological importance.

*

Present address : Eye Research Laboratory, University of Louisville, Ky. (U.S.A.). The authors are established investigators of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina.

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In an attempt to correlate some physiological parameters with ultrastructure, we have used several experimental approaches to this problem, which will be described under different headings.

Fig. 1. Electron micrograph showing part of an ependymoglial cell from the cerebralcortexofa freshly dissected frog brain. Microvillous projections (mv) extend from the free border to the ventricular cavity. The characteristic dense inclusion bodies (i) are seen. N = nucleus; mi = mitochondria. Arrow points to amorphous material, probably glycogen. References p . 21 71218

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Fig. 2. Part of the external portion of the cerebral cortex of a freshly dissected frog brain. The upper part shows the limiting membrane of the ependymoglial cells (glm). In the plexiform layer the size and aspect of the ependymoglial processes (gp) are seen. e = synaptic ending.

Properties of isolated frog brains in vitro

Since mammalian brain slices swell spontaneously in vitro the brain of cold-blooded animals was tested as experimental material. It was found that the spontaneous gain in water of frog brains in vitro (judged from the weight change) was small and compar-

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able to that of a thin muscle of the frog, the sartorius. When immersed in balanced Ringer’s solution, the increase in weight of frog brains was only 6 to 7 % of the initial wet weight after 2 to 5 h of immersion. The Na and K content of these brains did

Fig. 3. Plexiform layer of the cortex of a frog brain after 2 h of immersion in Ringer’s fluid at room temperature, bubbled with 5 % CO:!and 95 % 0 2 . Note the preservation of the structure, and the size and aspect of the ependymoglial processes (gp). Inside these, the dense inclusion bodies are found (i) as in the body of the ependymoglial cells. References p . 2171218

TABLE I N a A N D K CONTENT OF FROG B R A I N S Figures in brackets are numbers of brain (Zadunaisky et al., 1963)

W E I G H T C H A N G E S AND

Initial weight (mg)

Fresh controls 2-h Immersion in normal Ringer's solution 2-h Immersion in 70 mM/1 KCI-Ringer's solution 2-h Immersion in 10-4 M ouabainRinger's solution

95.5 & 3.4* (16) 94.6 f 3.2 (24) 94.1 & 3.0 (19) 94.6 & 3.4 (8)

Final weight (mg)

Weight change ( % initial wt)

101.9 & 3.4

7.7

131.3 & 3.6

40.6

127.7 & 3.2

33.6

Potassium content Initial wt (mM/kg)

81.8 f 3.1 (16) 80.7 & 3.4 (1 3) 138.0 & 8.1 (12) 37.0 & 2.9 (8)

Final wt fmMlkg)

Sodium content Initial wt (mMlkd

Final wt (mMlkg)

61.8 f 2.0 74.2 f 3.0

55.5 f 3.8

50.7 & 3.4

97.0 & 3.5

39.6 & 4.3

28.1 f 2.3

29.3 & 2.5

99.2 f 6.3

76.4 & 3.4

F

z

U

0

m

* Standard error.

Fj

OSMOTIC G L I A L C H A N G E S

201

Fig. 4. Part of the plexiform layer of the cortex of a frog brain, after 2 h of immersion in Ringer’s solution. Note the good preservation of the mitochondria (mi) and the synaptic endings (e) with the synaptic vesicles (sv). d = dendrite.

not vary significantly from the normal (Table I) and the oxygen uptake was constant during periods of up to 2 h in Ringer’s fluid (Zadunaisky and Curran, 1963). Spontaneous electrical activity of isolated frog brains can be recorded under very similar conditions (Libet and Gerard, 1939). Thus, this brain preparation offered the opReferences p. 21 71218

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portunity of making quantitative studies on the movements of ions and water, and at the same time to study the changes of the structure observed with the electron microscope (Zadunaisky et al., 1963). The normal structure of the tissue is well preserved after immersion in Ringer’s solution for 2 h. Figs. 1,2,3, and 4 show sections of the brain cortex, after immediate fixation and after 2 h in a Ringer’s solution. The submicroscopic structure of the incubated brains did not change appreciably as compared with freshly dissected tissue. In both cases the different layers observed by Cajal(l922) in the cerebral cortex could be recognized under the electron microscope. The ependymal layer consists of a continuous epithelium having a few cilia and numerous microvillous projections at the ventricular edge (Fig. 1). These cells contain numerous mitochondria, a few membranous elements of the endoplasmic reticulum and ribosomes, small Golgi membrane complexes and an amorphous material of low electron density similar to that described as glycogen by Revel et al. (1960). The variable amount of the latter determines a clearer or darker appearance of the cytoplasm. The most characteristic inclusions of these cells are ragged-shaped osmiophilic bodies, probably the lipids described by Oksche (1958) which can also be observed along the processes that penetrate the cortex towards the meninges. The glial processes emerging from the outer pole of these ependymal elements can be seen in continuity with the cell for a short length only. Because of the repeated branching and the thinness of the section, only the profile of these cut processes can be observed in a single section. In the pyramidal layer two distinct types of nerve cells, one having a much denser cytoplasm than the other, can be recognized. These dark and light neurons can be observed in the control as well as in the swollen brains. The plexiform layer occupies most of the cortex and its aspects vary with the depth. Near the pyramidal layer it contains numerous dendrites and nerve endings intermingled with fine axons and thin ependymal processes. In the outer portion the nerve components are separated by larger spaces occupied by the ependymal processes which tend to become thicker and form in the outermost layer a continuous limiting sheath (Fig. 2). All the fine dendritic and axonic branches, nerve endings and ependymoglial processes are packed together with their limiting membranes in close contact. As in the mammalian brain, intercellular clefts of only 100-200 A can be observed in between the different cellular processes. The ependymoglial processes can be differentiated in the normal and the incubated control specimens by their irregular profiles, the amorphous material with low electron density which they contain, and by the presence of the same ragged-shaped osmiophilic inclusions that were observed in the perikaryon of the ependymal cells. Many of these processes also contain mitochondria. The dendritic branches and some of the fine axons have fine tubular components, the neurotubules, of about 200 A, and the nerve endings contain the characteristic synaptic vesicles (Zadunaisky et al., 1963). The extracellular space of isolated frog brains The fraction of the tissue occupied by extracellular sodium has been determined in isolated frog brains (Zadunaisky and Curran, 1963).

203

OSMOTIC G L I A L C H A N G E S

0

I

I

2

1

I

3

I

4

Time ( h )

Fig. 5. Washout of 24Nafrom a frog brain. The brain was immersed for 45 min in a Ringer’s solution containing 24Na,and the loss of the radioisotope from the brain into isotope-free Ringer’s solution was followed during 4 h. The crosses represent the difference between the extrapolated straight line and the experimental points up to 1 h. The rapid line represents the exit of radiosodium from the extracellular space. The slower straight line given by the points indicates efflux from a compartment where obstruction to diffusion is present, most probably a cellular compartment (Zadunaisky and Curran, 1963).

The brains were immersed in a solution containing 24Na or 22Na for different periods and after reimmersion in inactive fluid, the loss or washout curve of tracer sodium was followed for 2 to 3 h. A typical result is shown in Fig. 5. The loss of radiosodium can be decomposed into two components, a fast and a slow one. Since very little volume change of the tissue takes place during the experiment, and the sodium content is apparently in a steady state, the kinetics of the sodium loss from the first and fast component and the evaluation of sodium fluxes from the slope of the second, were studied (Zadunaisky and Curran, 1963). Here we will refer mainly to the rapid component. In general the presence of this rapid phase allows the assumption that an extracellular space might be present in the system. To confirm this assumption it was necessary to show that the rate of loss of the ion involved, sodium in this case, was similar to the value of the diffusion coefficient of the ion in free solution. Secondly, it was important to verify if the solution of the diffusion equation for a similar geometrical shape as the one under consideration followed the same time course as the one obtained in the experiments with the isolated frog brain. The effective diffusion coefficient of Na in the extracellular space of frog brain, which is assimilated to a cylinder, can be obtained from the following equation (Hill, 1928): tt =

0.118 r2 DNa

where tt is the half-time of the straight portion of the rapid curve of Fig. 5 ; r is the mean radius of the frog brain, estimated by measuring the brain at 3 points and taking a mean value, and D Nis~the effective diffusion coefficient of Na. In 7 brains the value of D N a calculated from the above equation was 0.82 & 0.04 x 10-5 cm2/sec. This References p . 217/.218

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0

I

I

4

8

I

12 Tirne(rnin)

16

I

20

I

24

Fig. 6. Detailed representation of the rapid or extracellular component of 24Na washout from an isolated brain. The contribution of the slow component has been subtracted and the data replotted in terms of per cent of initial counts present in the fast or extracellular compartment. The line has been calculated from the solution of the diffusion equation (see text) and the points are those found experimentally. Note the reasonably good agreement between line and points which indicate movement of 24Na by free diffusion in water from a compartment thus characterized as extracellular (Zadunaisky and Curran, 1963).

value is equivalent to about half the diffusion coefficient of sodium in free solution (Herned and Owen, 1958) and agrees well with values found in other tissues (Harris, 1960). Thus, sodium seems to move freely in a compartment with characteristics of an extracellular space. With the value of D Nit~was then possible to solve the diffusion equation. Under our conditions, the solution of the diffusion equation has the following form (Jacobs, 1935):

where Q/Qo represents the fraction of the initial radiosodium remaining in the fast compartment at any time, and p n are the zeros of the Bessel function J (0).In Fig. 6, the solid line represents remaining radiosodium against time calculated with the radius and the DNa of an experimental frog brain, and the points are those obtained directly from the curve of washout. The line fits the experimental points relatively well, and this indicates that, as was assumed, this rapid component corresponds to the diffusion of radiosodium from an extracellular space. On the basis of a kinetic analysis and the total content of stable sodium in the brains, values for the size of this extracellular space could be computed (Zadunaisky and Curran, 1963). In Table I1 can be seen that the figure of 24.1 for the extracellular sodium of the frog brain arrived at is within the values reported in the literature for the extracellular space of mammalian brain and also for other tissues. There is apparently no question then that in vitro, the amphibian brain has a functional extracellular space, as any other tissue.

205

OSMOTIC G L I A L C H A N G E S

TABLE I1 D I S T R I B U T I O N OF S O D I U M IN ISOLATED FROG B R A I N

(Zadunaisky and Curran, 1963)

Experiment

1 2 3 4 5 6

Mean

Brain weight

(md

Total Ma content (pequiv)

73.6 69.0 73.5 78.3 61.4 71.5 71.2

3.12 3.08 3.12 3.25 3.08 3.00 3.12

Extracellular Extracellular Ma space (pequiv) ( % weight)

1.85 1.71 1.78 2.02 1.63 1.67 1.78

24.2 23.8 23.8 24-8

25.4 22.6 24.1

Cellular

Concentration of cellular Ma

(pwiv)

Ma

(pequivlg cell wafer)

1.37 1.37 1.34 1.23 1.45 1.33 1.35

25 26 24 21 32 24 25

The inulin space was also determined in frog brains. The total time of immersion in solution was short, and so, very little weight change interfered with the final calculations. The value found in 14 brains was 16.2 f 0.8% of the wet weight and the increase in weight during the experiment was 3.1% (Zadunaisky and Curran, 1963). Comparison of this value for extracellular space with the one obtained with sodium is compatible with observations in other tissues, where the radiosodium extracellular space is always greater than the one obtained with a larger molecule as inulin. Location of extracellular space. Since glial cells seem to be very permeable to water and also have a low membrane resistance (Hild and Tasaki, 1962) the possibility exists that they actually represent the extracellular space of the brain. To evaluate this hypothesis two types of experiment were done with isolated frog brains. In one case a metabolic poison, iodoacetic acid (2 mM), was added to the immersion medium while the brain was loaded with radiosodium and then the isotope loss curve was followed in inactive Ringer’s solution where iodoacetic acid was also present in the same concentration (Zadunaisky and Curran, 1963). It was assumed that if the ‘extracellular’ sodium was located inside a compartment limited by cell membranes, as the glial compartment, action of the poison should reveal some modification in the rate of loss of the radiosodium from the rapid component. As shown in Fig. 7 the time course of sodium loss is not affected by the inhibitor. Though diffusion from glial cells might not be affected by the interference of iodoacetate with the glycolytic cycle, nevertheless the result of this experiment tends to indicate that more probably sodium is lost from an actual extracellular than from a cellular pool under our conditions. Another experimental approach consisted in the measurement of the inulin space during swelling of the brains produced by penetration of KCl and water. Replacing part of the NaCl of the Ringer’s solution by KCI results in swelling of tissues in vitro (Boyle and Conway, 1941) because the potassium salt behaves as permeable while the Na salt is relatively impermeable. In experiments of this kind, the electron microscope revealed a great swelling of References p . 21 71218

206

J. A. Z A D U N A I S K Y , F. W A L D A N D E. D. P. D E R O B E R T I S 1AA x-x

D =,O ,

80 x lC5C m 2 / 5 e C

control t . D= .,o

81x 10-5crn2/sec

V "

aurn

&C

L E

10-

8-

6O

5

10 Tirne(rnin)

15

20

25

Fig. 7. The effect of iodoacetate on the fast component of 24Nawashout. The circles represent an initial washout with normal Ringer's solution and the crosses represent a subsequent washout of the ~ coefficient) were same brain in the presence of 2 mM of iodoacetate. The values of D N (diffusion computed from the equation given in the text. Note that the inhibitor does not modify appreciably the rate of loss of 24Na from the extracellular space (Zadunaisky and Curran, 1963).

Tirne(rnin1

Fig. 8. Determination of the inulin space in isolated frog brains. The control brains were immersed in 1 % inulin in normal Ringer's solution and the swollen ones in the same concentration of inulin but in a Ringer's solution where 70mM of NaCl were replaced by 70 mM of KCI. Total inulin content in the controls and in the brains swollen by penetration of KCI and water was determined after the immersion period and the inulin space expressed as per cent of the initial wet weight. As can be observed the swollen brains show a reduction in the inulin space. The points and circles represent the mean of 3 isolated frog brains.

the glial cells with almost no swelling of the neurons (Zadunaisky et al., 1963, see below for details). The inulin space was followed at different times during the swelling due to KC1 and water penetration and, as observed in Fig. 8 the values for the inulin space are actually lower in the swollen than in the normal tissues, when the spaces are referred to the initial weight of the brains. This shrinkage of the extracellular space during swelling of the cells of a tissue has been shown for the sartorius muscle of the frog (Boyle and Conway, 1941). A probable conclusion from this experiment is that inulin does not penetrate easily glial cells of the frog brain even during their swelling and that most probably this substance is distributed in intercellular spaces. In mammalian brain slices, there seems to be an increase of the inulin space during incubation (Pappius and Elliot, 1956) and probably the conditions of the tissues make the cell membranes permeable to inulin.

207

OSMOTIC GLIAL CHANGES

From the results of these two approaches it is difficult to consider the glial cells as having the properties of an extracellular space. Exchange of radiosodium by the frog brains In initial experiments with frog brains in vitro, it was found that the exchange of radiosodium proceeded slowly (Zadunaisky and Curran, 1963). This point was carefully examined nowywith brains of the South American frog Leptodactylus ocellatus (Zadunaisky and Chiarandini, 1963). The exchange of 22Na with the stable sodium of the tissues reached equilibrium between 60 and 90 min, but the fraction of exchangeable sodium after this time was only 64 % of the total sodium content of the brain in 35 cases, as shown in Fig. 9.

s

t

Exchange of sodium

I

1

Mean of 5 brains and r standard e r r o r

60

180 120 Time ( m i d

240

300

360

Fig. 9. Exchange of zzNawith stable sodium of isolated frog brains. Complete exchange for the total sodium of the brains is not obtained in 6 h of contact with radiosodium. The equilibrium reached in 1 to 2 h, is established at a level where only 64% of the total sodium content of the brains exchanged with the isotope (Zadunaisky and Chiarandini, unpublished observations).

As observed in this figure, the stabilization is reasonably good and does not indicate further exchange with the isotope after 2 h. To establish if this was due to actual impossibility of exchange of the stable sodium of the tissue, the total analytical sodium of frog brains was determined by flame photometry after immersion for different times in a modified Ringer's solution in which all the sodium had been replaced by choline. The loss of stable sodium in this condition is shown in Fig. 10. As can be observed 74% of the total sodium was lost, while the rest remained in the brains after 6 h of incubation. The stabilization of the loss was again reasonably good, so that if any more sodium was lost, its rate must have been very low. Two possibilities arise from these results. One could be that from 26 to 36% of the total sodium content of the brains is compartmentalized in a fraction of the tissue with extremely low sodium permeability. And the other possibility could be that some of the sodium is bound to cellular structures. References p . 217/218

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J. A. Z A D U N A I S K Y , F. W A L D A N D E. D. P. D E ROBERTIS

Frog brains

loot

Fig. 10. Loss of stable sodium from frog brains in sodium-free medium. Isolated frog brains were immersed in a medium where all the sodium had been replaced by choline, and their total sodium content determined after different periods of immersion. The values of the ordinate represent amount of sodium lost as per cent of the total sodium content of fresh dissected brains. The circles represent the mean (S.E. indicated by the bars) of 5 brains. After 6 h in sodium-free Ringer’s solution 24% of the stable sodium still remains in the brains (Zadunaisky and Chiarandini, unpublished observations)

-5

*Ot

H i ah KCI

c

151 15

L

,a 10 t n

MS-R

0

‘ 0 5

High KCH3S04 Low Na

Y

L U l

.-

P

0

5’

60

120 Time (rnin)

180

240

Fig. 11. Penetration of potassium chloride and impermeability into potassium methylsulfate. The points and circles represent the weight of one isolated frog brain each, as per cent of the initial wet weight. MS-R indicates a Ringer’s solution where all the chloride has been replaced by potassiummethylsulfate. After 90 min in this medium one brain (circles) was transferred to another medium, where 70 m M of NaCl were replaced by 70 m M of KCI. The other was transferred to a Ringer’s solution where 70 m M of NaCH3S04 were replaced by 70 mM of KCH3S04. As can be observed the one in high KCI and low Na medium gained weight due to penetration of KCl and water. The other brain in methylsulfate medium with high K and low sodium, did not swell, indicating the nonpenetration of the larger CH3S04 ion.

Penetration of KC1 into isolated frog brains As it was mentioned above, the replacement of part of NaCl by KC1 in the immersion medium produces a great swelling of the frog brains. In Fig. 11 the time course of swelling for one frog brain in high KC1and low Na Ringer is seen. Since animal cell membranes are in general permeable to K and C1 it was of interest to see the site’of swelling in frog brains, as observed with the electron microscope. In Figs. 12,13 and-14 the generalized swelling of the ependymoglial cells and processes can be seen. The neurons observed in the field show their normal appearance, with only some dilatation

OSMOTIC GLIAL CHANGES

209

Fig. 12. Penetration of KCI and water. Plexiform layer of the cerebral cortex of a frog brain after 2 h of immersion in high KCI and low NaCl Ringer. The ependymoglial processes Cpp) are swollen, while a dark and a clear neuron (nc) show the normal aspect. i = dense inclusion bodies.

of the sacs of the endoplasmic reticulum in advanced stages of tissue swelling. These neurons are surrounded by greatly enlarged and pale ependymoglial processes which have a round shape. Other components of the plexiform layer, such as fine axons and dendrites, show no volume changes. An interesting aspect is the disappearance of Referentex P. 217/218

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J. A. Z A D U N A I S K Y , F. W A L D A N D E. D. P. D E R O B E R T I S

the synaptic vesicles, a fact which indicates a probable depolarizing effect due to the penetration of KC1 into the tissue. These results could indicate that only the glial cell reacted to the penetration of the salt and water, and comparison with other tissues is significant in this case. Harris (1961) immersed sartorius muscles in Ringer’s solution where NaCl had been

Fig. 13. Penetration of KCl and water. Two nerve cells, a dark and a clear one, are surrounded by greatly enlarged ependymoglial processes (gp). i = inclusion bodies.

OSMOTIC GLIAL CHANGES

211

Fig. 14. Penetration of KCl and water. Section of the plexiform layer of the cortex of a frog brain incubated in high KCl and low NaCl Ringer for 4 h. g p = ependymoglial processes; i = inclusion bodies; er = endoplasmic reticulum. d = dendrite. Note that after prolonged incubation and great swelling the sacs of the endoplasmic reticulum of a neuron show dilatation.

replaced by KCI, and after the swelling occurred he examined electron micrographs of the muscle fibres. In this case all the fibres of the muscle were swollen and the cytoplasm showed in his pictures the watery appearance that we observed in the References p . 21 71218

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J. A. Z A D U N A I S K Y , F. W A L D A N D E. D. P. DE R O B E R T I S

ependymoglial cells and processes. Obviously there must be a peculiar property of the brain tissue as a whole, that is, a property due to structural arrangement of the cells or a special property arising from the neurons only, to account for this finding. One straightforward interpretation could be that the neurons are not easily distensible due to the possession of a rigid membrane or a rigid matrix. This will assimilate neurons to plant cells and unfortunately no direct evidence to support this point is available. Another possible interpretation could be that the ependymoglial cells are exposed to the immersion medium more directly than the rest of the structure and in this way the penetration of KC1 is done more easily into them than into the neurons. The anatomical picture of the ependymoglial cells in the frog brain, as mentioned, is one of a continuous cytoplasm which lines the ependymal cavity, extends through the cortex in the form of processes and finally forms a continuous limiting sheath in the outermost layer of the frog brain. This arrangement would favour the penetration of water and salts first into the ependymoglial cell. However, this interpretation would imply some other speculative concepts. One could be that the ependymoghal cells are in contact with extracellular fluid while the neurons are not, and all the regulation of the environmentof the neurons would be done by the ghal cells. Another consequence could be, also, that the properties of the membrane of the ependymoglial cell are different in regions of contact of the cell with extracellular fluid and in other regions in contact with the neurons. Though this might sound too speculative, in other systems, like frog skin, different properties of the outside and the inside parts of the cell membranes have to be postulated to account for the experimental results (Ussing, 1960). Eflective osmotic volume of the frog brain Since immersion of brain tissue in hypotonic solutions results only in swelling of glial cells, and also penetration of salt and water seems to increase only the size of glial cells, it could be predicted that the fraction of the tissue that responds to a change in the osmotic environment will be small. Also the evaluation of an effective osmotic volume could provide an approximate value for the size of the glial compartment. Analyses of the osmotic properties of single cell populations were carried out extensively in the past (Ponder, 1948; Dick, 1959), but detailed studies in entire tissues have seldom been made. The isolated frog skin has been utilized as experimental material to ascertain the effective osmotic volume and the probable concentrations of ions inside the cells of the epithelium (Mac Robbie and Ussing, 1961). A quantification of osmotic changes in isolated frog brains was made in an attempt to evaluate its effective osmotic volume (Zadunaisky et al., 1963). Obviously the results obtained are approximate and give only a guiding idea of the behaviour of the system. If the modifications in volume of the brains are followed by their weight changes, under conditions where only water moves from the immersion fluid to the tissue, then the value of the osmotically active fraction of the brains will be given by the following expression (Ponder, 1948): Weif = v - 1OO/( 1/T-1) References p . 2171218

213

OSMOTIC GLIAL CHANGES

2

1

4

3 Hours

Fig. 15. Weight changes of an isolated frog brain in hypotonic Ringer. The brain was immersed first in isotonic sulfate Ringer (SR) and after the weight was equilibrated it was immersed in the same medium but diluted to half its normal osmolarity (+ SR). The brain gained weight until it stabilized, and then again returned slowly to its equilibrium value. Note that the actual weight increase due to water penetration is very small (Zadunaisky et al., 1963).

in which Weif is the effective osmotic volume, V is the volume of the brains in hypotonic solution, T is the ratio of the osmolarity of the hypotonic to that of the isotonic medium and 100 represents the volume of the tissue in isotonic fluid. In order to ensure that only water would move from cell to immersion fluid and vice versa, the permeable chloride ion was replaced by the non-permeable methylsulfate ion, which does not penetrate into the tissue. In Fig. 11 is shown the effect of replacement of part of the NaCl of the Ringer’s solution by KC1 in one case and by KCH3S04 in the other. KC1 rapidly produces swelling due to its free penetration into the tissue, while KCH3S04 produces no swelling, actually a small shrinkage and equilibration is observed, probably due to a small difference in the osmolarity of the two Ringer’s solutions. T A B L E I11 (Weff) I N (Zadunaisky et al., 1963)

V A L U E S FOR EFFECTIVE OSMOTIC V O L U M E

Weight change at equilibrium in hypotonic fluid (wl

13.5 13.0 24.0 21.1 26.9 22.7 30.5 34.3 Average Rejerences p . 21 71218

ISOLATED FROG B R A I N

Weff referred to total weight

Weff referred to ‘cell water’

11.3 12.8 22.2 15.2 17.6 15.7 18.8 23.6

15.7 17.7 30.7 21 .o 24.5 21.7 26.0 32.7 23.7

17.1

214

J. A. Z A D U N A I S K Y , F. W A L D A N D E. D. P. D E R O B E R T I S i0Oy

I

I

1

I

I

2

3

,

4

I

5

Time (h)

Fig. 16. Effect of ouabain (10-4M)on 24Naefflux from a frog brain. The efflux is expressed as a rate (counts/min). The degree of inhibition was estimated by extrapolating the straight lines obtained before and after addition of ouabain, as shown. The effect was irreversible (Zadunaisky and Curran, 1963).

Under these conditions, then, if the brains are equilibrated in isotonic methylsulfate-Ringer and then immersed in the same Ringer’s solution with its osmolarity reduced to half, the increase in volume (or weight) will indicate the fraction of the tissue that reacted as an osmometer. In Fig. 15 the sequences of events in the case of one brain are observed. The actual increase in weight was small, and as it is reproduced in Table I11 the effective osmotic volume of the brain found was 17.1% of the total weight, When allowance was made for the inulin space and the values were referred to ‘cell water’ the effective osmotic volume was 23.7%. The conclusion from these results could be that the glial cells occupy this reduced fraction of the whole frog brain, and that the small effective osmotic volume confirms the findings of electron microscopy where only a fraction of the tissue showed volume changes during immersion in hypotonic fluids. Action of G-strophantin on sodium eflux and structure The addition of cardiotonic glucosides to isolated red cells produces the arrestlof the sodium transport mechanism (Schatzmann, 1953) which is accompanied by volume changes due to retention of salt (Tosteson and Hoffmann, 1960). In other tissues the effects of G-strophantin on sodium or potassium movements are also associated with interference with the active transport processes at the level of the cell membranes (Wildbrant and Rosemberg, 1961). In the case of the isolatedfrog brain, the addition of ouabain produced a reduction of 54.5 % in sodium efflux. Fig. 16 shows the effect of the glucoside on the slow component of sodium loss, which is irreversible under the experimental conditions. Simultaneously with the reduction in sodium efflux there is a swelling of the brains which amounts to 33.6 % over the initial wet weight, with a gain of sodium and loss of potassium (Zadunaisky et al., 1963). The changes in ultrastructure observed in these brains consisted of considerable swelling of the ependymoglial cells and their processes. The swelling of these processes could be seen throughout the different layers of the cortex (Figs. 17, 18). The neurons did not change in volume or in the density of the cytoplasmic matrix. Mitochondria, both in the

OSMOTIC GLIAL CHANGES

215

Fig. 17. Section through several bodies of ependymoglial cells after swelling of a frog brain by ouabain. The cytoplasm (gc) appears very pale and mitochondria (mi) show some vacuolization. i = inclusion bodies; N = nucleus; nu = nucleolus.

nerve cells and in the enlarged glial processes, showed different degrees of swelling. The fine structure of synaptic endings, axon and dendrites was almost normal though in some zones the synaptic endings were discrethy swollen with clustering of the synaptic vesicles. Again, then, the picture of a preferential osmotic reaction by the References p. 2I7/218

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J. A. Z A D U N A I S K Y , F. W A L D A N D E. D. P. D E R O B E R T I S

glial cells, in contrast to the apparent morphological normality of the neurons was observed. It is possible that ouabain does not penetrate into the whole tissue, and only did so in ependymoglial cells. However, the clear-cut reduction in sodium efflux and the effect on the mitochondria of the nerve cells would indicate good penetration

Fig. 18. Section of the plexiform layer o# a frog brain after effect of ouabain. Note the swelling of ependymoglial processes (gp), the effect on mitochondria (mi) and the synaptic endings (e). i = inclusion bodies.

OSMOTIC G L I A L C H A N G E S

217

of the agent into the tissue. Since this swelling has to be interpreted as the consequence of electrolyte and water retention, the picture is again one of cellular hypertonicity, without reaction of a part of the system, the nerve cells. G E N E R A L CO NCL USION S A N D S U M M A R Y

The gain in water of isolated brain tissues in vitro is due to retention of salts and water in the glial cells, which behave as osmometers and can retain appreciable quantities of fluid. The neuronal elements do not seem to be so reactive to water and salt modifications, due probably to an extremely low membrane permeability, to the existence of a rigid cell membrane or matrix or to a lack of contact with the extracellular fluid. Functionally, there is an extracellular space of a considerable size, which is apparently located outside the glial components of the brain, since swelling of the glial cells is not accompanied with corresponding increases of the inulin space of isolated frog brain, and interference with metabolism does not induce changes in the rate of sodium loss from the extracellular space of the brains. The electron-microscopical findings which showed swelling of only the glial fraction of the cell population of brain tissue in vitro correlate well with the small osmotically active fraction of frog brains. It is conceivable that the water and electrolyte movements could be regulated by glial cells. The glial cells could be in direct relationship with the functional extracellular space on one side and in contact with the neurons in other parts of their membranes. With a low salt and water permeability of the neurons, any electrolyte and water exchange between neurons and extracellular fluid could be effected through the glial cells, which will ultimately regulate thus the activation of the neurons. If, as proposed by other authors, these glial cells contain a high concentration of electrolytes, they could provide the neurons with an appropriate environment for the production of the resting potential and for excitation. ACKNOWLEDGEMENTS

Part of this work was supported by the Consejo Nacional de InvestigacionesCientificas y TCcnicas, Argentina and the National Multiple Sclerosis Society.

REFEREN C ES BOYLE, P. J., AND CONWAY, E. J., (1941); Potassium accumulation in muscle and associated changes. J. Physiol., 100, 1-63. S. RAMONY, (1922); El Cerebro de Zos Butrucios. In Libro en Honor de S. Rambn y Cajal. CAJAL, Madrid, Publicaciones de la Junta para el Homenaje a Cajal. CONWAY, E. J., GEOGHEGAN, H., AND MACCORMACK, J. I., (1955); Autolytic changes at zero centigrade in ground mammalian tissues. J. Physiol., 130, 427-437. DAVSON, H., AND SPAZIANI, E., (1959); The blood-brain barrier and the extracellularspace of brain. J. Physiol., 149, 135-143. G. B., (1955); An electron-microscope study of the blood-brain DEMPSEY, E. W., AND WISLOCKI, barrier in the rat, employing silver nitrate as a vital stain. J . biophys. biochem. Cytol., 1, 245-256.

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DE ROBERTIS, E. D. P., (1955); Submicroscopic organization of some synaptic regions. Acta neurol. 1at.-amer., 1, 3-15. DICK,D. A. T., (1959); Osmotic properties of living cells. Znt. Rev. Cytol., 8, 388-488. FARQUHAR, M., ANI) HARTMANN, F., (1957); Neuroglial structure and relationships as revealed by electron microscopy. J. Neuropath. exp. Neurol., 16, 18-39. GERSCHENFELD, H. M., WALD,F., ZADUNAISKY, J. A., AND DE ROBERTIS, E. D. P., (1959); Function of astroglia in the water-ion metabolism of the central nervous system. Neurology, 9, 412425. HARRIS,E. J., (1960) ; Transport and Accumulation in biological Systetm. London, Butterworths Scientific Publications. HARRIS,E. J., (1961); The site of swelling in muscle. J. biophys. biochem. Cytol., 9, 502-504. HERNED, H. S., AND OWEN,B. B., (1958); Thephysical Chemistry of electrolytic Solutions. New York, Reinhold Publishing Co. I., (1962); Morphological and physiological properties of neurons and glial HILD,W., AND TASAKI, cells in tissue culture. J. Neurophysiol., 25, 277-304. HILL,A. V., (1928); Diffusion of oxygen and lactic acid through tissues. Proc. roy. SOC.B, 104,41-96. HORSTMANN, E., (1957); Die Struktur der molekularen Schichten im Gehirn der Wirbeltiere. Naturwissenschaften, 44,448-456. JACOBS, M. H., (1935); Diffusion processes. Ergebn. Biol., 12, 1-160. LEAF,A., (1956); On the mechanism of fluid exchange of tissues in vitro. Biochem. J., 62, 241-248. LIBET,B., AND GERARD, R. W., (1939); Control of the potential rhythm of the isolated frog brain. J . Neurophysiol., 2, 153-169. MACROBBIE, E. A. C., AND USSING, H. H., (1961); Osmotic behaviour of the epithelial cells of frog skin. Acta physiol. scand., 53, 348-365. OKSCHE, A., (1958); Histologische Untersuchungen uber die Bedeutung des Ependyms, der Glia und des Plexus chorioideifur den Kohlenhydratstoffwechseldes Z.N.S. Z. Zeluorsch., 48, 74-1 29. PAPPIUS, A. H., AND ELLIOT,K. A. C., (1956); Water distribution in incubated slices of brain and other tissues. Cunad. J. Biochem. Physiol., 34, 1007-1022. PONDER, E., (1948); Hemolysis and related Phenomenon. New York, Grune and Stratton. J. P., NAPOLITANO, L., AND FAWCEIT,D. W., (1960); Identification of glycogen in electron REVEL, micrographs of thin tissue sections. J. biophys. biochem. Cytol., 8, 575-589. SCHATZMANN, H. J., (1953); Herzglykoside als Hemmstoffe fur den aktiven Kalium und Natriumtranspart durch die Erythrocytenmembran.Helv. physiol. pharmacol. Acta, 11, 346-354. TOSTESON, D. C., AND HOFFMANN, J. F., (1960); Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J. gen. Physiol., 44, 169-194. USSING, H. H., (1960); The frog skin potential. J. gen. Physiol., 43, 135-147. WILDBRANT, W., AND ROSEMBERG, T., (1961); The concept of carrier transport and its corollaries in pharmacology. Phurmacol. Rev., 13, 109-184. WYCKOFF, R. W. C., AND YOUNG,J. Z., (1956); The motor-neuron surface. Pruc. ruy. SOC.B, 44, 440450. ZADUNAISKY, J. A., AND CHIARANDINI, D. J., (1963); unpublished observations. P. F., (1963); Sodium fluxes in isolated frog brain. Amer. J. ZADUNAISKY, J. A., AND CURRAN, Physiol., 205, 949-956. ZADUNAISXY, J. A., WALD,F., AND DE ROBERTIS, E. D. P., (1963); Osmotic behaviour and ultrastructural m3difications in isolated frog brains. Exp. Neurol., 8, 290-309.

219

Some Aspects of Amino Acid Transport in the Central Nervous System E. LEVIN, G . J. N O G U E I R A

AND

C. A. G A R C I A ARGIZ

Department of Biochemistry, FacuIty of Pharmacology and Biochemistry, University of Buenos Aires Buenos Aires (Argentina)

I want to report some studies we are carrying out on transport of amino acids from the ventricular cavities to the nervous tissue. The preliminary results point to the presence of a barrier, the fluid-tissue barrier for amino acids in the central nervous system. We have applied the technique of ventriculo-cisternal perfusion to study the disappearance of amino acids from the cerebrospinal fluid (CSF) avoiding the unspecific bulk flow through the arachnoid villi. Pappenheimer et al. (1961) in the goat, have shown that Diodrast and phenolsulfonphthalein can be removed from the ventricular system into the blood by an active process, located in the fourth ventricle. Davson et al. (1962) in the cat and later in the rabbit (Pollay and Davson, 1963) pointed to a similar process for p-aminohippuric acid (PAH), iodide and thiocyanate. These substances when injected into the blood, reach in the CSF a concentration less than 1 % of that in the plasma. Thus, there is an active unidirectional movement of these substances from CSF to blood through the choroid plexuses. It is known that most of the amino acids show a restriction to their passage from blood to the nervous parenchyma and to the CSF(Lajtha, 1962). The present study was undertaken to establish if the blood-CSF barrier could also be explained as an active process which removes the amino acids from the CSF to blood through the epithelium choroid plexuses. Methods. In anesthetized cats the ventricular system was perfused with Merlis (1940) artificial CSF at a constant speed using a slow infusion pump at a rate of 66 ,ul/min, that is 4 ml/h. For amino acids the rate of infusion was 0.25 ,uM/min which makes 15 pM/h. The effluent fluid was collected at 15-min intervals during a period of 2 h controlling the inflow and outflow volumes. The percent of recovery was determined by the formula: vol. out vol. in References p . 223/224

conc. out conc. in

*

100

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E. LEVIN et

al.

which takes into consideration the dilution produced by the newly formed fluid during the experiment. To estimate the absolute value of recovery and absorption during the first hour, the cat was perfused for 60 min with Merlis fluid plus the amino acid, and then the ventricular system was washed for 30 min with Merlis solution alone. All the fluid was collected and the percent recovery was calculated by the above mentioned formula. Analytical. Inulin was determined by the method of Hubbard and Loomis (1942). PAH by the method of Bratton and Marshall (1939) and the amino acids were estimated after paper chromatographic separation using butanol : acetic acid : water (12 : 3 : 5 ) as solvent, staining the spots with ninhydrin and eluting with ethanolcadmium sulphate according to Cook and Luscombe (1960). The radioactivity of 14C-labelledamino acids was measured in a micromil window automatic flow counter (Nuclear Chicago).

- -

15

I

30 45 60 75 90 105 120 Time in min

Fig. 1. Ventriculo-cisternalperfusion of glutamic acid. Recovery in the effluent as per cent of that in the inflowing fluid.

Results. Fig. 1 reproduces the results of one experiment with glutamic acid to show the type of curve obtained by plotting per cent recovery in each sample against time during the 2-h perfusion. During the first hour, the lower rates of recovery could be attributed to dilution due to the outflow of the CSF preexisting in the system and also to an increased absorption by the tissue. A certain rate is achieved at 45 to 60 min after which the rate of absorption and consequently the rate of recovery becomes constant. In Table I the values obtained with several amino acids are shown. For these experiments, 3 to 4 amino acids were perfused together and occasionally inulin or PAH were included for comparison. The values correspond to the second hour of perfusion when a steady level of concentration was reached in each sample. When the steady state was attained, the per cent recovery of the amino acids varied from 74 to 100% which corresponds to an absolute disappearance from 0 to 4pMlh according to the amino acid considered. The disappearance of the amino acids could be produced by two routes: (I) to the

22 1

AMINO A C I D TRANSPORT I N C N S

TABLE I P E R C E N T A G E R E C O V E R Y OF A M I N O A C I D S AFTER VENTRICULO-CISTERNAL PERFUSION

Comparison with p-aminohippuric acid and inulin; figures give means f standard deviations; number of experiments in parentheses. Single values where less than 3 experiments were done. Data from the second hour perfusion. p-Aminohippuric acid: 41 f 11 (8)

Inulin: 85 f 8 (5)

Glutamic acid y-Aminobutyric acid Glutamine Leucine , ! I Alanine -

84 f 10 84 f 9 96 f 9 81 f 2 90 f 7

Glycine Phenylalanine Lysine Valine Tyrosine

88-100 83-86 78-79 74-85 86

(5) (4) (5) (3) (3)

blood through the choroid plexuses; (2) to the nervous tissue through the ependymal lining of the ventricles. One would expect a high rate of disappearance at least by the second route because of the high uptake capacity of the nervous tissue as shown by in vitro studies (Tsukada et al., 1963). The route towards the blood apparently was not functioning as for PAH, consequently there is not an active process of removal of the amino acids from CSF to the blood. The low rates of absorption by the tissue could be due to a competitive process because of the simultaneous presence of several amino acids. To check this possibility, glutamic acid, glutamine and y-aminobutyric acid (GABA) were perfused separately with their corresponding isotopes to see also if some exchange mechanism of the type described by Lajtha and Toth (1962) was present. It is possible to see that for glutamine and glutamic acid there is a competitive process in their uptake by the nervous tissue when they are present in a pool of amino TABLE I1 P E R C E N T A G E OF RECOVERY AFTER VENTRICULO-CISTERNAL PERFUSION OF A SINGLE AMINO ACID

Mean of 3 experiments f S.D. Data from the second hour perfusion.

% Recovery Amino acid

Chemical determination

'41

counts

y-Aminobutyric acid

85 f 15

60 f 6

Glutamine

78 f 10

78 f 10

Glutamic acid

67 f 4

73 f 8

References p . 2 1 / 2 4

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et al.

acids. For glutamine we obtained a rate of 3.8 pM/h during the second hour when perfused alone, against 0.6 pMlh when injected together with glutamic acid and GABA. Glutamic acid gave a rate of absorption of 4.9 pM/h when perfused as a single amino acid against 2.4 pM/h when it was in the pool. GABA remained at the same slow rate of absorption, namely 2.25 pM/h after the first hour of perfusion. For this amino acid the per cent recovered for the isotope was lower than the value obtained by the chemical method, showing an increased turnover when the amino acid has a lower rate of net absorption by the tissue. This exchange mechanism is not evident for glutamine and glutamic acid. To measure the maximum rate of absorption which occurred during the first hour i.e. before the saturation level was attained, a perfusion was done during 60 min. Glutamine was chosen because it is one of the amino acids considered to pass the blood-brain barrier (Schwerin et al., 1950) and GABA because of its high uptake by brain slices (Elliott and Van Gelder, 1958). Inulin was included for comparison. After 60 min the system was washed as described in Methods. TABLE I11 PERCENTAGE RECOVERY OF AMINO ACIDS WASHING A N D I N U L I N AFTER PERFUSION O F T H E V E N T R I C U L A R SY STEM

+

Amino acids Inulin

Chemical determination

14C counts

y-Aminobutyric acid 98

108

67

96

43

103

41

Glutamine

By this procedure there is a complete recovery of the inulin. We previously got a value of 85 % after reaching the equilibrium level, which means that 15% was diffusing into the tissue surrounding the ventricles, After washing we were able to recover all the inulin (100 %) which came by diffusion from the tissue to the washing fluid. With the amino acids we have different results because they are taken by the tissue where they could be metabolized, stored by some cell organelles, or adsorbed to membranes. We like to use the expression ‘occluded’ proposed by Elliott and Van Gelder (1958) to say that they are not free, not available for diffusion. The sum of radioactivity of the effluent fluid plus the radioactivity of the brain after washing, gave 100% of recovery showing that the 1% was kept in the nervous parenchyma.

AMINO A C I D TRANSPORT I N CNS GLUTAMINE

223

GABA

10 p M/h

Ih

2h

Ih

2h

Fig. 2. Absorption and recovery of glutamine and GABA after perfusion and washing of the ventricular system.

Fig. 2 summarizes the final results. If we compare the results of absorption for glutamine (9.0 pM/h) and for GABA (4.6 ,uM/h) we can speak of a restriction to the uptake of GABA by the tissue in relation to glutamine; and to both of them if we take as reference in vitro studies carried out with several amino acids (Tsukada et al., 1963; Lajtha and Toth, 1962; Schwerin et al., 1950; Elliott and Van Gelder, 1958; Neame, 1961). The results from in vitro incubation of brain slices show a n active accumulation of the amino acids by the brain against a concentration gradient. For GABA the uptake oscillates between 15 to 20 pM/g tissue/h (Elliott and Van Gelder, 1958). However, in our in vivo conditions there is an important restriction of the uptake by the tissue. Thus, we postulate the existence of a barrier for amino acids between the CSF and the nervous parenchyma, the fluid-tissue barrier, at the level of the ventricular cavities. Such a barrier could explain also why the free amino acids of the brain do not pass into the CSF, maintaining a differential concentration gradient between the tissue and the fluid. REFERENCES BRATTON, A. C., AND MARSHALL, R. O., (1939); A new coupling component for sulfanilamide determination. J. biol. Chem., 128, 537-542. M. J., (1960); Estimation of aminoacids in serum. J . Chromatog., 3, 75. COOK,E. R., AND LUSCOMBE, DAVSON, H., KLEEMAN, C. R., AND LEVIN,E., (1962); Quantitative studies of the passage of different substances out of the cerebrospinal fluid. J . Physiol. (Lond.), 161, 126-142. N. M., (1958); Occlusion and metabolism of y-aminobutyric ELLIOTT,K. A. C., AND VANGELDER, acid by brain tissue. J. Neurochem., 3, 2 8 4 0 . T. A., (1942); The determination of inulin. J . bid. Chem. 145,641-647. HUBBARD, R. S., AND LOOMIS, LAJTHA,A., (1962); Cerebral passage of free aminoacids. Amino Acid Pools. J. T. Holden, Editor. Amsterdam-New York, Elsevier (pp. 554-563). LAJTHA,A., AND Tom, J., (1962); The brain barrier system. 111. The efflux of intracerebrally administered aminoacids from the brain. J. Neurochem., 9, 199-212.

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MERLIS, J. K., (1940); The effect of changes in the calcium content of the cerebrospinal fluid on spinal reflex activity in the dog. Amer. J. Physiol., 131,67-71. NEAME, K. D., (1961); Uptake of aminoacids by mouse brain slices. J. Neurochem., 6, 358-362. PAPPENHEIMER, J. R., HEISEY, S. R., AND JORDAN,E. F., (1961); Active transport of diodrast and phenolsulfonphtalein from cerebrospinal fluid to blood. Amer. J . Physiol., 200, 1-10. POLLAY, M., AND DAVSON, H., (1963); The passage of certain substances out of the cerebrospinal fluid. Brain,86, 137-143. SCHWERIN, P., BESSMAN,S. P., AND WAELSCH, H., (1950); The uptake of glutamic acid and glutamine by brain and other tissues of the rat and mouse. J. biol. Chem., 184, 37-42. TSWKADA, Y.,NAGATA, Y., HIRANO,S., AND MATSUMATI, T., (1963); Active transport of aminoacid into cerebral cortex slices. J. Neurochem., 10, 241-256.

225

Second Discussion Period

TASAKI: Now we have come to the time for discussion. I would like to suggest that the members of the panel make comments on the subject we have heard today or ask questions of the speakers who have presented their views already. To begin with, I would like to raise a question as to the problem of intra- and extracellular space in the central nervous system. This problem has been discussed by Dr. Lasansky who told us that the extracellular space is somewhat larger than previous electron microscopic studies have indicated. About 10 years ago, when we started to investigate the neuronal activity in the central nervous system with microelectrodes, we believed that there was a large space between individual neurons, a space filled with extracellular fluid. I wonder what per cent of the total space occupied by the brain can now be attributed to the extracellular space. Is there anybody who wants to comment on this point? DE ROBERTIS : With the improvement in techniques for electron microscopy, and specially with the use of the new embedding techniques, like Epon, the intercellular space has increased a little over the figure which we had before. I would say this is very difficult to calculate, because the brain tissue is so complex that to map out the whole extracellular space is almost impossible. I understand that some people are doing that work, specially in Dr. Kuffler’s laboratory for invertebrates. I don’t know the figure which they have right now. I suggest that this figure will be probably below 10%, approaching maybe more to the physiological figures with inulin or sucrose. However, I think that the figures obtained with other methods like chloride and sodium are much greater and are not indicating a true extracellular space. I would like your comments, maybe from Dr. Davson on this matter.

DAVSON: In English there is an expression that one has an axe to grind, and if you understand that you will understand also when I say that I myself have no axe to grind on this matter, in that it could be 5 or it could be 10 or it could be 20%. It wouldn’t alter any general ideas. But the most enlightening note is that given by Dr. Zadunaisky. There, he was studying the tissue in vitro of a cold-blooded animal that did not suffer from the progressive oedema that takes place in warm-blooded tissue. And there I think he showed us really conclusively that the inulin space is large, of the order of 15 % or more, and it is almost impossible to conceive of a cell that is able to behave like a cell, that is to restrict its sodium concentration and to accumulate potassium, it is barely conceivable that such a cell should be permeable to inulin. So I would like to tread hard on this thought that a cell can be a cell and can be an extracellular space at the same time.

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SVAETICHIN : This morning Prof. De Robertis presented some beautiful electron micrographs showing that some synaptic endings make laterally an intimate contact with glial cells, and Dr. Lasansky demonstrated similar structures, the so-called ‘tight junctions’ in the retina. The ‘tight junctions’ which Prof. De Robertis demonstrated at the synaptic endings possibly represent the structural counterpart to Eccles ‘presynaptic inhibition’, which we believe depends on glial cell activity. In certain places the apposed plasma membranes of adjacent cells appear as fused and one sees in permanganate fixation not 6 but 5 layers. This kind of structures have been described by Sjostrand for the mitochondrial double membranes and they are also found at the contacts between the cells in the smooth muscle networks. Possibly, this ‘tight junction’ forms a special structure for functional interaction between adjacent cells. We have suggested (Proceedings Int. Congr. Psychology, Washington, 1963 and IVIC Anniversary, Acta cient. venez., Suppl. I, 1963, 135-153) that the double membrane unit formed by the apposed plasma membranes of functionally interacting cells is a ‘mitochondrial equivalent’ in which the intercellular energy transfer is based on the activity of respiratory chains which we believe form a structural component of these double membranes. Possibly, the ferrocyanide space shown by Dr. Lasansky in the retina is the ‘drain’ for the water formed within this double membrane structure in connection with the respiratory chain function. TASAKI: Is there any comment on the subject of the extracellular space? GALAMBOS: I would make just two short comments, the first on Dr. Tasaki’s question of what an electrode with a tip of 4 microns or so actually contacts in the brain. No matter how large the extracellular spaces are, such an electrode must be immersed in an area of destroyed cells. Neuroglia and neuropil must be disrupted in the vicinity of the electrode tip, and it’s a real surprise that anything at all happens under an electrode in such an abnormal situation. The second point would be the way we seem to have divided ourselves here into those interested in the amount of separation between cells in the brain and those interested in the closeness of the contacts. The first group visualizes fluids moving back and forth through membranes with transfer of substances across the membranes of neurons and glia into this space. The other group seems interested in the smallness of this space and in the way one cell influences an adjacent one through direct membrane-to-membrane transfer of energy and substances. LASANSKY: I would like to direct my questions to Dr. Pappius. Firstly, I was very surprised that inulin seems not to penetrate within the undamaged parts of a brain slice because the inulin space of the retina, as measured by Ames and Hastings, is 29 % and of course there are no damaged surfaces in this thin layer of tissue. Besides, Dr. Zadunaisky’s data also indicate that inulin penetrates in brain tissue and in this case also most of the surface is probably undamaged. Therefore, I wonder about the reason for these differences. The second question is about the sucrose space in brain slices. I do not know whether

SECOND DISCUSSION PERIOD

227

there is any significant amount of white matter in your slices and if there is, whether you would consider the possibility that the sucrose space includes myelin because, according to Shanes and Berman, myelin sheaths are part of the sucrose space in peripheral nerve. PAPPIUS:Referring to the last question first, we found in a limited number of studies, that the distribution of sucrose in the white matter is very similar to that in the cortex. In other words, there is a considerable sucrose space but there is also a space in the white matter that is not penetrated by sucrose (see Fig. 2, p. 138). As far as the inulin results in the mammalian cerebral cortex are concerned if you agree with the assumption that the protein space is the same as the inulin space, then the photomicrographs which I showed indicate clearly that inulin equilibrated only with the damaged tissue elements and it did not penetrate any further. The differences between the cerebral cortex and the retina may be due to the fact that these are after all different tissues. The difference between my results and Dr. Zadunaisky’s may be due to the fact that he is working with frogs and I am working with mammalian tissue. POLAK:Practically all the speakers in this Symposium have considered glial cells, particularly astrocytes, the main fluid reservoir in cases of in vitro experimental brain oedema. I will show some slides of human pathologic material belonging to surgical and autopsy specimens. Such material will show the cell changes which are observed in cases of cerebral herniation, peritumoral oedema and inflammatory oedema. We have seen similar changes in experimental brain oedema in dogs. The picture observed in these cases should be analyzed in the light of common pathologic knowledge. The changes observed in connective tissue and epithelial cells in cases of oedema are similar to those seen in brain tissue. One should remember what happens in mechanical and inflammatory oedema in the cellular and intercellular compartments of connective tissue and what is seen in cases of spongiosis and intracellular vacuolation of the epidermis in many pathologic conditions of the skin. In brain tissue oedema hypertrophy of the glial cells can be seen in astrocytes, oligodendrocytes and glioepithelial cells, but liquid accumulation can also be observed in microglial and nervous cells. In cases of oedema of great intensity and long duration the astrocytic elements break down and liquid accumulates in the intercellular spaces. Such space is not large in brain tissue but is large enough to allow some fluid accumulation. 1 will project only 6 slides. The first shows what happens with astrocytes. These increase in size and their processes become long and flexous. These findings are similar to those found with the electron microscope (Fig. 1). The second slide shows a case of oedema of longer duration and is similar to the previous picture but here the thickening of the footplates is conspicuous (Fig. 2). Fluid in the intercellular space created by cellular rupture can also be observed. We can see in the next slide the classical acute swelling of the oligodendroglia as described by Penfield, del Rio Hortega and others, which is the expression ofthe accumulation of fluid in this type of glial cell (Fig. 3).

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In the next slide we can see the thickening of the processes as well as enlargement of the cellular body of microglial cells (Fig.4). The last two slides show the increase in size of the neuronal bodies, their blunt limits and the spherical and hypertrophic nuclei (Figs. 5 and 6). The picture we have just shown led us to the conclusion that in human pathologic material studied with light microscopy as contrasted with in vitro artificially provoked brain oedema, the accumulation of fluid occurs in all the cellular elements and in the intercellular space of the nervous tissue. KLATZO: I would like to re-emphasize my belief, though not original itself, that brain oedema is a process of an extremely diverse nature. It can be related to a variety of pathogenic mechanisms, as has been demonstrated in Dr. Bakay’s and our own studies. It is also interesting that the target of the oedematous process may be centered in a preferential or selective way on different basic cellular constituents of the nervous parenchyma. Thus, for example, Dr. Polak has just shown us a reactive swelling of astrocytes in his most beautiful metallic impregnations. The almost selective reaction of astrocytes in some types of experimental brain oedema has been demonstrated in our previous investigations. Penfield, some time ago, described a classic picture of acute swelling of oligodendroglia which in certain conditions appear to be the only element affected. In a widely studied tin poisoning Aleu, Katzman and Terry have shown by application of electron microscopy the accumulation of the fluid almost exclusively in the clefts between the separated myelin sheaths. Finally, we were able recently to produce an almost selective swelling of the neurons by alum phosphate intoxication. BAKAY:I would like to ask a question primarily directed to Dr. De Robertis. We have seen striking features of different types of oedemas of the white matter like traumatic and inflammatory oedemas. Although these are primarily oedemas of the white matter they seem to be extremely different in nature. One is a protein-rich oedema fluid and the other one is an almost entirely protein-free ultrafiltrate. Obviously the white matter is involved in different types of oedemas. Furthermore, it has been lately suggested not only by myself but by other investigators that there might be an innate peculiarity of the extracellular space of the white matter, that makes it more prone to oedema than the grey matter. I was wondering whether Dr. De Robertis has any observations or any comments about this.

DE ROBERTIS: It is very interesting that in all these cases of traumatic oedema, the white matter is more affected than the grey matter. My suggestion is that there is much less knowledge of the electron microscopy of white matter than that of the grey, because of the difficulties in preparation. I think, however, that with the new perfusion fixation I have described, we would solve these problems and learn more about what is going on in the white matter. I would suggest, however, that one important fact is that the myelin sheath can very easily swell by disruption of the leaflets of the myelin sheath, and a great deal of water can then be coordinated or

SECOND DISCUSSION PERIOD

Fig. 1. See text.

Fig. 2. See text.

229

230

SECOND DISCUSSION PERIOD

Fig. 3. See text.

Fig. 4. See text.

SECOND DISCUSSION PERIOD

Fig. 5. See text.

Fig. 6. SeeItext.

23 I

232

S E C O N D DISCUSSION P E R I O D

integrated in between the membranes. We have seen in some cases that these myelin leaflets may swell considerably and make large cystic-like cavities in the brain, in certain conditions, and I think this may be a very important reservoir for water in white matter.

BAKAY:In connection with this, I want to mention that in electron microscopic studies recently performed on the triethyl tin type of oedema, we found as other authors have tdat there was a splitting of the lamellae and an increased water content. TASAKI:W& does the radioactive material spread along the myelinated fiber in the white matter? Why does it not spread toward the grey matter which is much closer to the site of injury? What is the mechanism for this particular mode of transport? Is there any explanation to the phenomena you mentioned? BAKAY: This is very hard to say because all we can see is that in this traumatic oedema, although the trauma is applied to the surface of the cortex, the oedema is exclusively located in the white matter. However, it originates at the subcortical layers from the damaged vessels which are in the vicinity of the lesion. I don’t know why they propagate into the white matter, but I thought that perhaps for an accumulation of fluid and electrolytes the myelin sheath would be a natural tubing along which they could much more easily propagate. In the grey matter they would have to circumvent and go around tightly packed cells, which is like going through a stone wall that’s put together brick by brick. I think that there is a certain conductivity by the fact that the myelin fibers are running parallel. With regard to proteins and dyes, which is probably the same, one can actually see, 6 hours after the injection, the dye or the protein, following certain accurate fiber systems. I think that this is more a mechanical than an actually chemical phenomenon. KLATZO: With regard to the striking preference of the extravasated proteins to spread through the white matter, some ultrastructural features regarding the availability of some ‘potential’ spaces in the white matter could offer a simple explanation of this phenomenon. Personally, I have some doubts in this respect in view of our recent findings iqdicating that from the cerebral ventricles serum proteins penetrate deeper into the grey than into the white matter; a finding which appears to contradict the assumption that ultrastructural features of the white matter alone are essential in facilitating the spread of the serum proteins. I would appreciate hearing if anybody can offer any clues for the interpretation of these intriguing differences in protein penetration. BAKAY: I can’t give you the answer but this is true for other substances like histamine, which has been shown by other investigators to be absorbed presumably by active transport from the cerebrospinal fluid, through the ventricular wall into the periventricular grey and this process apparently stops at the edge of the grey matter. But no explanation has been given and I don’t think anybody could answer your question at the present time.

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PAPPIUS : Just going back to the previous discussion about the location of the oedema fluid in the white matter, there has recently been published a very interesting report from Chicago by Raimondi et al. They confirmed the earlier findings of glial changes in cerebral oedema, but they also showed abnormal extracellular space in the white matter associated with oedema induced by inflation of an extradural baloon.

ZADUNAISKY: I just want to bring the discussion to our observations in osmotic oedema. In isolated frog brains or mammalian brain slices we never get swelling of the neurons. I would like to ask Dr. Tasaki if he has some comments on why a cell won't gain water when exposed to a hypotonic or to a high KCl-low NaCl medium. TASAKI: I do not know.

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Excitability of Neurons and Glial Cells ICHIJI TASAKI Laboratory of Neurobiology, National Institutes of Mental Health, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. ( U.S.A.)

Electrophysiological properties of mammalian neurons and glial cells have been investigated in this laboratory by using the tissue culture technique and by introducing microelectrodes into thin brain slices. The cells are observed with a phase microscope when the microelectrodes are inserted. The results from tissue culture materials, obtained in collaboration with Dr. Hild, were published elsewhere (Hild and Tasaki, 1962). Our experiments on brain slice preparations from newborn guinea-pigs are still in progress. In this article we wish to review the present state of our knowledge concerning mammalian neurons and glial cells as examined by various microphysiological techniques. Electrophysiological properties of neurons

Since about 1952 a large number of papers have been published dealing with the resting and action potentials of single nerve cells in the mammalian nervous system (Eccles, 1957; Frank and Fuortes, 1961). Almost all of these investigations were carried out by introducing hyperfine (glass or metal) recording electrodes into various parts of the mammalian central nervous system without direct visual observation of the cells under study. Although this method of 'blind' recording yielded a large volume of important knowledge of the function of the nerve cells, it has become increasingly clear that the information about the exact position of the microelectrode relative to the cell under study is indispensable for proper interpretation of the results obtained (Spyropoulos and Tasaki, 1960). Our study of tissue culture material was designed to examine electric responses of the neuron soma and dendrites under direct visual observation. Cultivation of the neural elements used for the present investigation was done partly in the Tissue Culture Laboratory of the University of Texas Medical Branch in Galveston and partly in the Laboratory of Neuroanatomical Sciences of the National Institute of Neurological Diseases and Blindness. A phase-contrast microscope was used to view various parts of neurons and glial cells, as well as the tips of the stimulating and recording electrodes. When a hyperfine glass microelectrode is pushed into the cell body of a neuron,

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there is a sudden D.C. shift representing the resting potential of the cell. By delivering brief pulses of either inward- or outward-directed currents through the stimulating microelectrode, it is possible to evoke all-or-none action potentials in the cell. Action and resting potentials of such impaled neurons deteriorate with time. It is possible to record the electric response of the cell body and dendrites extracellularly with a microelectrode that makes contact with the cell surface. In recording action potentials from the surface of neurons, there is no progressive deterioration of the responses. In some neurons in tissue culture, there are repetitive responses in the absence of external stimuli. Fig. 1 shows three examples of such spontaneously fired action potentials recorded from the neuron surface. Note that the configurations of the action potentials of these three neurons are very different from one another. The differences in configuration arise mainly from variations in the pressure one has to apply to the neuronal surface with the recording microelectrode

A

B

c

3

<

Fig. 1. Several types of spontaneously fired action potentials recorded with glass microelectrode making contact with the surface of neuron somata. Upward deflections represent positivity of recording electrode. Records A were taken from neuron in 13-day-old culture of rat cerebellum, records B from 10-day-old culture of rat cerebellum, and record C from 15-day-old culture of rat cerebellum. Sudden change in configuration of recorded action potentials in record C was induced by pushing recording electrode harder against neuron surface. Oscilloscope sweep speed was same for all records; deflection sensitivity for B was same as that for C. (From J. Neurophysiol., 25, (1962) 284). References p . 2411242

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in order to record properly the action potentials. If the tip of the recording electrode is not making direct contact with the neuronal surface, the recorded responses would be too small in amplitude to be recognized. If, on the contrary, the tip of the recording electrode presses too strongly against the neuronal surface, the chance of injuring the neuron is great. It is extremely difficult to control the pressure applied to the neuronal surface by the tip of the recording electrode. This situation illustrates how risky it is

5 msec Fig. 2. Action potentials recorded at surface of dendrite on stimulation of neuron soma. Stimulating shocks were 0.05 msec in duration, approximately 20 ,uA at threshold and repeated at rate of 7/sec. Arrangement of electrodes (both extracellular) and major, clearly discernible, portion of dendrite are shown. Ten-day-old culture of cat cerebellum. Ax = axon; S = stimulating electrodes; R = recording electrode. (From J. Neurophysiol., 25 (1962) 290).

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to interpret the configuration of electric responses recorded without direct visual observation of the neurons under study. The electric responses recorded from the surface of neurons show a strong negative phase. This negativity indicates that there is an inwardly directed current in the portion of the membrane under the recording microelectrode. There is little doubt that the surface membrane of the cell body of a neuron in tissue culture is electrically excitable. It is possible to elicit action potentials of the cell body by stimulating the neuron directly or by delivering electric shocks to the dendrites. A recording microelectrode pressing the surface of a dendrite often picks up spontaneously fired action potentials. If, under such circumstances, stimulating electric shocks are applied to the neuron soma or to some portion of the dendrites of the same neuron, it can be shown that ‘dendritic potentials’ obey the all-or-none law, At least in large cerebellar neurons under tissue culture, the basal portions of the dendrites-are capable of carrying nerve impulses without decrement. Fig. 2 illustrates an example of the all-or-none responses induced by stimulation of the cell body and recorded from the surface of a dendrite. It is interesting to note that the conduction velocity along the basal portion of the dendrite is of the order of 0.1 msec, that is, roughly 1/1000 of the velocity in the largest mammalian myelinated nerve fiber. There has been controversy among neurophysiologists concerning the excitability of the neuron soma and dendrite. Some investigators believe that the membranes of the cell body and dendrite do not participate in production of action potentials (Freygang, 1958; Furshpan and Furukawa, 1962). Others believe that the surface membrane of the neuron is excitable (Fatt, 1957; Araki and Terzuolo, 1962). As far as neurons in tissue culture are concerned, the problem is now settled: the soma and dendrite membranes are electrically excitable. Some physiologists, however, seem to believe that the neuronal membrane is inexcitable in vivo because of the presence of numerous synapses which are absent in tissue culture material. Physiological properties of glial cells

It is well-known that glial cells in tissue culture show spontaneous pulsatory movements (Canti et al., 1935; Pomerat, 1951; Hild, 1954; Chang and Hild, 1959). We have seen that a mass of glial cells in tissue culture respond to strong electric shocks with a slow contraction. The phase of active contraction is 1 to 3 min in duration and the phase of relaxation is of the order of 10 min. Occasionally, we have seen synchronous twitches in the mass of glial cells in the absence of external stimuli. For demonstration of contractability of glial cells, it is necessary to use the timelapsewmovie technique because of the sluggishness of the process. A preliminary report on glial contractability has been published by Chang and Hild (1959). Fig. 3 is reproduced from their paper. Attempts were made to demonstrate contractions of glial cells in freshly excised brain slices. Two methods were devised for this purpose: the first method is to detect slow changes in optical properties of the brain slice in response to electric stimulation. References p . 241/242

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Fig. 3. Three selected film frames of a motion picture sequence showing the effect of electrical stimulation on an isolated astrocyte. Eight-day-old culture of kitten cerebellum. Phase contrast. Stimulating electrode with a tip diameter of approximately 12 p ; distance from the cell body approximately 24 p. (a) Immediately before stimulation. The cell body shows normal size and configuration; (B) 1.7 min after stimulation. The cell body is markedly contracted and the processes slightly thickened due to shifting of cytoplasm from the cell body into the processes; (C)7.5 min after stimulation. The cell body has expanded again and regained its original configuration. The total duration of this contractile response was 7.5 min which represents the shortest duration observed in the course of this investigation. (From J. cell. comp. Physiol., 53 (1959) 142).

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If a dense layer of glial cells undergo a slow contraction following electric stimulation, one might expect a change in the light intensity scattered by the tissue; measurement of the intensity of the scattered light with a photomultiplier tube will then give information about the contractability of the glial cells. An alternative method is to measure the electrical impedance of a brain slice at the time when a slow contraction of the mass of glial cells is expected to take place. The results obtained by these two different methods were consistent; there was a clear indication thzt the light-scattering property undergoes a transient change following a strong electric shock. The duration of the ‘contraction phase’ as detected by the optical method was close to that seen in tissue culture material. During the ‘contraction phase’ there was a rise in the electric impedance of the brain slice measured with alternating current of 60 to 120 cycles/sec. The time course of ‘relaxation’ was obscured by a slow, continuous change in optical and electric properties of the slice. The resting membrane potential of a glial cell can be determined by piercing the cell with a glass microelectrode. Both with tissue culture material and brain slices of the cat, values between 50 and 70 mV (negative inside) can be obtained in clean penetrations. The resting potential can be reversibly reduced by raising the potassium-ion concentration in the surrounding medium. The membrane resistance of the glial cells can be determined byrintroducing two glass microelectrodes into a single glial cell in tissue culture. The surface membrane of a glial cell of about 15 p diameter shows a resistance of 0.6 MQ to 1.7 MQ to penetrating electric current. The membrane obeys Ohm’s law in a wide range of current intensity under these experimental conditions. Multiplying the surface area of a glial cell by the resistance mentioned above yields a membrane resistance for a unit area of the order of 10 Qcm2. This value is far smaller than the generally accepted value of the membrane resistance of various neurons. The estimation of the surface area of a glial cell is difficult; therefore our estimation of the membrane resistance for a unit area is subject to a large error. The time constant of the glial membrane is of the order of 0.5 msec. This value, combined with our estimation of the membrane resistance, leads to an unexpectedly large membrane capacity. It is at present impossible for us to evaluate the effect of the glial cell processes upon the electric measurements. It is possible, therefore, that a large correction has to be introduced when the effects of these numerous processes are taken into proper consideration. A strong electric shock applied to a glial cell with a large extracellular electrode is known to bring about a reversible, transient reduction in the resting potential of the cell. The degree of reduction depends on the intensity of the applied shock. There is evidence indicating that this reduction in the resting potential is associated with a simultaneous reduction in the membrane impedance. The intensity of current needed to induce such a potential variation seems too large to link the phenomenon with physiological events in living brain tissue. However, the ‘electric responses’ of glial cells appear to signal the beginning of a sequence of events leading to contractile responses. The temporal relationship between the electric and contractile processes in the glia1:cells is in some respect similar to that observed in smooth muscles. References p . 2411242

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Some observations on amoeba and slime molds With a view to elucidating electrophysiological properties of single cells that demonstrate primitive contractile movements, various measurements were made on Amoeba proteus, Chaos chaos and on Physarum polycephalum. There were a number of similarities between mammalian glial cells and these primitive cells. The resting potentials of the amoebae have been determined with glass microclectrodes and were found to be between 40 and 70 mV (Bingley and Thompson, 1962). The membrane resistance and capacity of the amoebae were determined with two intracellular microelectrodes; the values of approximately 1 to 3 K O cm2 and 10 pF/cm2 were obtained (Tasaki and Kamiya, 1964). In response to strong electric shocks, a kind of localized response could be induced. In the absence of external stimuli, both amoebae and slime molds showed slow but vigorous electric activity.

I

I

0

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10 15 TIME (Min)

I

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Fig. 4. Time course of potential difference across an amoeba (upper trace) and simultaneous hydrostatic pressure difference required to keep the amoeba stationary (lower trace). (From Tasaki and Kamiya, 1964).

Fig. 4 is an example of records showing the time course of the potential difference across an amoeba (upper trace) and the hydrostatic pressure difference required to keep the cell in a small capillary (lower trace). The upper trace of the figure shows a succession of rapid potential changes repeated at irregular time intervals. There are simultaneous changes in the membrane impedance associated with the potential changes. The potential changes, produced by depolarization localized in limited areas of the membrane, are suppressed by application of anesthetics, KC1, etc. Attempts are now being made to see if there are spontaneously induced potential changes in mammalian glial cells. The lower trace in Fig. 4 can be regarded as a measure of the motive force of amoeboid movement. It is interesting that these movements in amoebae can not be suppressed by application of anesthetics or of solutions of MgClz or CaC12. This situation is analogous to that in the mammalian glial cell; previously, we have seen

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that glial cells in tissue culture continued to pulsate under the action of anesthetics and divalent cations. SUMMARY A N D CONCLUSION

Various technical difficultiesare encountered in the electrophysiological investigation of neurons and glial cells in the mammalian central nervous system under direct visual control. Using a tissue culture technique and a method of preparing thin brain slices, progress has been made in research along this line. The surface membrane of the soma and dendrites of neurons in tissue culture is capable of responding to electric stimuli with all-or-none action potentials. Electric responses have been recorded from neurons in brain slices of newborn guinea-pigs. Glial cells in tissue culture sometimes respond to strong electric stimulation with a slow contraction followed by a gradual relaxation. The duration of the contractile phase is 1 to 3 min and that of relaxation is of the order of 10 min. Experimental evidence is presented indicating the existence of contractile responses in the glial cells in brain slices. In a dense mass of glial cells, there are occasionally synchronous contractions in many cells in the absence of external stimuli, suggesting the presence of some kind of physiological interaction among the cells. It is interesting that the duration of this slow contractile process in glial cells is comparable to that of Leao’s spreading depression in the cerebral cortex. The resting potential of glial cells in tissue culture and in brain slices has been determined with the use of glass microelectrodes. The membrane resistance and capacity have been estimated by introducing two microelectrodes into single glial cells. The membrane of the glial cell appears to be far more permeable to various cations than the neuronal membrane. Strong electric shocks applied to glial cells with an extracellular electrode produce a reversible reduction of the resting potential; this potential change appears to be the first step in the sequence of events leading to a contractile response. Similarities are pointed out between mammalian glial cells and fresh water amoebae. REFERENCES

T., AND TERZUOLO, C. A., (1962); Membrane currents in spinal motoneurons associated with ARAKI, the action potential and synaptic activity. J. Neurophysiol., 25, 772-789. BINGLEY, M. S., AND THOMPSON, C. M., (1962); Bioelectric potentials in relation to movement in amoebae. J . theor. Biol., 2, 16-32. D. S., (1935); Tissue culture of gliomata. CinematoCANTI,R. G., BLAND,J. 0. W., AND RUSSELL, graph demonstration. Ass. Res. nerv. Dis.Proc., 16, 1-24. CHANG,J. J., AND HILD,W., (1959); Contractile responses to electrical stimulation of glial cells from the mammalian central nervous system cultivated in vitro. J . cell. comp. Physiol., 53, 139-144. ECCLES, J. C., (1957); The Physiology of Nerve Cells. Baltimore, Johns Hopkins Press. FATT,P., (1957); Electric potentials occurring around a neuron during its antidromic activation. J . Neurophysiol., 20, 27-60. FRANK,K., AND FUORTES, M. G. F., (1961); Excitation and conduction. Ann. Rev. Physiol., 23, 357-386. FREYGANG, W. H., JR., (1958); An analysis of extracellular potentials from single neurons in the lateral geniculate nucleus of the cat. J . gen. Physiol., 41, 543-564.

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FURSHPAN, E. J., AND FURUICAWA, T., (1962); Intracellular and extracellularresponses of the several regions of the Mauthner cell of the goldfish. J. Neurophysiol., 25, 732-771. &D, W., (1954); Das morphologische, kinetische und endokrinologische Verhalten von hypothalamischem und neurohypophysarem Gewebe in vitro. Z. Zellfrsch., 40,257-312. HILD,W., AND TASAKI, I., (1962); Morphological and physiological properties of neurons and glial cells in tissue culture. J. Neurophysiol., 25, 277-304. POMERAT, C.M., (1951); Pulsatile activity of cells from the human brain in tissue culture. J.nerv. ment. Dis., 114, 4 3 W 9 . SPYROPOULOS,C . S., AND TASAKI,I., (1960); Nerve excitation and synaptic transmission. Ann. Rev. Physiol., 22, 407432. TASAKI, I., AND KAMIYA, N., (1964); A study on electrophysiological properties of carnivorous amoebae. J. cell. comp. Physiol., 63, 365-380.

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Nervous Function Based on Interactions Between Neuronal and Non-Neuronal Elements G. SVAETICHIN, K. NEGISHI, R. F A T E H C H A N D , B. D. D R U J A N A. S E L V f N D E TESTA

AND

Department of Neurobiology, Instituto Venezolano de Investigaciones Cientifcas (IVIC) , Caracas (Venezuela)

1. I N T R O D U C T I O N

Until recently, theories for nervous function have solely been based on the neuron doGtrine which largely reflects the view of Cajal deduced from his silver impregnated sections. The millisecond spike process, the ion theory and the pumps, electrical excitability and ionic current flow, synaptic transmission, reverberating circuits, etc., refer to properties of the neuron, and present day neurophysiology is in fact exclusively the physiology of the neuron. According to this orthodox view, the function of the glial cells, which outnumber the nerve cells in nervous tissue, is restricted to the mechanical support and electrical isolation of the neurons. However, the early suggestions by histologists of a nutritional role of the glial cells, and the recent suggestions based on electron microscopy that the glial cells form the extraneuronal ionic space (De Robertis and Gerschenfeld, 1961; Sjostrand, 1960, 1961), have contributed to elevate the importance of the neglected glial cell. Further, the studies by HydCn and Pigon (1960) and Cummins and HydCn (1962), on the biochemical systems of neuronal and glial cell fragments separated from each other by microsurgery, demonstrate the existence of complementary metabolic systems in the two different types of cells, while the histochemical studies of Friede and his co-workers (1954, 1962a, b, 1964), have produced important information concerning the enzyme distribution in glia and neuron. All this is strong evidence that the glial component is functionally as important as the neuronal. At the symposium on Neurophysiology and Psychophysics of the Visual System held in Freiburg (Laufer et al., 1961; Mitarai et al., 1961; Svaetichin et al., 1961) and also at conferences given in the U.S.A., we presented our electrophysiological studies on the behaviour of different types of glial cells in the fish retina (the S-potentials). We also outlined the significance for nervous function in general of these glial structures, which display characteristics very different from the ‘all or none’ behaviour of the neuron. The presentation of our experimental evidence for a functional interaction between References p . 263-266

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glial cells and neurons produced some repercussions among neurophysiologists. Galambos (1961) wrote an exciting article with the title ‘A glia-neuronal theory for brain function’, and subsequently, with an open mind, he has been accumulating information which supports the functional importance of the glial cells. It should also be mentioned that Rushton (1962, 1963) has emphasized the importance of the glial cells in several reviews on the physiology of vision. 2.

RETINAL

S-POTENTIALS, AND THEIR LOCALIZATION EL E M E N T s (co N TR o L LER c E L L S)

TO NON-NEURONAL

Neurophysiology is to a great extent dependent on the information obtained by electrophysiologicalmethods (microelectrodes), since, so far, this is the only technique available for studying at the cellular level the rapid, millisecond range events, which reflect the functional interactions between intact and normally functioning cells. The lack of localization on the cellular level of the site of recording of the microelectrode, introduced into the depth of central nervous tissue, has for a long time hindered the interpretation of the recordings obtained. An electrophoretic dye method for the marking of individual cells (Svaetichin, 1951) was used by MacPherson et al. (1957)for the identification of the origin of the S-potentials in the fish retina. Similar methods were employed by Mitarai (1958), Oikawa et al. (1959), Tomita et al. (1959), Gouras (1960) and Brown and Tasaki (1961). The use of lithium carmine for the marking dye, introduced by Mitarai (1958), represents an important improvement. Two classes of S-potentials have been described in the fish retina, namely the L (Luminosity) and the C (Chromatic) responses, both being graded and non-linearly related to the intensity of the light stimulus. The different types of S-potentials, and their significance to retinal physiology, are discussed in the recent reviews by Motokawa (1963) and Tomita (1963), a field to which these authors have so substantially contributed. The hyperpolarizing L-responses originate in the horizontal cells (which are 50-100 times larger than the bipolar cells); the C-responses (4 different types observed), which are hyperpolarizing or depolarizing depending on the wavelength of the stimulus light, originate in different types of large glial cells located in the amacrine cell layer and at the level of the ganglion cells. One type of C-response, showing hyperpolarization responses for short wavelength stimuli, seems to originate in the radial Miiller fiber. However, the complex structure of the retina in this particular region revealed in recent microscopical studies in this department (Selvin de Testa, 1965) indicates that additional microelectrode localization experiments are necessary to establish definitely the site of origin of this C-response. It is interesting to mention, that by the use of the superfine micropipette electrodes developed by Naka et al. (1960) in Tomita’s laboratory, we have obtained (Svaetichin and Negishi, unpublished) at the ellipsoid level of the receptors transmembrane potentials which have characteristics similar to the S-potentials (Svaetichin, 1953). These apparently intracellular recordings within the receptor layer, have many properties in common with the Spotentials, and with the receptor potentials recorded across the receptor layer after

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NH3 application (Fatehchand et al., 1962), which are restricted to the illuminated area (Mitarai et al., 1961). As regards the classification of the above mentioned cells which produce the Spotentials, we would like to make the following comments on the basis of the recent morphological studies by Selvin de Testa (1 965) using various silver methods, etc., and on the ultrastructural studies of Gloria Villegas (1961) at this Institute. One of Cajal’s requirements for calling a cell a neuron, was the presence of an axon. The cells which give rise to the C-types of S-potentials have no axons and are morphologically non-neuronal, possibly corresponding to interstitial amacrine and displaced elements (amacrine cells of Cajal, 1952). Like the horizontal cells, these cells form networks which fill up the space around the typical neuronal structures of the retina. The nuclei of these non-neuronal elements are elongated and the cytoplasm is generally fibrillar, being seen in the electron microscope as astrocytic filaments. The cytoplasm of these non-neuronal cells does not stain with methylene blue using Ehrlich’s vital or supravital method (not even after anoxia or after fixation of the tissue). The methylene blue technique of Ehrlich is specific for neuronal structures (Cajal, Dogiel, Retzius). The ganglion cells, bipolars and neuronal type of amacrine take the stain well, while the cytoplasm of the non-neuronal cells remains colourless. No dendritic, axonal, or synaptic structures of the horizontal cells in the fish retina have been observed with electron microscopy (G. Villegas, 1961; G . Villegas and R. Villegas, 1963) or light microscopy (Selvin de Testa, 1965). Concerning the middle horizontal cell H2 (Figs. 1 and 2) it is particularly evident, that it can receive its specific spectral information (Section 7) only by means of non-synaptic contacts with radial elements, which pass through the mesh formed by the tangentially oriented horizontal cells. In our earlier studies on the localization of the microelectrode tip (Svaetichin et al., 1961), a shorttime fixation of the tissue in Bouin solution was followed by freeze-sectioning without dehydration, followed by mounting in glycerol. This method proved to preserve the large ‘watery’ non-neuronal structures without shrinking, the cells showing an ‘almost live’ appearance, while dehydration methods, staining with heavy metals, etc. produce pronounced deformations of the non-neuronal structures and the intercellular relations. Fig. 1 is a schematic diagram of the fish retina, drawn from a radial section. The microphotographs seen in Figs. 2, 3, and 4 illustrate the neuronal (dark) and non-neuronal (light) structures of the fish retina as seen in tangential sections. Fig. 2, at level of the internal horizontal cells, corresponds to layer H2 of Fig. 1. Fig. 3, at the interstitial amacrine cell level, corresponds to layer AM4 of Fig. 1. Fig. 4, at level of the displaced amacrines, corresponds to layer DAm of Fig. 1. The isolated retina (Mugil brasiliensis) was fixed for 1-2 h in Bouin solution and the sections (freeze microtome), after being washed in water were stained by methylene blue, and mounted in glycerol. In these reproductions from colour prints, the neuronal elements, which appear dark, were blue in the original, whereas the nonneuronal elements, appearing light, were colourless in the original colour positives. We are well aware of the difficulty in classifying cells as neurons and glial cells (in the fish retina) on the basis of morphological studies alone. The radially oriented receptor-bipolar-ganglion cell chain is a typical neuronal conductor line, and in the References p . 263-266

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Fig. 1. Schematic diagram of the fish retina (from Selvin de Testa, 1965). The scheme is based on light microscopical studies in this laboratory. The retina is seen in a radial section. m.1.e. = membrana limitans externa; e.p.1. = external plexiform layer; H1, H2, H 3 = horizontal cells; Am1 = a new type of amacrine cell described by Selvin de Testa; Am2 = glial amacrine (G. Villegas, 1961); Am3 = neuronal amacrine; Am4 = interstitial amacrine; i.p.1. = inner plexiform layer; DAm = displaced amacrine; GC = ganglion cell; m.1.i. = membrana limitans interna; M = Muller fiber.

amacrine layer there exists an abundance of fiber structures running tangentially, which undoubtedly are nerve fibers originating from nerve cells in this layer. In addition to these neuronal structures, there are in the fish retina large cells which form networks of a tangential orientation, and further there are the radially oriented Miiller fibers which are classified as glial (or supporting) cells. These large cells forming the tangential networks cannot on a morphological basis be classified as neurons (Selvin de Testa, 1965). In the retina from fish which inhabit turbid water, and which do not have a high visual acuity, the cell size is unusually large; there are large cones,

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Fig. 2. Microphotograph reproduced from colour positive, showing horizontal cells (H2, Fig. 1)

in tangential section of fish retina (Mugil brasiliensis).Notice inthe center of the picture the elongated nucleus of the large horizontal cells, the cytoplasm of which is unstained. Neuronal elements passing through meshes formed by horizontal cells are deep blue in the colour positive.

Fig. 3. Microphotograph reproduced from colour positive showing tangential section through amacrine cell layer of fish retina (Mugil brasiliensis). The network is formed by the large interstitial amacrine cells (AM4, Fig. l), the cytoplasm of which is fibrillar and unstained by methylene blue. The neuronal structures seen in the meshes of the network are deep blue in colour positive, while Miiller fiber and glial amacrines (M andAM2, Fig. 1) are unstained. References p . 263-266

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Fig.4. Microphotograph reproduced from colour positive showing slightly oblique tangential section through ganglion cell layer of fish retina (Mugil brasiliensis). The network is formed by displaced amacrine cells @Am, Fig. 1). The cytoplasm of these cells is fibrillar, and unstained in colour positive. Four ganglion cell bodies are seen in the meshes of the network, the cytoplasm being deep blue in the colour positive.

large horizontal cells (Fig. 1: HI, H2, H3), few bipolars grouped around the Muller fiber (Fig. 1: M), few ganglion cells of large size, and tangentially oriented cells of an impressive size located at the external (Fig. 1 : Am4) and internal (Fig. 1: DAm) borders of the inner plexiform layer. The retina of clear water fish, having high visual acuity, contains a large number of small cells per unit volume. In such a retina, which rather looks like a frog retina, the tangentially oriented non-neuronal elements have minute dimensions, the bipolar layer is filled with small cells, and the grouping of the bipolars around the Muller fiber cannot be observed. As pointed out by Mitarai (1958) the recording of S-potentials from such a retina is not possible, apparently due to the small size of the cells. The part of the retina which is close to the receptors, say the bipolar and the horizontal cell layers, could possibly be considered a special retinal structure, while on the other hand the rest of the retina must be considered as corresponding to the central nervous system. Therefore, it can be concluded that the two fundamentally different types of cell, the neuronal and the non-neuronal, together form in the retina

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a functional system which also exists in the gray matter. Because of the uncommonly large size of the non-neuronal elements in the retinae of fish inhabiting turbid water, it has been possible to obtain intracellular recordings from these peculiar cells, these cells being accessible to studies in light microscopy. Non-neuronal elements of this kind having a giant size occur rarely in other parts of the central nervous system. The non-neuronal elements identified in the fish retina as horizontal cells, amacrine cells, displaced amacrine cells (Cajal, 1952), have by a combined electrophysiological and biochemical approach been shown to have characteristics very different from typical neurons. We have reached the conclusion that extracellular recording techniques cannot be used for studies of the electrical activity of these cells. We believe this is the reason why the functional activities of such non-neuronal elements have not been observed in other parts of the central nervous system, where the small size of these cells makes intracellular recordings difficult. Most likely, corresponding non-neuronal networks form an important functional part of the gray matter, being represented by elements mainly of a tangential (horizontal) orientation (horizontal cells, ‘short axon’ cells, etc.; see Cajal, 1952). The functional organization of the colour vision system of fish is based on the interaction between the typical neuronal and receptor elements with the non-neuronal controller elements described above. We have shown in our electrophysiological studies (Svaetichin et al., 1961) that the colour system of the fish retina is identical with that of primate and man. On the basis of the wavelength information obtained from three different cones (Young-Helmholtz theory) the opponent colour processes are created in the subsequent retinal stages (Hering-Miiller-Schrodinger-JuddHurvich theory). The spectral response curves registered from the different nonneuronal elements located on both sides of the inner plexiform layer, viz. DAm, Am4 and probably Am2 (Fig. l), agree with the predictions of the stage theory for colour vision which is based on psychophysical studies of man. This is further support for our view that corresponding non-neuronal (controller) networks exist also in the retina of man, although the cells are minute in size. It is interesting that the horizontal cell network described by GloriaVillegas (1961) in the extra-foveal region of the human and primate retina has dimensions below the resolving power of the light microscope. It is thus obvious that in the fish retina there are several types of cells giving electrical responses to light stimulation, but which nevertheless are impossible to classify as neurons. These cells are in intimate membrane to membrane contact with the receptors and neurons, thus supporting their classification as glial elements, and can be termed controller cells, for reasons which will become evident. 3.

ELECTROPHYSIOLOGY OF GLIAL CELLS I N NON-RETINAL TISSUE

If the retina is not considered, the only electrophysiological studies on structures known to be glial cells of which we are aware are those by Hild and Tasaki (1962) on tissue culture, by Villegas et al. (1962) on what they assume to be the Schwann cell of the squid giant axon, and recently by Kuffler and Potter (1964) on the leech central nervous system. However, Hild and Tasaki (1962) studied glial cells from tissue culture References p. 263-266

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in which the normal structural relationship between cells is lacking. Under these conditions it is probable that the glial-neuronal interaction would not be observable. We will first discuss the microelectrode experiments by Kuffler and Potter (1964) on the large glial cells in the leech central nervous system. In these studies intracellular recordings were difficult to maintain unless the spontaneous movements (glia or muscle) of the tissue were prevented by the following methods. We quote from Kuffler and Potter (1964, p. 293): 'The most successful of these agents was 7-8% ethanol. Lowering the temperature to 5-7" probably also helped'. As regards ethanol, we have found that even if much smaller concentrations than those used by Kuffler and Potter (1964) were applied to the retina, the glial membrane potential immediately increased and the responses to light were abolished. Concerning the temperature, as we point out subsequently, the glial cells are very unlikely to be operative when cooled down to 7-8", which in Kuffler's experiments depressed the contractions. As regards the experiments by Villegas et al. (1962), the glial satellite has a 'cell thickness varying from 0.1-0.2 ,u near the ends to 0.8-0.9 ,uin the region of the nucleus' (Villegas et al., 1962, p. 75). The (lithium carmine-KC1 filled) electrode used had a resistance of 5-15 megohm, and it can be deduced that the electrode tip diameter was of the same order as the cell thickness. This makes it most unlikely that a functioning glial cell was studied. In this connection we should mention our experience with satellite (glial) cells surrounding the dorsal root ganglion cell body. These glial cells have a thickness of about 2 ,u, and we found that even with 500 megohm KC1 (2.5 M) filledelectrodes, it was impossible to obtain recordings of the membrane potential (frog). 4.

I N F L U E N C E OF G L I A L F U N C T I O N I N G O N N E U R O N A L PR OC ESSES

Systematic studies have recently been carried out in this laboratory by Negishi et al. (1965) on typical neurons (cell bodies of the dorsal root ganglion and sciatic axons) showing that the effects of temperature change, anoxia, HCN, N3H, Diamox and NH3 etc. are basically different in the neuron and in the glial controller cell of the retina. Preliminary work along these lines was carried out by Laufer et al. (1961). In the retina, controller cell resting potential and responses to light appear to be directlyrelated to aerobicmetabolism (see Figs. 4,7 and 10 in Svaetichin et al., 1963), and electric current does not affect responses (Watanabe et al., 1960), whereas the opposite is true for the neuronal membrane. As an example, we have found that anoxia immediately blocks glial activity in the retina, while the cell recovers almost instantaneously when oxygen is resupplied. On the other hand the 'all or none' spike process of the neuronal cell body and axon continues for at least half an hour in the absence of oxygen. Further, in the retina the glial cell function is critically dependent on the temperature being kept within a narrow optimum range (for tropical fish 18-23"), whereas neuronal function is very little influenced over a large temperature range. However, within a temperature range corresponding to the previously mentioned optimal range for the glial cell, temperature change does have a small but definite effect on neuronal resting level and on the hyperpolarizing after-potential, which we believe is related to a temperature effect on the glial satellite cell. The small but

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definite changes of the neuronal membrane potential and the hyperpolarizing afterpotential observed under anoxia, pC0z changes, NH3 application, etc., also appear to reflect changes of the glial satellite function (Laufer et al., 1961; Negishi et al., 1965). 5.

STEADY POTENTIALS

Under different adaptational states of the retina and under different experimental conditions, simultaneous recordings of the membrane potential changes in the different types of retinal non-neuronal (controller) cells, and the transretinal D C level changes, show a clear relationship between the two variables. Fig. 5 illustrates

A

B

C

Fig. 5. Simultaneous transretinal (R) and glial (G) recordings illustrating glial-control of retina, excitability. The 6 mvcalibration to the left corresponds to the transretinal (R)recordings, and the scale040 mV is for the glial membrane potential (G). At the beginning of the record (A) the transretinal D C level is +0.5 mV, showing small upward deflections evoked by white light stimuli; the glial membrane potential is -30 mV with 10 mV responses (downwards) to light stimuli. Exposing the retina to a minute amount of NH3 (arrows) immediately affects the transretinal and glial potentials. When the glial membrane potential depolarized to about -20 mV, it caused a doubling of both the glial (G) and the transretinal (R) responses to light. Within about 40 sec after NH3 application, the glial function was abolished, resulting in large increases of the transretinal D C level and the responses to light (records B, C). These large responses to light are produced by the photoreceptors only. A concentration of NH3 in tissue of less than 0.001 % is sufficient to produce the effect. The two abrupt upward steps in A and C (tracing R) are due to manual shifts of the DClevel.

the effect of a very low concentration of NH3 gas on the isolated fish retina. The transretinal DC level (R) and the light-induced responses increase simultaneously with the reduction of the controller cell membrane potential (G). When the membrane potential decreased, the light-induced responses increased, indicating a diminished control of the retinal excitability. The NH3 gas finally blocked the controller cell function, the membrane potential showing a slow decay towards zero. The transretinal recordings of the light-induced responses are in the normal untreated retina nodinearly related to the light intensity (semi-logarithmic relationship) whereas the large amplitude transretinal responses, produced after blocking the controller cell function show a linear relationship to the stimulus light intensity (Fatehchand et al., 1962). It was shown in earlier studies from this laboratory that the large light-induced responses isolated by NH3 application originate across the receptor layer only (within 80 microns from the surface of the exposed receptor layer), and that the transretinal References p . 263-266

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DC potential is then also restricted to the receptor layer. The transretinal DC level changes produced by NH3 and COe gases are in opposite directions, agreeing with the opposite effects of these gases on the membrane potentials of the controller cells (Laufer et af., 1961). A relationship between the transretinal potential and the membrane potential of the controller cells has also been found in connection with adaptation to light and darkness, temperature change, etc. From such experiments we conclude that the non-neuronal elements participate in the control of excitability in the retina (increment thresholds, non-photochemical adaptation, Weber-Fechner law), as also discussed by Rushton (1962, 1963). It would also seem that in the retina there is a feedback control system, in which the receptor forms the linear input element and the neuronal conductor the subsequent forward element, while the non-neuronal cells constitute the feedback element, as suggested by Vallecalle and Svaetichin (1961). The time lag in the feedback path would account for the oscillations often recorded in the controller cell (Laufer et af., 1961). It is interesting that cocaine increases the depolarizing response (C-response cell, Fig. 6) and also produces a tendency to oscillation, while it has also been shown

COCAINE 4 8min

Fig. 6 . Effect of cocaine on L- and C-types of response recorded from non-neuronal elements in fish retina. In A and B (C-type response), red light stimulus (R) induces depolarizing response and blue light stimulus (B) hyperpolarizing response. Record B, obtained 8 min after application of cocaine on the exposed receptor layer, shows a large increase and an oscillation tendency of the depolarizing response, while the hyperpolarizing response is unaffected. Records C and D, obtained from horizontal cell (Gtype response), show only hyperpolarizing responses to the alternate light stimuli, the cocaine having no influence on the behaviour of this cell.

that cocaine produces oscillation of the b-wave of the ERG (see Fig. 8, p. 142 in Ottoson and Svaetichin, 1952). Studies by Negishi et al. (1963) and Selvin de Testa et al. (1963) on the transcortical DC levels and the EEG under different experimental conditions make us believe that the transcortical DC level also reflects the interaction between neuronal elements and non-neuronal controllers. Both in the retina and cortex the DC level changes are associated with changes in excitability. We suggest that the activity of the non-neurona1 controller elements is only indirectly reflected by the DC level. The effect on the

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DC level is possibly due to a metabolic interaction between the neurons and the controller cells which induces a gradient in the membrane potentials of the radially oriented neurons (difference in membrane potential between dendrites, soma and axon). 6.

M E T A B O L I C D E P E N D E N C E OF C O N T R O L L E R C ELL F U N C T I O N I N G

The membrane potential level of the non-neuronal (controller) cell in the fish retina shows a dependence on the functional state of the retina. Studies in our neurochemistry laboratory by Drujan et al. (1964) show pronounced changes in the metabolites related to changes in the adaptational state of the retina. Hence, it appears reasonable to assume that the different membrane potential levels observed depend on different metabolic equilibria in light and dark adaptation. This suggestion is supported by experiments in which the controller cell membrane potential was changed by application of COz and NH3, by subjecting the retina to anoxia (Nz), to CO plus 10% carbogen, or to temperature changes. Under such experimental conditions the controller cell membrane potential and the light-induced responses were rapidly and pronouncedly affected, whereas corresponding experiments on neuronal structures reveal a different behaviour of the nerve cell membrane (see Section 4). Fig. 7A illustrates the high sensitivity to temperature changes of a horizontal cell membrane potential and its light-induced responses. Heating the retina from 23" to 32" increases the membrane potential by 42 mV ( 5 mV/degree), indicating that the TOC mV

Fig. 7. Recordings in A show effect of temperature change (tracing T) on the membrane potential and the hyperpolarizing light-induced responses of a non-neuronal (controller) cell in fish retina. B shows that a similar effect to temperature increase is produced on the controller cell by exposure to COZgas. (Fig. B from Laufer et at., 1964). References p . 263-266

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membrane potential increases when the metabolic systems are accelerated (enzymes). Fig. 7B shows a corresponding increase of the membrane potential produced by C02, this gas apparently also (in certain concentrations) accelerating the metabolic systems. The temperature effect and the C02 effect were immediate, indicating that the membrane potential is directly coupled to metabolism. The same record shows that the horizontal cell membrane potential is reduced by cooling and increased by heating, a constant finding for this type of cell. However, there is another kind of controller cell, found in the amacrine layer, which shows responses to light having a polarity which depends on the wavelength (C-type). The membrane potential and the lightinduced responses of this type of cell have a more complex dependence on temperature. Depending on the state of the retina, the membrane potentials can be either reduced or increased by cooling. The behaviour of this kind of controller cell indicates that the two kinds of light-induced responses (depolarizing and hyperpolarizing) are due to the interaction between two adjacent controller cells. We conclude from this and other experiments that the hyperpolarizing response originates in the cell which has been penetrated by an electrode, and that the opposite depolarizing response is impressed on this cell from the adjacent one. We would also suggest that the membrane potential change of the adjacent cell is reflected in an opposite direction into the other cell. This would account for the complex behaviour, the direction of the shift caused by temperature changes being determined by which of the two metabolically dependent membrane activities is dominating. The complex temperature dependence of this type of controller cell is illustrated by the recordings in Fig. 8. We show in Fig. 12 the suggested interaction between the two adjacent cells. This interaction, involving reciprocal excitation and inhibition, is suggested to hold in general for the nervous system.

Fig. 8. Effect of temperature change on C-type of response recorded from two different non-neuronal elements in fish retina. Trace (T) is the temperature recording, and (G) is the record of membrane potential and light-induced responses. Alternate short and long wavelength light stimuli respectively produce hyperpolarizing and depolarizing responses of the controller cell membrane. In one cell (A-B) the non-neuronal membrane responds to cooling with a hyperpolarization, while in the other cell (C-D) the membrane responds to cooling with a depolarization.

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The behaviour of the controller cell membrane potential and the light-induced responses under temperature change show that there is a temperature optimum for the function of the cell which is rather narrow (15-25"), corresponding to the environmental temperature range of tropical fish. This indicates that the electrical activity of the controller cell directly reflects metabolic rate (enzyme temperature dependence). We have observed that the receptor potential (recorded across the receptor layer after NH3 application) is also strongly temperature dependent, showing the same optimum range. Other studies in which the retina has been subjected to anoxia, or to CO plus 10% carbogen, show that the glial membrane potential is affected within seconds, the responses to light being abolished. Following anoxia, the membrane potential shows practically no temperature dependence (Fig. 9). We conclude that the glial membrane T°C

mV

-25sec

-

Fig. 9. The flow of N2 into the chamber started in A (arrow), and the controller cell membrane potential (tracing G) reacted immediately to the reduced 0 2 pressure. Within 1 min (B) when most of the 0 2 was replaced by Nz,the membrane potential increased to 55 mV, the light-induced responses being abolished. In B a temperature change (tracing T) still produced a small effect on the potential (cf. Fig. 7),while in C and D the influence was negligible. The potential showed a decay towards zero in C and D, while under 0 2 the membrane potential keeps stable. Under anoxia the controller cell potential behaves as a diffusion potential. Fast recovery was obtained if OZwas resupplied within 5-7 min, while anoxia for 1 C 1 5 min was irreversible.

potential is directly coupled to aerobic metabolism (Krebs' cycle, respiratory chain) and that after blocking the aerobic system, the membrane potential corresponds to a diffusion potential. This diffusion potential level is generally higher than the membrane potential under aerobic conditions. The addition or removal of hydrogen ions seems to be a powerful factor determining the functional state of the retina. Mitarai et al. (1961) showed that the glial membrane References p. 263-266

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potential is low (5-10 mV) in dark adaptation, and high in light adaptation(30-40mV). Laufer et al. (1961) reported that COz increased and NH3 decreased the glial membrane potential, while the effect on the neuron was opposite. Sickel (1961) demonstrated that an increase of pH produced dark adaptation while a decrease gave light adaptation. Comparative findings have been reported by Negishi et al. (1963) in our laboratory on the EEG of the cat. They found that CO2 provoked activation pattern while NH3 produced synchronization. The effect of COz can be prevented by application of Diamox; in other words, COz carries hydrogen ions to the metabolic system. Superficial anaesthesia with urethane or barbiturates can be arrested by COa and deepened with NH3. Negishi et al. (1965) have also recently found thar pC0z changes do not affect the membrane potentials recorded from the nerve cell bodies of the dorsal root ganglion dissected from Diamox treated frogs. This is significant since the carbonic anhydrase has been shown by Giacobini (1962) to be present in the glial cell but not in the neuron. Our experimental findings strongly suggest that COz and NH3 are the prime regulators of cellular respiration in the retina. It is known that CO2 and NH3 rapidly penetrate plasma membranes, while the corresponding ionic forms do not (Svaetichin et al., 1963). The position of COz as: (1) final product of metabolism, and as (2) regulator of the rate of the respiratory chain, make us think that C02 is a critical parameter in a feedback system which controls the glia-neuron functional interaction. A significant fact is that there is an optimal value (5-6 %) of pC02 for maximum rate of respiration (see Fig. 11 in Svaetichin et al., 1963). The horizontal cell and other non-neuronal (controller) cells in the retina must strongly depend on oxidative metabolism, since their membrane potentials and responses are very sensitive to oxygen lack and to the application of metabolic agents which interfere with aerobic metabolism. The carbonic anhydrase, located almost exclusively in the glia, is probably structurally associated with oxidative enzymes. Quastel (1957, 1959) showed that in cerebral cells, the oxidative enzymatic systems are localized in the plasmatic membrane, as well as in the mitochondria. The horizontal cell has extremely few mitochondria, and this suggests that the respiratory enzymes are mainly located in the plasma membrane. We consider the double membrane formed by the opposed glial and neuronal membranes (or the glial cell membrane alone) to be equivalent to the mitochondria1 membrane. In this connection, it is relevant that Cummins and HydCn (1962) have shown that 25 % of the total ATPase of the neuron is attached to the cytoplasmatic side of the neuronal plasma membrane, while the neuronal mitochondria are concentrated near the nucleus. Support for our view that the respiratory enzymes are mainly located in the plasma membrane of the glial cell is given by irradiation experiments. We have carried out preliminary experiments in co-operation with Dr. J. A. Velandia of this institute (unpublished) concerning the effect of y-irradiation of the retina from a cobalt source. It is known that mitochondria in muscle cells are destroyed by y-irradiation, while the plasma membrane and its potential are practically unaffected (Portela et al., 1960, 1963). Miquel (in this Volume) has demonstrated that irradiation causes swelling

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Fig. 10. Suggested scheme of a structural and biochemical framework for glial-neuronal interaction. Interaction occurs by means of the glia-neuron double membrane structure. An oxidation-reduction (redox) battery formed by the respiratory chain is shown as spanning both membranes, to schematically explain our findings of associated glial and neuronal membrane potential changes. The neuronal membrane potential is mainly determined by a ‘Donnan’ equilibrium (also shown by a membrane battery) although a small fraction (about 20 %, probably due to glial influence) is dependent on aerobic metabolism. The glial membrane potential strongly reflects the rate of aerobic metabolism, and abolition of aerobic metabolism reveals a membrane potential which is apparently a diffusion potential. The spike process and active ion transport processes (pumps) are shown on the right. The pumps are probably associated with the aerobic metabolism of the glial cell. The Krebs’ cycle is structurally associated with the respiratory chain and is therefore indicated as being located in the globular proteins attached to the plasma membrane. Cytoplasmic systems of aerobic glycolysis (glucose to pyruvate) and pentose cycle are indicated. The tube-like structure represents the endoplasmic reticulum and contains certain anabolic systems (e.g. protein synthesis and the glutamine pathway). The sites of action of the physiological regulators NH3 and COz are indicated. The rate of turnover of the Krebs’ cycle is increased by COz fixation (Berl et al., 1964); our experiments indicate that the carbonic anhydrase is indispensable for the effect of C02 on the glial cells. Dashed arrows associated with NH3 indicate anabolic pathways, while unfilled arrows associated with COZ and/or the absence of NH3 indicate catabolic pathways. Filled arrows symbolize pathways whose direction and rate can be changed depending on surrounding conditions. (From Svaetichin et al., 1963.)

only of the non-neuronal elements in the brain. We found that the membrane potentials and the light-induced responses of the glial cells were rapidly abolished by y-irradiation, whereas considerably higher dosages (8 times) had no effect on the membrane potential and spike process of the cell body of spinal ganglion, while the afterpotential was reduced. This would agree with our belief that the glial membrane is distinctly different from the neuronal membrane, and can be considered a mitochondrial equivalent. The biochemical literature supports our view that CO2 and NH3 are critical agents References p . 263-266

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which control the flow of substrate towards either protein synthesis or oxidative metabolism as is indicated in our diagram (Fig. 10) taken from Svaetichin et al. (1963). For example, it has been demonstrated that any detectable amounts of NH3 are sufficient to divert DPNH from oxidative metabolism respiratory chain towards protein or glutamine, etc. synthesis (Tower, 1960). We propose that such an action, reducing the rate of transfer of hydrogen and electrons in the respiratory chain would consequently produce a fall in the glial membrane potential. COz accelerates the Krebs’ cycle and the respiratory chain due to a production of hydrogen ions in the presence of carbonic anhydrase or due to fixation (Berl et al., 1964), and consequently increases the glial membrane potential. The close experimental relationship between glial membrane potential and rate of oxidative metabolism, and the fact that the glial membrane behaves as a linear resistance (Watanabe et al., 1960) strongly suggest that the membrane potential reflects the rate of electron transfer in the respiratory chain located in the plasma membrane, as we have earlier pointed out (Svaetichin et al., 1963). For a discussion of electronic conduction through membranes we refer to Jahn (1962, 1963). That the action of COZis to accelerate the Krebs’ cycle and that of NH3 to deaccelerate has recently been suggested (Berl et al., 1964) on the basis of biochemical studies of nervous tissues. We suggest that the inhibitory effects of the biogenic amines on the central nervous system (Marazzi, 1957) are possibly due to liberation of NH3 and its consequent effects on glial metabolism. Drujan et al. (1964) have recently demonstrated in our department that there is a significant decrease in the catecholamine content of the retina in light as compared to dark adaptation. The inhibitory processes seen in the retinal non-neuronal controller cells only appear in light adaptation (Mitarai et al., 1961),while the breakdown of dopamine increases in light adaptation (Drujan et al., 1964). Further, monoamine oxidase, which is responsible for the breakdown of the catecholamines, is inhibited by Marsilid, which is a known antidepressant drug. All the major tranquillizers such as reserpine, tetrabenazine, chlorpromazine, haloperidol, etc. are catecholamine releasers, or, as suggested by Carlsson et al. (1963), potent inhibitors of the uptake mechanism of catecholamines in tissue. In connection with our view of the role of glia in the above phenomena, it is significant that Mitarai (1962, personal communication) has shown that when the retina is light-adapted the activity of the monoamine oxidase in the Miiller fiber is increased. Since our evidence strongly suggests that there are significant changes in the glial cell metabolic rate when the retinal adaptational state is altered, it may be expected that there will be associated changes in the ATP content in the retina. Drujan et al. (1964) have found that this is in fact the case. Further, we have found (Svaetichin et al., 1963) pronounced and specific effects of drugs such as strychnine, cocaine, etc. on the retinal non-neuronal controller cells, indicating that similar structures are probably the sites of action of many neurotropic drugs on the central nervous system, as might be expected from the direct dependence of these non-neuronal cells on aerobic metabolism for correct functioning. In this connection it is significant that Mitarai et al. (1964) have recently shown that a small concentration of insulin applied to the retina has a pronounced influence on the behaviour of the glial membrane potential,

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while Kojima et al. (see Mitarai et al., 1964) have demonstrated that after insulin injection glycogen granules accumulate in the Muller fibers of the rabbit retina. It is significant that Friede (1954) has found changes in the glycogen content of the leech glial cells under stress.

7.

THEORETICAL EXPLANATION OF EXCITATION SPREAD I N CONTROLLER CELL N E T W O R K S

The light-induced response of the horizontal cell spreads a t 0.35 m/sec from cell to cell in each tangential network (Mitarai et al., 1961) of the fish retina in spite of the fact that there are no synaptic contacts joining the horizontal cells, and although the plasma membrane of these cells is electrically inexcitable. Each of the three horizontal cell networks (see Fig. 1) has its own characteristic response. Each of these characteristic responses has a different spectral response maximum, these maxima being in perfect agreement with the three different cone pigment absorption curves measured in the same fish (Svaetichin et al., 1965). The activities of these different controller cell networks influence the general excitability of wide areas in the retina surrounding a restricted illuminated spot (Mitarai et al., 1961). We therefore suggest that the levels of excitability in the central nervous system depend on similar spreading in the controller cell networks. One might even suggest that for instance in the optic nerve there is in addition to the transmitted neuronal spike information also a spreading of excitation control along the glial cell membrane (possibly reflected in the slow optic nerve potential, ventral root potentials, etc.). All this indicates the existence of excitation and propagation processes which cannot be accounted for by ‘local current flow’ and ‘synaptic transmitter’ theories valid for propagation and transmission in neuronal networks. The behaviour of the horizontal cell and other non-neurona1 controller cells is uninfluenced by ionic current flow. However, these cells are profoundly and directly dependent on the function of aerobic metabolic pathways and the respiratory chain. For the interpretation of such and similar phenomena in the central nervous system, the following theory is proposed (Fig. 11). The glialneuronal double membrane (or the glial membrane alone) is a mitochondria1 equivalent, of which the respiratory chain forms a fundamental structural component. The straight, oriented respiratory chains, which are polarized energy dipoles, cross the plasma membrane at regular spacings of 25-50A, the distances between the different cytochrome components of the individual chains also being about 25 A. Hydrogen free radicals (electron supply) are produced by the Krebs’ cycle in glia. The energy transfer across the membrane occurs by unidirectional (Forster) resonance transfer between nonidentical pairs of molecules within a respiratory chain, and between molecules of adjacent chains. The main lateral interaction is due to fast excitation transfer which tends to produce the same molecular excitation level within each individual crystal layer formed (laterally) by identical molecules of adjacent respiratory chains. The lateral interaction triggers the release of additional free hydrogen radicals from the Krebs’ cycle, the rate of electron transfer thus tending to be equal in all chains (Green, 1956) of the same cell membrane. This is a probable mechanism for References p . 263-266

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et al.

NEURON

[

B

!

GLlA Fig. 11. Schematic diagram of concept of molecular excitation spread along controller cell membrane, possibly also involving adjacent neuronal membrane.

excitation spread along electrically inexcitable membranes. However, such a system can only be based on a structural network relationship between respiratory chains and the Krebs’ cycle which enables a stepwise compensation of the energy degradation process of lateral transfer by an additional energy supply from the catabolic Krebs’ cycle. Such a mechanism could conceivably apply to photoreceptors, the initial excitation of one retinene molecule causing excitation of the whole receptor double lamella (Jahn, 1962,1963), this spreading along the ciliary process down to the plasma membrane of the ellipsoid and the proximal portion of the receptor. The lamellar portion of the cone probably does not need much extra energy supply from the Krebs’ cycle as it operates with an abundance of photons and free photopigment biradicals. Intercellular transfer of excitation (glia-glia, ellipsoid-ellipsoid, Muller fiber and receptor, glia-neuron, and possibly also in smooth and heart muscle networks) occurs only at specialized regions (‘transferapse’), where the opposed plasma membranes are partly fused (seen as 5 layers in permanganate fixation, Sjostrand, 1961). The above outlined theory offers a reasonable explanation for non-synaptic cellular interactions. Such a fast spread of molecular excitation over the whole cell membrane would make it difficult to record an extracellular signal, agreeing with our observations that the glial cell potentials are not directly reflected in the extracellular recordings and in the ERG in the retina. As mentioned above, the velocity of spreading in one of the horizontal cell layers in the retina has been measured to be 0.35 m/sec. The propagation of potential changes along the surface of the cerebral cortex is of the same order of magnitude (Lilly and Cherry, 1955). The dimension of a horizontal cell in the middle layer of the fish retina is 60-70 p, which gives a conduction time across the cell of only 0.2 msec. We suggest that possibly such a molecular excitation spread along electrically non-excitable plasma membranes of non-neuronal elements is important in controlling the spread of nervous excitability and inhibition in the gray matter and the basal nuclei, where the electrical insulation provided by myelin sheaths is largely

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lacking. Under such circumstances Kirchoff’s laws for the distribution of electrical currents would not provide sufficient specificity. Propagation of slow potentials correlated with neuronal excitability changes may be related to the non-neuronal excitation spreading. The diencephalic and mesencephalic reticular systems may also largely employ such mechanisms. Transcortical and transretinal DC level changes produced by externally applied currents have a very small effect on neuronal and photoreceptor excitability. Creutzfeldt et al. (1962) showed that the transcortical potential had to be increased by 10 times its normal value (by externally applied current) before affecting the neuronal firing rate. We have observed that the light-induced responses recorded across the receptor layer in the retina were not affected by large externally produced changes of the DC level which normally exists axoss the receptor layer. CONCLUSIONS

According to the views developed on the basis of our studies on the isolated retina, central nervous structures of the cat, isolated nervous tissue, and morphological studies, we propose that nervous function is based on an interaction between the neuronal conductors and the non-neuronal elements, the non-neuronal elements exerting an excitability and inhibition control on the neurons. The neuronal conductors function according to the classical concepts (Hodgkin-Huxley system) involving ionic current flow, membrane electrical excitability, and synaptic transmission. The non-neuronal elements form networks and make specific plasma membrane to membrane contacts with the neuronal elements. The plasma membrane of the nonneuronal controller cells is electrically inexcitable, and the membrane activity depends

Fig. 12. Schematic diagram of concept of the mechanism for reciprocal excitation-inhibition which involves the interaction between controller cells (Gl, G2) and neuronal elements ( N l , N2). NF indicates an afferent fiber which is assumed to influence the general excitability control level of the non-neuronal networks (non-specific activating system). References p . 263-266

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on the molecular excitation spread which can continue from cell to cell at specialized membrane to membrane contact regions. In isolated nervous tissue, and in the fish retina, we have obtained evidence that the non-neuronal elements control the excitability of the neurons in a specific way. In the retina, the reciprocal inhibition*xcitation system, which is responsible for the wavelength dependent firing pattern of the neurons, is apparently dependent on the controller cell system. Possibly the extensor-flexor control in the spinal cord is due to a similar mechanism. The scheme in Fig. 12 shows how we visualize such reciprocal excitatory-inhibitory systems.

SUMMARY

The cells originating the S-potentials in the fish retina are morphologically distinct from neurons, do not stain with methylene blue (Ehrlich method), are identified as horizontal cells, amacrjne cells and Miiller fibers, and form networks occupying the extraneuronal spaces. These cells also have functional characteristics which differentiate them from neurons : they are electrically non-excitable, their electrical behaviour (membrane potential and graded light-induced responses) has high temperature sensitivity within a narrow optimum temperature range (1 8-23' for tropical fish), and strongly depends on the maintenance of aerobic metabolism, while in anoxia the high temperature sensitivity is abolished. Retinal intracellular recordings indicate that the non-neuronal elements may be termed 'controller cells', since they interact with the receptors and neurons. The functional organization of the colour vision system of fish is based on such interactions. Three kinds of cones with differing spectral absorption characteristics give rise to opponent colour processes in subsequent retinal stages, the spectral responses of the non-neuronal cells agreeing with deductions from the psychophysics of human colour vision, and supporting the view that such controller cells also exist in the human retina. The hydrogen-ion concentration is an important factor in the functioning of the non-neuronal elements and their control of retinal excitability. The membrane potentials of the controller cells are reduced by NH3 and increased by CO2 in small concentrations, while these gases have opposite effects on neurons. An increase of pH changes the retinal state from light to dark-adapted, accompanied by reduction in the membrane potentials of the non-neuronal elements which now exert a diminished control on retinal excitability. The fall in membrane potential is accompanied by a shift in the transretinal steady potential (SP). Evidence that similar controller cells also affect the excitability of non-retinal nervous tissue is discussed. Experiments on dorsal root ganglion of frog indicate a probable coupling between satellite glial cells and the neuron, affecting the neuronal hyperpolarizing after-potential and a fraction of the membrane potential. Studies in cat of EEG and transcortical SP indicate that shifts in the SP are associated with excitability changes, and reflect, as in the retina, interactions between neurons and controller cells. CO2 provokes

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activation pattern of EEG, and NH3 produces synchronization, while these gases also produce shifts of opposite polarity in the SP. The dependence of the functioning of controller cells on aerobic metabolism, and their high temperature sensitivity (much reduced in anoxia), suggest that the electrical activity of these cells directly reflects metabolic rate; the opposite effects of small COZ and NH3 concentrations on the membrane potentials are explained by the influence of these gases on the rate of aerobic metabolism. It is postulated that for the nonneuronal cells, the oxidative enzymes are primarily located in the plasma membrane, and that the double membrane formed by the opposed neuronal and non-neuronal membranes (or by the non-neuronal membrane alone) is a mitochondria1 equivalent. This view is supported by irradiation experiments on muscle cells, brain tissue and the retina. The non-neuronal cells are, as are mitochondria, much more sensitive to irradiation than neurons, the results indicating that the non-neuronal plasma membrane is distinctly different from the neuronal membrane. Using the concept that the membrane potential of a non-neuronal element reflects the rate of electron transfer in the respiratory chain located in the plasma membrane, a lheoretical scheme is given for excitation spreading in controller cell networks, the tangential networks in the retina influencing the general excitabilityof wide areas surrounding a restricted illuminated spot. It is suggested that similar mechanisms underlie the propagation of slow potential changes in the cerebral cortex, etc.

ACKNOWLEDGEMENTS

We are grateful for the help of Dr. P. Witkowsky (present address: Dept. Ophthalmology, Columbia University) in the experiments described in Figs. 5, 6, 8 and 9). The neurohistological experience of Mr. V. Parthe of the University of' Carabobo has been invaluable to us in our studies of retinal structure, and we gratefully acknowledge his aid. We would also like to record our appreciation of the skilful help by our technician Mr. C. Muriel in the construction of our experimental equipment, and in the preparation of the illustrations.

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NAK.4, K.9 INOMA, s.,KOSUGI, y.,AND TONG,c.,(1960); Recording of action potentials from single cells in the frog retina. Jap. J. Physiol., 10, 436-442. NEGISHI,K., SELV~N DE TESTA, A., HERNANDEZ, J. A., AND D ~ A BORGES, Z J. M., (1963); Potencial estable transcortical: Su interpretacion en terminos de la interaccibn metabolica glia-neuronal. I. Estado de despertar y sueiio. Acta cient. venez., Suppl. 1, 154-162. NEGISHI, K., AND SVAETICHIN, G., (1965); Effect of anoxia, COZand NH3 on S-potential producing (controller) cells and on neurons. Arch. psychiat. Neurol., In the press. T., AND MOTOKAWA, K., (1959); Origin of so-called cone action potential. OIKAWA, I., OGAWA, J. Neurophysiol., 22, 102-1 11. OTTOSON, D., AND SVAETICHIN, G., (1952) ; Electrophysiological investigations of the frog retina. Cold Spr. Harb. Symp. quanf.Biol., 12,165-173. PORTELA, A., HINES,M., PEREZ, J. C., BRANDES, F., BOURNE, G. H., STEWART, P., AND GROTH,D., (1960); Effects of X-irradiation on muscle membrane. Exp. Cell Res., 21,468-481. PORTELA, A., PEREZ,J. C., STEWART, P., HINES,M., AND REDDY,V., (1963); Radiation damage in muscle cell membranes and regulation of cell metabolism. Exp. Cell Res., 29, 527-543. QUASTEL, J. H., (1957); Metabolic activities of tissue preparations. Metabolism of fhe Nervous System. D. Richter, Editor. London, Pergamon Press (pp. 267-285). QUASTEL, J. H., (1959); Enzymatic mechanisms of the brain and the effects of some neurotropic agents. Biochemistry of the Central Nervous System. F. Briicke, Editor. London, Pergamon Press (pp. 90-1 14). RUSHTON, W. A. H., (1962); The retinal organization of vision in vertebrates. Biological Receptor Mechanisms. J. W. L. Beament, Editor. XVIth Symposia of the Society for Experimental Biology. Cambridge, Cambridge Univ. Press (pp. 12-31). RUSHTON, W. A. H., (1963); Increment threshold and dark adaptation. J. Optical SOC.Amer., 53, 104-1 09. SELV~N DE TESTA, A., (1965); Morphological studies on the horizontal and amacrine cells of the teleost retina. Vision Res., In the press. SELV~N DE TESTA, A., NEGISHI,K., HERNANDEZ, J. A., AND DiAz BORGES, J. M., (1963); Potencial estable transcortical: Su interpretacion en terminos de la interaccibn metabblica glia-neuronal. 11. Estado anoxico. Acta cient. venez., Suppl. 1, 163-168. SICKEL, W., (1961); Stoffwechsel und Funktion der isolierten Netzhaut. The Visual System: Neurophysiology and Psychophysics. R. Jung and H. Kornhuber, Editors. Berlin, Springer-Verlag (pp. 80-94). SJOSTRAND, F. S., (1960); Electron microscopy of myelin and of nerve cells and tissue. Modern Scientific Aspects of Neurology. J. N. Cummings, Editor. London, Edward Arnold Ltd. (pp. 188-231). SJOSTRAND, F. S., (1961); Topographic relationship between neurons, synapses and glia cells. The Visual System: Neurophysiology and Psychophysics. R. Jung and H. Kornhuher, Editors. Berlin, Springer-Verlag(pp. 13-22). SVAETICHIN, G., (1951) ; A combination of microscopes and micromanipulators for electrophysiological investigations on single nerve cells. Acta physiol. scand., 24 (Suppl. 86), 15-22. SVAETICHIN, G., (1953); The cone action potential. Acfaphysiol. scand., 29 (Suppl. 106), 565-600. SVAETICHIN, G., FATEHCHAND, R., DRUJAN, B. D., LAUFER, M., WITKOVSKY, P., NEGISHI, K., AND SELV~N DE TESTA, A., (1963) ; Interaccibn glia-neuronal: Su dependencia metabblica. Una nueva teoria acerca del funcionamiento del sistema nervioso. Acfa cient. venez., Suppl. 1, 135-153. SVAETICHIN, G., LAUFER, M., MITARAI,G., FATEHCHAND, R., VALLECALLE, E., AND VILLEGAS, J., (1961): Glial control of neuronal networks and receptors. The Visual System: Neurophysiology and Psychophysics. R. Jung and H. Kornhuber, Editors. Berlin, Springer-Verlag (pp. 445-456). SVAETICHIN, G., NEGISHI, K., AND FATEHCHAND, R., (1965); Cellular mechanisms of a Young-Hering visual system. Ciba Symp. on Colour Vision, Physiology and Experimental Psychology, London. In the press. TOMITA, T., (1963); Electrical activity in the vertebrate retina. J. Optical SOC.Amer., 53,49-57. TOMITA, T., MURAKAMI, M., SATO,Y.,AND HASHIMOTO, Y.,(1959); Further study on the origin of the so-called cone action potential (S-potential). Its histological determination. Jap. J. Physiol., 9, 63-68. TOWER, D. B., (1960); Neurochemistry of Epilepsy. Springfield, Thomas. G., (1961); The retina as model for the functional organization VALLECALLE, E., AND SVAETICHIN, of the nervous system. The Visual System: Neurophysiology and Psychophysics. R. Jung and H. Kornhuber, Editors. Berlin, Springer-Verlag (pp. 61M12).

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Introductory Discussion on Glial Function ROBERT G A LA MBOS Department of Psychology, Yale University, New Haven, Conn. ( U.S.A.)

I have been asked to introduce the general discussion period of this Symposium which I shall do by making comments, observations and speculations about the role of glial cells in the functioning brain. I hope in this way to provoke from you still further discussion regarding the morphological, biochemical and physiological properties of these cells that interest us so much. Apparently, these ubiquitous glial cells do many different things in the brain. I have summarized in Table I cei tain classes of problems which current research upon TABLE I GLIA CELL A C T I V I T Y I N :

1. 2. 3.

4. 5. 6. 7.

Brain response to trauma, radiation, drugs (swelling, edema). Neuron metabolism. The electrical phenomenon of retina, brain, and peripheral nerve (excitability; the standing D. C. potentials; membrane and action potentials). Brain development, degeneration, and regeneration. Myelination, demyelination and allergic responses of brain. Synaptic events. Storage and retrieval of innate and learned behavioral responses.

glia seems trying to resolve. Of the 7 problem areas enumerated there, the first 3 have already received considerable attention in this Symposium, and I shall not undertake to deal further with them. The last 4, however, represent current research interests touched only tangentially in our discussions so far, and I shall devote my attention mainly to them. Brain development, degeneration and regeneration

In doing so let me start by asking you to examine your own ideas of the structure of a living brain. Do you think of it as an organ which at the microscopic level is continuously in motion, or as a stable, unchanging morphological entity which is permanently wired together? Both old and recent evidence seems to me to favor a dynamic concept of brain morphology according to which moment-to-moment changes in its internal References p . 275-277

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structure take place, and I should like to assemble some of this evidence here and touch upon its functional implications. It is, of course, self evident that much mechanical activity of cells must go on while the mammalian brain develops embryologically. Such events as invagination of the neural plate, thickening of the walls of the neural tube, formation of brain flexures, local enlargements of hindbrain, midbrain and forebrain to give a brain its characteristic adult morphology - all these developments require active cell division, motion and migration. Indeed, the two statements, morphological change in embryology, and widespread mechanical rearrangements among brain cells, are equivalent. More or less cell activity of this sort must be presumed to continue so long as the brain increases in size, which in man means up to about the 10th year of life (Magoun et al., 1960). Conel's description of the morphological changes that occur in the infant brain between 6 and 15 months shows the cortex to thicken, the interneuronal spaces to widen, the nerve cells to enlarge, and myelin to be more widely encountered (Conel, 1955). One recognizes from these facts that the human brain is to some extent at least, an active, moving structure during infancy. One can get some idea of how turbulent and violent this activity may be in some circumstances by studying the recently published electron microscope pictures of kitten cortex (Voeller et al., 1963) which illustrate dramatic changes during the first postnatal weeks in the size and distribution of apical dendrites, and in the number of glial processes that insinuate themselves among the neuronal material. The facts revealed in this particular study require us to suppose the kitten to pass through this period of life with a cortex literally seething with cells that change their shape, move about, and compete with one another for the space available. As one of my colleagues puts it, living gray matter may more closely resemble a squirming bag of worms than the static collection of nerve cells and fibers one deduces from text book drawings of silver stained sections. From what we know of the process of myelination, mechanical activity within the fiber tracts of the brain must take place during this process also. Whether or not the glial cell actively wraps itself around and around its axon, spinning a train of myelin lamellae behind itself (as so many observers now argue), the simple fact of myelin deposition means increased diameter of axons followed by mechanical rearrangement as they adjust to their larger size. Hence, whenever a fiber tract myelinates - or demyelinates - the spatial relations of its components must be in a state of flux. Tissue culture observations have from their inception clearly supported the above more or less self-evident arguments and inferences for brain motility. In tissue cultures neurons display amoeboid-like activity which, along with the varied and persistent motions of oligodendroglia, astrocytes, Schwann cells and ependymal cells has been amply documented by investigators using a variety of techniques, animal material, and culture media (Lumsden and Pomerat, 1951; Pomerat and Costero, 1956; Hild 1957a, b; Pomerat, 1959; Peterson and Murray, 1960; Weiss, 1961). Such data cannot be dismissed as irrelevant even though a clear demonstration that neurons and glia visibly move under the special conditions set up in a tissue culture flask does not prove, of course, that they also move and migrate when in vivo. If we are correct in inferring from all this indirect evidence that the brain is to some

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extent a mechanically active object, many morphological and functional questions arise which future experiments must solve. Does brain motility ever cease during life? When the human brain attains its adult size at age 10, do its neurons and neuroglia also stop their movements? Is the amount of brain motility related to brain region, or body activity, or time of day, or mental state? As judged from the indirect evidence, glial cells move about more vigorously than neurons, and under certain conditions they actively reproduce themselves by cell division in situ (Altman, 1962); in the normal brain does the commotion these glial cells create periodically remove axon terminals from, and permit their reformation upon, the dendrites and somata of postsynaptic elements? To put these questions succinctly, how is it that the brain motions do not prevent kittens and children from displaying the many stable and constant features we recognize in their behavior? Does this mean that the brain organization upon which their behavior depends does not require a stable morphological base in the mechanical sense? A very long list of questions such as these can easily be assembled by anyone wishing to do so. Perhaps someone will soon actually implant a light source and a microscope into a brain, take time-lapse photographs at high magnification of the living cells in situ, and thus provide us with some clear answers. Until this happens, unfortunately, we must remain in the same state of ignorance. Schafer summarized so clearly in 1900 when he wrote the following (1900, p. 607-608): “It has been conjectured that the extent of contact of the adjacent nerve cells at the synapses may vary from time to time, such variations being brought about by a contraction or expansion of the ramified processes by which the contact is effected, analogous to the amoeboid movements of protoplasm in general; and, fuither, that the effects of drugs in diminishing or increasing the resistance of the nerve centres to the passage of nervous impulses might be produced in a similar way. This conjecture opens up a wide field of speculation, since it is possible to extend it so as to embrace the explanation of many physiological and psychical conditions. Such speculations, however, unless they have been tested experimentally, do not lie within the scope of this work. No one has been able to see any amoeboid movement in the nerve cells of vertebrates, and the only direct evidence of such movement in nerve cells, that we have any knowledge of, has been furnished by Wiedersheim, who observed slow changes of form to occur in ganglion cells of the central nervous system of a minute crustacean. It is true that various observers have described in Golgi preparations, appearances in the processes of nerve cells after excitation, or after the action of anaesthetics and other drugs, which they interpret as indicating a withdrawal or retraction of the finer processes. But it cannot yet be accepted as proved that the changes which are described are invariable concomitants of alterations in the functional condition of the cells, still less that they are the cause of the functional conditions.” In this quotation, Schafer cites papers published between 1890 and 1900. Today’s uncertainty regarding the true morphological relationships between neurons and glial cells in a living brain has a history, therefore, that goes back more than 60 years. References p. 275-277

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Myelination, demyelination and allergic responses of brain

1 should like now to turn to the topic of glial activity in myelination and demyelination. Many recent experiments have dealt with how myelin forms around axons, is preserved there during life, and comes to be destroyed in disease. Glial cells appear to be mechanically and biochemically involved in all these events, and to be indispensable in some of them. Thus both the Schwann and the oligodendroglial cell has been directly observed in tissue culture to surround and invest its respective axon and then to lay the myelin down about it (e.g. Peterson and Murray, 1955; Hild, 1957a; Bornstein and Murray, 1958), and electron microscopic evidence in abundance argues for exactly similar processes in vivo (Geren, 1954; Causey, 1960; De Robertis and Gerschenfeld, 1961; Bunge et al., 1961) and in vitro (Ross et al., 1962). The major outlines of how myelin normally forms upon central and peripheral axons appear, then, to be in hand, though certain differences of detail in the two cases are not yet understood. The question of whether myelin is a stable entity which, once laid down, remains unchanged during the life of the axon, appears to be clearly answered by studies with radioactive cholesterol; the sterol molecules isolated from myelin sheaths of the adult rabbit turned out upon analysis to be those injected into the immature animal a year or more earlier (Wright, 1961). On the other hand, myelin can be removed from axons within a matter of hours, and it rapidly forms in substantial amounts in tissue cultures (Bornstein and Appel, 1961) and, of course, it forms in a definite sequence in the normally developing brain (Jacobson, 1963). All this evidence of brisk activity in myelin formation, dissolution and reformation contrasts sharply with the fact that the myelin, once laid down, persists thereafter in an unchanged state. Let me turn specifically to the demyelination problem. Demyelinating diseases like multiple sclerosis, because they represent important clinical problems, have provided the impetus for much laboratory activity aimed at discovering their cause. During this Symposium we may wish to examine the question of the degree to which demyelination is primarily a glial cell disease. If so, what follows will serve as an introduction. The literature on myelinolytic mechanisms grows rapidly (Kies and Alvord, 1959 and Roy. SOC. Med., 1961) with studies.on experimental allergic encephalomyelitis (EAE) playing a central role. This disease, as you know, develops in adult rabbits and other animals given homogenized brain or spinal cord by injection. Extensive demyelinationof the central nervous system comes on within a few weeks in favorable cases along with clinical symptoms that resemble the human diseases having similar neuropathological findings. A consensus seems to have been reached regarding the essential nature of the disorder in these animals; it is an allergic disease for which isolation of the specific antigen in the material injected has not yet been accomplished. Besides EAE, a second allergic neurological disorder, produced by Waksman and Adams (1956) and named by them experimental allergic neuritis (EAN) is recognized, The antigen in this case is contained in homogenized peripheral nerve injected into the experimental animals. Demyelination of spinal ganglia and peripheral nerves

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follows such injections, with no loss of myelin in spinal cord or brain. However, the lesions found in the two diseases, EAE and EAN, seem to be basically identical; demyelination, sparing of axis cylinders and supporting elements, and perivascular exudates containing histiocytes and lymphocytes. Bornstein and his colleagues have carried the search for these antigens a significant step forward in their tissue culture experiments (1961, 1962; Ross and Bornstein, 1961;Yonezawa et al., 1962; Ross et al., 1962). Thus Bornstein and Appel(l961) report that the serum of a rabbit afflicted with EAE will demyelinate axons from explants of central nervous system (rat cerebellum), but not from explants of peripheral neural structures (dorsal root ganglia), an experimental demonstration by still another technique of the different antigenetic properties of central and peripheral nervous tissue pointed out by Waksman and Adams (1956). The inverse experiment, applying serum from an animal with EAN to appropriate cultures of peripheral and central myelinated axons, has not tozmy knowledge been reported. As to what transpires when EAE serum produces demyelination in a tissue culture, Bornstein’sidescription (1962) is highly informative; the neuroglia swell, and some of them die, while the myelin sheaths develop fusiform swellings, break away from their axon to lie free in the culture, and eventually become reduced to scattered fat droplets. Neurologists concerned with human demyelinative disorders have been impressed by Bornstein’s demonstration that serum from patients with acute multiple sclerosis also acts upon these tissue cultures and in an exactly similar way. By means of fluorescent antibody techniques, the target for the globulins in the EAE serum has been identified as the neuroglial membranes and cytoplasm, along with the myelin itself (Appel et al., 1962). Throughout the demyelinative process, which can proceed to completion in a few hours in the presence of high antibody titers, the neuron soma, axon and dendrites show little evidence of disturbance. The last point I would make about these fascinating experiments is that if the gliotoxic EAE serum is removed from the culture flask when demyelination is complete, or nearly so, and replaced with normal culture medium, the damaged glial cells may recover and even proceed to remyelinate the naked axons. Bornstein (1962) reports cultures that have been demyelinated and remyelinated several times in this manner. Discovery of these antibodies specific for glial cells opens new research avenues for exploitation. Consider, for instance, this demonstration of different effects of EAE and EAN serum upon satellite cells of central and peripheral origin. What we know so far persuasively argues for a fundamental difference in the antigenic properties of Schwann and oligodendroglial cells. Can appropriate experiments demonstrate still further subclassification of glial types, with the glia of, say, caudate nucleus showing different immunological specificity when compared, say, to the glia of the hippocampus? An opening wedge toward an answer for this question may well be provided by the experiment of Mihailovic and Jankovic (1961), with which I shall conclude this section of the discussion. These experimenters injected into rabbits the caudate nucleus removed from cat brain, thereby producing anti-caudate antiserum in the rabbit. Intiaventricular injection of this immune serum into a normal cat produced prolonged and dramatic References p. 275-277

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changes in the brain waves - including complete disappearance of the spontaneous rhythms - which was limited to the caudate nucleus. When antibodies similarly developed in rabbit to cat hippocampus were injected intraventricularly into another normal cat, comparable EEG changes did not appear in the caudate. From this report, one may conclude, therefore, that the antigenic properties of caudate nucleus and hippocampus actually do differ. Whether the specific antigen involved derives from the neurons, the glia, the blood vessels or some combination of them is not settled yet. Since, however, the experiment so closely resembles the EAE studies where neuroglia are attacked, it seems as reasonable to suppose the antigen resides in the glial cells as that it comes from the neurons. In any event, whatever the final story will turn out to be, these studies where immune specificities are sought for and discovered in different brain regions cannot fail to provide useful information on glia function. Synaptic events

One question I repeatedly find myself asking is whether glia participate in the process of transmitting impulses across synapses. Since cells that lie close together in the body usually work together, the exceedingly close and numerous contacts between glial cells and neurons at synapses, a remarkably constant feature revealed by electron microscopic studies, cannot help but suggest some unsuspected functional interactions between them. Wherever impulses are generated, at central synapses, autonomic ganglia, motor endplates, or in the sense organs, satellite cells are almost invariably intimately associated with the neurons. What do they do there? I cannot present to you a tightly reasoned, convincing argument that glia actually participate in the synaptic processes at these various sites. At the present time this probably cannot be done. There are, however, scattered bits of evidence no one seems to understand that point in this general direction, and it is my impression that when further experiments place these curious facts in their proper setting glial participation in synaptic transmission will be counted in or ruled out. Let us consider first the paper by Koelle (1962) which assembles the data against the idea that synaptic events involve merely presynaptic secretion of excitatory substance (e.g. acetylcholine) which combines with postsynaptic receptors to initiate the ultimate nerve reaction (Fig. 1). Instead of this conventional view of a straightthrough process at synapses Koelle shows how certain facts are more consistent with this chain of events: (1) presynaptic secretion of a small amount of transmitter substance; then (2) re-excitation of the presynaptic element by this substance; with (3) presynaptic secretion of a large amount of transmitter chemical which (4) crosses the synapse to unite with receptor substance in the usual way. The distinguishing feature of this hypothesis (schematized in the figure) is the notion of an initial presynaptic event which, through positive feedback, evokes still another presynaptic process which is the one crucial for transmission. Koelle’s synthesis of the extensive work from his and other laboratories thus postulates two presynaptic events as you can see, both taking place in the nerve fiber approaching the synapse. Yet one of these events could just as well be contributed

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CONVENTIONAL

KOELLE

7 Fig. 1. Models of synaptic action.

by the glial cells; there are no facts known to me that eliminate this possibility. Koelle also recognizes this, pointing out that glia certainly exist at synapses, and that glia contain large quantities of nonspecific cholinesterase for which no function is known. What I have just been saying can be phrased negatively: do not eliminate glia from consideration until some experimental facts oblige you to do so. On the positive side, which always provides more convincing arguments, some presumptive evidence that glia might well be active at synapses exists, and this brings me to the rather startling demonstrations (Katz and Miledi, 1959) of Schwann cell activity at denervated frog motor endplates. Studied in detail by Birks et al. (1959, 1960b), the phenomenon is simply this : miniature endplate potentials (m.e.p.p’s) can readily be recorded from striated muscle even several weeks after its motor nerve has completely disappeared, and at a time when only Schwann cells cover the muscle in the endplate region. These m.e.p.p’s evoked by Schwann cells are notably resistant to the forms of stimulation: electrical, chemical, and mechanical, which so drastically alter the m.e.p.p’s generated at normal endplates. Curare abolishes them just as it abolishes the normal ones, but botulinum toxin in a concentration that eliminates normally generated m.e.p.p’s does not affect the Schwann cell type (Thesleff, 1960). Hence, there is convincing evidence from these and related experiments for dual but closely related mechanisms that generate m.e.p.p’s at muscle endplates. In the normal case the presynaptic neuron presumably releases acetylcholine, doing this spontaneously or under the influence of arriving nerve impulses. At the denervated endplates, which anatomically appear to be only Schwann cell-muscular junctions, a transmitter (acetylcholine?) also seems to be released, but only spontaneously and never in response to electrical stimulation. References p. 275-277

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As for the mechanism generating these m.e.p.p’s, it is currently popular to hold synaptic vesicles in nerve terminals to be responsible for the normal ones. Electron microscopy shows the Schwann cell at both normal and denervated endplates to possess such vesicles, though they are relatively sparse and more varied in size than those seen in neurons (Birks et al., 1960a,b). Do these Schwann vesicles liberate acetylcholine to produce m.e.p.p’s in denervated muscle? If not, what is the mechanism by which Schwann cells, in the complete absence of an axon, evoke postsynaptic responses? Are we to suppose that this mechanism, whatever it may be, goes into action only after denervation, or does denervation simply unmask and make visible for us a relevant Schwann cell process at synapses normally unnoticed in the presence of the much more impressive activity of nerve terminals? All these questions, of course, merely state in different words the proposition that glial cells might participate in synaptic transmission. This problem can be summarized as shown in Fig. 1,which schematizes possible interactions between neurons and glial cells. The nature of the interactions, if any, between the various synaptic elements remains for the future experiments to define. Storage and retrieval of innate and learned behavioral responses

In this final section I wish to discuss this role of glia. Because of time limitations I shall deal only with glia and acquired memory, and refer, in fact only to the recent paper of HydCn and Egyhazi (1963). Though you may already know of this work, let me briefly outline the main points of this remarkable contribution toward understanding memory storage, that outstanding puzzle of brain function. The paper in question is the most recent of a long series that describe methods for dissecting single neurons out of the brain, isolating small volumes of purely glial protoplasm, separating neuronal nuclei from nerve cells, microchemical analysis for RNA, DNA, protein, and enzyme activity of the brain cells, and, finally, separation of the 4 purine and pyrimidine bases from RNA with quantitative measurement of the amount of each (HydCn and Pigon, 1960). This last analysis, which permits HydCn and his colleagues to measure absolute concentrations of each of the 4 RNA bases to an accuracy of the order of 20 ppg, is the crucial item for his demonstration that glial RNA changes accompany learning in the rat (Hydtn and Egyhazi, 1963). As is true of cells in general, ribonucleic acid (RNA) molecules of many types are synthesized by the nuclei of neurons and glial cells, the types under synthesis at any particular moment varying with the functional requirements of the cell. For the first time, as we shall see, HydCn and Egyhazi have identified a particular RNA characteristic of glia in trained rats. In their experiment rats learned to climb a thin wire stretching from the cage floor to a platform upon the wall. At first incapable of getting to the platform even once in a 45-min period, the hungry rats within 8 days became so skilful they travelled up and down 20 or more times in order to obtain the food placed on the platform by the experimenter. An earlier study by these authors described specific RNA changes

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in neurons dissected from Deiter's vestibular nucleus of these trained rats (Hydtn and Egyhazi, 1962); in this paper (Hydtn and Egyhazi, 1963) they direct their attention to glial RNA resulting from the training. The glia in question originally surrounded neurons in Deiter's nucleus and presumably lay in metabolic equilibrium with them there. In untrained rats as well as T A B L E I1 C O M P O S I T I O N O F R N A I N G L I A C E L L S I N P E R C E N T OF TOTAL

(From Hydtn and Egyhazi, 1963) RNA base

r '$1

Adenine Quanine Cytosine: Uracil

Control rats

Trained rats

5 animals, 33 analyses

6 animals, 42 analyses

25.3 f0.16 29.0 5 0.24 26.5 f 0.43 19.2 0.27

28.3 5 0.45 28.8 f 0.31 24.3 f0.36 18.6 f0.21

0.001

P

=

P

= 0.01

in control rats given passive vestibular stimulation, these glial cells contained RNA of a particular composition as indicated in Table 11. Glial cells analyzed from the trained rats, by contrast, possessed RNA of unique chemical constitution in that more adenine and less cytosine appeared here than in the controls. This demonstration of an altered base ratio in the RNA of glial cells would appear to be the first clear evidence that glia actively participate, along with the neurons, in learning and memory storage functions. SUMMARY

The search for the functions glial cells perform in the brain is under active investigation in many laboratories in the world. Besides the glial activities examined in detail several other lines of investigation not represented by specialists here should be cited. The survey presented here briefly considers the role ofglial cells in myelination and demyelination, their possible participation in events occurring at synapses, and the new biochemical information implicating them in the storage and retrieval of memories. REFERENCES

ALTMAN, J., (1962); Autoradiographic study of degenerative and regenerative proliferation of neuroglia with tritiated thymidine. Exp. Neurol., 5, 302-318. APPEL,S. H., BORNSTEIN, M. B., SEEGAL, B. C., MURRAY,M. R., (1962); The application of tissue culture to a study of experimental allergic encephalomyelitis: Immunological observations. ZV. International Congress of Neuropathology. H. Jacob, Editor. Stuttgart, Thieme (pp. 283-285). BIRKS,R., HUXLEY, H. E., AND KATZ,B., (1960a); The fine structure of the neuromuscularjunction of the frog. J. Physiol., 150, 134-144. BIRKS,R., KATZ,B., AND MILEDI,R., (1959); Electron-microscopic observations on degenerating nerve-muscle junctions of the frog. J. Physiol., 146,4546P.

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BIRKS,R., KATZ,B., AND MILEDI,R., (1960b); Physiological and structural changes at the amphibian myoneuronal junction, the course of nerve degeneration. J. Physiol., 150, 145-168. BORNSTEIN, M. B., (1962); A tissue-culture approach to demyelinative disorders. Symposium on Organ Culture, Washington. Monograph No. 11. National Cancer Institute, Washington. BORNSTEIN, M. B., AND APPEL,S. H., (1961); The application of tissue culture t o the study of experimental ‘allergic’ encephalomyelitis. J. Neuropath. exp. Neurol., 20, 141-1 57. BORNSTEIN, M. B., AND MURRAY, M. R., (1958); Serial observations on patterns of growth, myelin formation, maintenance and degeneration in cultures of newborn rat and kitten cerebellum. J. biophys. biochem. Cytol., 4, 499-504. BUNGE,M. B., BUNGE,R. P., AND RIS, H., (1961); Ultrastructural study of remyelination in an experimental lesion in adult cat spinal cord. J. biophys. biochem. Cytol., 10, 67-94. CAUSEY, G., (1960); The Cell of Schwann. Edinburgh and London, Livingstone. CONEL,J. L., (1955); The cortex of the human fifteen month infant. The Postnatal Development of the Human Cerebral Cortex. Vol. 5 . Cambridge, Harvard University Press. H. M., (1961); Submicroscopicmorphology and function DEROBERTIS, E. D. P., AND GERSCHENFELD, of glial cells. Znt. Rev. Neurobiol., 3, 1-65. GEREN,0. B., (1954); The formation from the Schwann cell surface of myelin in peripheral nerves of chick embryos. Exp. Cell Res., 7 , 558. HILD, W., (1957a); Myelogenesis in cultures of mammalian central nervous tissue. 2.Zelvorsch., 46, 71-95. HILD,W., (1957b); Ependymal cells in tissue culture. 2.Zellforsch., 46,259-271. HYD~N H., , AND EGYHAZI,E., (1962); Nuclear RNA changes of nerve cells during a learning experiment in rats. Proc. nut. Acad. Sci., 48,1366-1373. HYDBN,H., AND EGYHAZI,E., (1963); Glial RNA changes during a learning experiment in rats. Proc. nut. Acad. Sci., 49, 618-624. H., HYD~N , AND PIGON,A., (1960); A cytophysiological study of the functional relationship between oligodendroglial cells and nerve cells of Deiter’s nucleus. J. Neurochem., 6, 57-72. JACOBSON, S., (1963); Sequence of myelinization in the brain of the albino rat. A. Cerebral cortex, thalamus and related structures. J. comp. Neurol., 121, 5-29. KATZ,B., AND MILEDI,R., (1959); Spontaneous subthreshold activity at denervated amphibian endplates. J. Physiol., 146, 4 4 4 5 P . KIES,M. W., AND ALVORD, E. C., (1959); Allergic Encephalomyelitis. Springfield, Thomas. G. B., (1962); A new general concept of the neurohumoral functions of acetylcholine and KOELLE, acetylcholinesterase. J. Pharm. Pharmacol., 14, 65-90. LUMSDEN, C. E., AND POMERAT, C. M., (1951); Normal oligodendrocytes in tissue culture. J. exp. Cell Res., 2, 103-114. MAGOUN, H. W., DARLING, L., AND PROST,J., (1960); The evolution of man’s brain. The Central Nervous System and Behavior. M. A. B. Brazier, Editor. Transactions of the thud conference. New York, Josiah Macy Jr. Foundation (pp. 33-126). MIHAILOVIC,L. J., AND JANKOVIC, B. D., (1961); Effects of intraventricularly injected anti-N. caudatus antibody on the electrical activity of the cat brain. Nature, 192, 665-666. PETERSON, E. R., AND MURRAY, M. R., (1955); Myelin sheath formation in cultures of avian spinal ganglia. Amer. J. Anat., 96, 319-356. PETERSON, E. R., AND MURRAY, M. R., (1960); Modification of development in isolated dorsal root ganglia by nutritional and physical factors. Develop. Biol., 2, 461476. POMERAT, C. M., (1959); Rhythmic contraction of Schwann cells. Science, 130, 1759-1760. POMERAT, C. M., AND COSTERO, I., (1956); Tissue cultures of cat cerebellum. Amer. J. Anat., 99, 211-248. Ross, L. L., AND BORNSTEIN,M. B., (1961); Symposium on disseminated sclerosis and allied conditions. Proc. roy. SOC.Med., 54, 1 4 2 . Ross, L. L., AND BORNSTEIN, M. B., (1962); The application of tissue cultures to the study of experimental ‘allergic’ encephalomyelitis. 111. Electron microscopic observations of demyelinization and remyelinization. ZV. International Congress of Neuropathology. H. Jacob, Editor. Stutigart, Thieme (pp. 285-287). Ross, L. L., BORNSTEIN, M. B., AND LEHRER,G. M., (1962); Electron microscopic observations of rat and mouse cerebellum in tissue culture. J. ceN. Biol., 14, 19-30. SCHAFER, E. A., Editor, (1900); Textbook of Physiology. Edinburgh and London, Young J. Pentland. S., (1960); Supersensitivity of skeletal muscle produced by botulinum toxin. J. Physiol., THESLEFF, 151, 598-607.

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VOELLER, K., PAPPAS, G. D., AND PURPURA, D. P., (1963); Electron microscope study of development of cat superficial neocortex. Exp. Neurol., 7 , 107-130. WAKSMAN, B. H., AND ADAMS,R.D., (1956); A comparative study of experimental allergic neuritis in the rabbit, guinea-pig and mouse. J. Neuropath., 15,293-332. WEISS,P., (1961); The concept of perpetual neuronal growth and proximo-distal substance convection. Regional Neurochernistry. S. S . Kety and J. Ekes, Editors. Oxford, Pergamon Press (pp. 220-242). WRIGHT, G. P., (1961); Metabolism of myelin. Proc. roy. SOC.Med., 54, 2C30. YONEZAWA, T., BORNSTEIN, M. B., PETERSON, E. R., AND MURRAY, M. R., (1962); A histochemical study of oxidative enzymes in myelinating cultures of central and peripheral nervous tissue. J. Neuropath. exp. Neurol., 21,419487.

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General Discussion

DAVSON: We have with us in our Symposium a man who is not only an authority on recent work on the glial cells but also an authority on nervous conduction itself. It is Dr. Tasaki and I wonder if he would mind just telling us a little bit about the requirements of the neurons insofar as they must be kept separate from each other; that is, there must be a space between them. Could he just indicate to us what are the consequences of having, say, a glial cell just a bit too close to the neuron; what would be the consequences of these junctions that apparently consist of a fusion of membranes between the neuron and the glial cell. TASAKI:When we introduce a microelectrode into a brain slice or into a mass of cells in tissue culture, the tip of the electrode would probably be in one of three possible locations :inside a dead cell (neuron or glia destroyed by the electrode),in a glial cell, or in a neuron. Glial cells seem to have large resting potentials; clean penetration of a glial cell often gives rise to a D. C . shift of more than 50 mV. Penetration of a neuron often induces an injury discharge of impulses. Now, coming to the problem of the chemical composition of the fluid in the extracellular space, we believe that both divalent cations (calcium, magnesium) and monovalent cations (sodium, potassium) are required to maintain normal excitability of the neuron. I do not think there is unequivocal evidence indicating that calcium and sodium ions are present inside glial cells. In the light of Dr. Lasansky’s evidence in the retina, it is most probable that these ions exist in the extracellular space; i.e. in the small space between glial cells and neurons. The normal chemical composition of the extracellular fluid could be maintained by the process of diffusion and possibly by the function of the glial cells. DAVSON: Well then, would there not be a limit to the smallness of this? We know that when the nerve conducts an impulse it loses potassium, and that this will accumulate in the space. If the space is too small, the potassium concentration will rise to such a height that after-potentials will occur and lead to permanent depolarization. TASAKI:I think it is possible that glial cells play some role in the process of action potential production. In the squid giant axon, at least, we know that there is an enormous interdiffusionof ions across the surface membrane during activity; strong fluxes of extracellular cations (calcium and sodium)into the axons and, simultaneously, a strong flux of intracellular cation (potassium) into the extracellular space takes place during action potential. I believe that such an interdiffusion should set up a potential field of appreciable magnitude in the extracellular space and in the layer

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279

of surrounding satellite cells. (Dr. Portela has some evidence along this line in skeletal muscle fibers.) For this reason, I believe that the existence of glial cells around neurons can affect the falling phase of the action potential and the process of recovery from repetitive activity. In addition, there is a possibility that metabolic products of glial cells affect the excitability of neurons. DAVSON: Now, we have opened completely all the problems amongst us. I don’t know if the morphologists would now just like to emphasize their opinions, derived from what we have heard throughout the Symposium. Would one of the electron microscopists like to tell us his impressions? ZADUNAISKY: From the morphological standpoint, it still seems that there is very little space between the cells. From the physiological viewpoint we all see a reasonable amount of fluid that will permit diffusion through the tissue in an extracellular space. The point which I make now is that there might be contacts between neurons and glia, which might not have interspace between them or the neurons have a rigid membrane or matrix. We arrive to this conclusion from our own evidence, thinking that the penetration of some substances might be made first into the glia, due to contact with the extracellular fluid. We can not explain, for instance, the lack of penetration of some salts into the neuron. In this respect I would like to have some opinions from the members of the Symposium. DAVSON: The point you are emphasizing is the fact that your neurons refuse to join in on osmotic exchanges. We really need the opinion of a morphologist here on the fact that the neuron seems to be unable to respond to a difference of osmotic pressure. A red blood cell swells as soon as you reduce osmotic pressure outside. Why is it that the neuron does not? Is it feasible that there is enough structural rigidity to resist quite large differences:of osmotic pressure? DE ROBERTIS: It is difficult to answer if this rigidity of the nerve cell and incapability to osmotic changes might depend only on the membrane or on the whole structure of the cell. The cell should be considered as a unit, and I would be inclined to think that the neuron has a very rigid cytoplasm because of the great amount of material represented by intracellular membranes - the so-called endoplasmic reticulum and Golgi complex in the cytoplasm - and also the rich amount of other material of fibrillar and tubular nature in the matrix of the cytoplasm. If one looks under the electron microscope at a nerve cell, the whole cytoplasm appears filled with material. There is a‘greater electron density than in other cell types, particularly in astroglial cells. FRIEDE:I don’t think that it is generally true that neurons do not respond to osmotic changes. Conceivably, the neuron is so extremely well protected in its normal environment that osmotic changes do not reach it under normal conditions. It is very easy to produce swelling of neurons by hypotonic solutions in vitro and to produce shrinkage of neurons by hypertonic solutions.

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Also, it has been shown that minimal tissue injury produces, in the brain, shrunken, hyperchromatic nerve cells which suggests that the volume of nerve cells can change and that it can change quite rapidly. Thus, the scarcity of neuronal osmotic changes may not represent a specific property of the nerve cell as such, but rather the degree of protection it has in the living tissue. TASAKI: In the squid giant axon, it seems to me that the volumeoftheaxonisprobably maintained at a relatively constant level by virtue of the mechanical properties of the axoplasm and the surrounding connective tissue layer. The axoplasm of the axons we have investigated is a kind of solid gel. It is unlikely that the axon readily changes its volume in response to a rise or fall in the internal hydrostatic pressure. PAPPIUS:I think, in conjunction with what Dr. Friede said, it is only fair to point out that in our in vitro work with cerebral cortex slices, we can modify osmotically the extent of the fluid compartment which we think represents the neuronal space. The space can be modified by incubation in either hypotonic or hypertonic media (see our Fig. 5, p. 142).

KLATZO:With regards to Dr. Friede's comment that the neurons in vivo might be protected from osmotic effects by their environment I would like to mention that neurons grown in tissue culture also seem to be more resistant than other nervous tissue cellular elements to osmotic disequilibria. Lowering the osmolarity of the media we could observe that the glial cells, and particularly the astrocytes, were the first to succumb and become acutely swollen, whereas the nerve cells could withstand greatly lowered osmolarity without apparent changes in their morphological appearance (unpublished observations). DAVSON: May I make an exception for a former colleague of mine, Dr. Levin, who is on the floor but he would like to talk and I am sure with profit.

LEVIN:I just wish to raise a question. Apparently we are accepting the proposition that the neurons do not swell and are not influenced by oedema. But I remember the pictures in Dr. Polak's presentation in human cerebral oedema where he showed by light microscopy a swelling in neurons, in oligodendrocytes, in astrocytes, in microglia and an enlargement of the extracellular space. Dr. Klatzo also showed slides demonstrating swelling of neurons. Furthermore, Dr. Pappius spoke about three possible compartments where oedema could be produced, one of them being the neuronal compartment. Thus I am really wondering whether, with our experimental procedures, we are producing more favorable conditions for the development of an oedema of the glial cells, but in the neuropathology of humans and experimental animals other ways could be possible for the production of a generalized oedema beginning in the true extracellular space. The contributions of the Symposium to this problem are very important but I still think this remains an open question.

G E N E R A L DISCUSSION

28 1

DE ROBERTIS : I would like to answer to what Dr. Levin has said in a very general way. I think we have to differentiate between processes which are physiological or near to physiology and processes which are clearly within the pathological range; otherwise we can make some terrible confusion. The fact that nerve cells are not osmotically active in comparison to astroglial cells has been very well demonstrated. The experiments of Dr. Zadunaisky were done in the most physiological way that one can do in in vitro experiments. The experiments in tissue culture that Dr. Klatzo has mentioned are also physiological. However, when we speak of neuropathology we refer to the cells which we don’t know if they are dead or alive and by what mechanisms the changes have been produced. I would like to bring a word of caution to the discussion and that is not to confuse the things which are more in a physiological range with those that are strictly pathological. Of course, sometimes the boundaries are difficult to establish but I think it is always very important to take this point into consideration. KLATZO:I would like to add that we have recently been studying the effects of alum phosphate poisoning on the nervous system. This intoxication produces a striking and almost selective swelling and vacuolization of the neurons. Perusal of the literature, however, reveals that neuronal swelling as a true ante-mortem alteration is a rather rare phenomenon found only in a few pathological conditions, mostly related to some type of intoxication. DAVSON: To summarize our position so far as we have discussed matters in the last fifteen minutes, that is, the fluid relationships between the blood and the neurons, we have satisfied ourselves that there must be some space surrounding the neurons. We have seen that it is necessary that they should have a space of some magnitude in order that the resting and action potentials may be manifest. It is still an open question as to whether the exchanges that do take place between blood and this fluid-filled space occur by way of the glial cells. I think this is a very likely possibility, in that the glial cells are really looking after the fluid, making sure that it is renewed as soon as there is too high an accumulation of potassium or too low a concentration of sodium. Furthermore, I think Dr. Tasaki’s explanation of the failure of the neuron to show large changes in volume is adequate; if there is quite a considerable portion of the cytoplasm in a jellyish form, well then, we can expect it not to show such large changes in volume as if it were purely fluid. Now, could we go on to some of the aspects that have been interestingly brought up by Dr. Galambos. He has mentioned that the glial cells might be concerned with memory. We owe that idea originally to him some years ago now. Would he have time just to give the uninitiated an idea of how it could possibly work. GALAMBOS: If I am to give the uninitiated an idea of how glial cells might be involved in memory, it’s going to be a case of the blind leading the blind, because I do not know. There are, however, two considerations that seem to be relevant here. The first is related to the remarkable developments in genetics and their application to the cells

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GENERAL DISCUSSION

of the brain. The demonstration that nuclear DNA manufactures RNA which then goes into the cytoplasm to command all the cellular activity of the body applies to the nervous system as well. It’s this DNA to RNA machinery that makes a cat look different from a dog, and in some way still obscure, this same genetic mechanism makes the behaviour of a cat differ from the behaviour of a dog. When any new-born animal displays his repertoire of species-specific behavioural responses, this means, however poorly we understand the details of the process, that his brain has been organized to do so by the DNA-RNA mechanism. Now my second point. I find it very difficult to make a large distinction between an innate behavioural response like breathing or suckling or whatever other behavioural response the new-born organism comes equipped with and the things that the animal learns during the course of its lifetime. The capacity to speak, which in man is learned, and the capacity to breathe, which is innate, seem equally complicated behavioural responses to me. Since one of them is made possible through the DNA-RNA mechanism in the brain cells, it seems probable to me that nature would use exactly the same mechanism for taking care of the other of these complicated responses. Reading memories into and out of neurons and glial cells by way of the genetic code is still very much an idea which few facts support. As I tried to indicate, evidence is still very meager that glial cells are involved in learning. Yet the evidence that only neurons are involved in learning is meager also. We are just starting on the question of whether the neurons and the glial cells, working together, are both involved. The most impressive evidence that I know of consists of the single cell microanalyses of HydCn and Egyhhzi whose essential feature is that the genetic code provides the key. SVAETICHIN: I liked very much the talk of Bob Galambos. Concerning the role of glia in the mechanism of memory I would like to mention that we now have additional evidence from the studies on the retina supporting the idea that the neurons are passive conductors only, whereas the control of excitability and inhibition depends on the glial cells. The neuron produces all or none of the spikes which last for a millisecond or two, whereas the glial cell shows properties very different to the ones of the neuron. Since the glial cells show long lasting graded changes in connection with different functional states (e.g. dark and light adaptation), and since the glial cells exert a control of excitability and inhibition on the neuron, we suggested in 1960 at the Freiburg Symposium (Springer-Verlag, 1961, Ed. Jung-Kornhuber) that also the memory process would depend on the glial cell and not on the neuron.

Luco: First, I will refer to one of the problems discussed by Dr. Galambos, the problem of memory. Recently, we have been working on a learning process in Blatta orientalis. Normally, they hold the anthena with one of the forelegs in order to clean it with the palpus. After the forelegs were removed, they learn to hold the anthena with one of the middle legs. It takes about a week to achieve this reaction and it remains for a long period. The comparison between the reactivity of the central nervous system of a group of normal cockroaches and of a group of insects that have learned this new motor situ-

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283

ation permits us the following conclusion: A nervous pathway normally closed becomes permeable during the process of learning. The time course of the electrophysiological reactions seems to indicate that we are dealing with a monosynaptic pathway. Its mechanism is unknown but according to the suggestion of Dr. Galambos we can think that some modification of the glial cells may contribute to the opening of this pathway. In the paper we read yesterday it was suggested that in a nude terminal of a cholinergic myelinated fiber, the chemical mediator was released during stimulation and so, the glial cells do not seem necessary for this normal function. MIQUEL:Although the study of astroglia-neuron relationship in retinal tissue has yielded interesting results, we must keep in mind that extrapolation of this information to cerebral cortex might not be valid. It is well known that the normal retina contains more glycogen than cerebral cortex. Furthermore, retinal tissue shows more active glycolysis. Consequently, the metabolic relationships between astroglia and nerve cells in the retina may differ from those that we assume exist in normal cortical tissue.

DAVSON: Before Professor Morea officially closes this Congress, may I be permitted to say just one or two words on behalf of myself and my colleagues, the visitors to this Symposium. It would be quite invidious for us to mention by name any of you who have been so incredibly generous and hospitable to us. The generosity has extended from the highest professors down to the youngest beginners in neurosurgery. May I then, on behalf of my colleagues here, express my sincere thanks for this hospitality and generosity and the hope also that you will one day, all of you, individually, come to our laboratories and give us the chance to try to return this hospitality.

284

Conclusions of the Symposium EDUARDO D. P. D E ROBERTIS Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires (Argentina)

Truly I was not prepared for drawing the conclusions of this Symposium. I believe that all will agree that this has been a Symposium of high scientific standard and of great interest to the Latin-American Congress of Neurosurgery because several of the problems treated are intimately related with those that neurosurgeons encounter in their daily practice. Since it was an interdisciplinary Symposium, different aspects of the structure and function of neuroglia were considered. The morphological viewpoint, particularly the work of the Spanish school of Cajal, del Rio Hortega, Achucarro in the early part of this century, was presented by Professor Polak. Other morphological aspects at the electron microscope level were presented by several authors, demonstrating that this technique has made important and new contributions. I would like to mention, among others, the demonstration that the intercellular spaces between glia and neurons are very small against what was supposed with optical microscopy, i.e. that there is little intercellular material and no space which could amount to a considerable size. In spite of this, it has been shown in this Symposium, particularly by the work of Dr. Lasansky, that this extracellular space is indeed permeable to ions. This demonstration is most important to clarify the concepts of the physiology of glia and to explain some of the differences that there were between physiologistsand morphologists regarding the problem of the extracellular space. It is now evident that in spite of being small, a space does exist which is active in diffusion and through which neurons may interchange with the extracellular medium. The contribution of electron microscopy was also important in the demonstration of direct attachments of the cell membrane around the capillaries and in certain synaptic regions. It is evident that astroglia tend to make tight junctions between their own processes and with those of neurons. This may be of important significance in explaining the blood-brain barrier mechanism and a synaptic barrier and points toward the function of astroglia in this mechanism. Related to this problem, the work of Dr. Miquel was of considerable interest. He demonstrated that under the action of radiation, astroglia undergoes considerable change. This cell becomes filled with glycogen, a fact also shown by Estable Puig with the electron microscope. These findings show that there are metabolic changes induced by radiation and

CONCLUSIONS

285

indicate the way of access of some metabolites. Glucose, which is so important to the neuron, probably reaches it through the cytoplasm of astroglia. The work of Dr. Friede shows cytochemical differences between astroglia and oligodendroglia and puts forward a series of problems of great interest regarding intercellular relationships. His demonstration that the number of satellite cells around certain neurons varies with the length of the axon, is of considerable biological interest. The fact that in animals of larger size there are more satellites, indicates that the perineuronal oligodendrocytes are metabolically linked to the neurons. This fact has also been demonstrated by the cytochemical work of Professor HydCn and co-workers in Sweden. A series of interesting papers were given on the problem of brain oedema and water surcharge of this tissue. Through the presentations of Dr. Klatzo, Dr. Pappius, Prof. Bakay, Dr. Zadunaisky and Dr. Schadt, several concepts related to the movement of liquids in brain and the participation of different compartments in fluid exchanges have been clarified. However, as I previously mentioned in the discussion, we have to differentiate the more pathological aspects from those that can be considered physiological to the brain. The lesions produced by freezing and shown by Klatzo and Bakay are evidently of pathological interest. In these conditions the blood-brain barrier is broken and there is necrosis of the capillaries. Similar findings frequently occur in trauma and brain tumors. These observations show that capillaries are altered in the more superficial part of the brain and that there is a passage of blood proteins which penetrates into the white matter. These and the observation of a difference between white and grey matter regarding swelling, are of practical interest. Some evidence was mentioned that myelin lamellae could be important in the accumulation of fluid in pathological conditions. Returning to the extracellular space, Professor Davson gave us a very lucid explanation of modern concepts on this problem. He demonstrated that there is a small space which can rapidly be filled in vivo in opposition to a second larger extracellular space which can be filled only through other ways in which the blood-brain mechanisms are not active. This puts the emphasis again on the glia cells. The presentation of Dr. Zadunaisky and co-workers shows in a conclusive way that the ependymoglia cells -which can be considered as a primitive astroglia - have different osmotic properties than the neurons. In all the experiments in which the osmotic pressure or the ionic content of the medium was changed (i.e. increasing the potassium) and finally in the experiments with ouabaine which inhibits the sodium pump, it was observed that ependymoglia reacted osmotically while the neurons remained mainly unchanged. Both the papers of Dr. Tasaki and Dr. Svaetichin who studied the physiological aspects of neuroglia and the considerations made by Professor Galambos were of considerable interest. All of us have followed the work of Dr. Tasaki, when he showed for the first time, a membrane potential in glia cells cultured in vitro and together with Chang and Hild demonstrated a contractile reaction of glia cells with stimulation. The knowledge of the electrical properties of glia cells is of value to interpret the possible interrelation with neurons.

286

CONCLUSIONS

The paper of Dr. Svaetichin showing the existence of decremental potentials in certain regions, corresponding to the Muller and to the horizontal cells indicates the possibility of a direct relationship between neuroglia and nerve cells of the retina. In this relationship it was postulated that glia could have a controlling effect on synaptic phenomena. This presentation and some of the concepts of Professor Galambos on glia-neuronal interactions showed some of the most interesting physiological aspects of this component of the nervous system. The h d i n g of tight junctions between membranes makes possible an interaction with transference of energy and brings us into concepts which can only be explained by solid state physics. It is evident that nowadays we have to consider more and more the possibility of direct energy transfers through thin membrane systems. The presentation of Professor Galambos was an excellent summary of the present state and the future relared to the problem of glia. He mentioned the work of Professor H y d h showing a relationship between glia and cellular mechanisms of memory. These findings and the hypotheses presented are very attractive, but I think that we do not have to exaggerate too much in one sense or in another the importance of the different components of the nerve tissue. The truth is generally found in an intermediary line and in this case this should be found between the neuron, as primary component of the brain, and the glia in order to define better the total function of the nervous system.

AUTHOR INDEX

Author Index* Adams, C. W. M., 38, 42 Adams, R. D., 270,271 Aleu, F., 176, 178 Allen, J. N., 49, 60 Allweis, C., 110 Altman, J., 105, 107, 269 Alvarez, N., 253, 258 Alvord, E. C., 270 Ames, A., 49, 62, 132 Andriezen, W., 19 Appel, S. H., 270, 271 Aprison, M. H., 162 Araki, T., 237 Arnold, A., 105 Ashby, W., 54 Austin, G. M., 166

Bradbury, M. W. B., 124-134, 147 Brandes, F., 256 Bratton, A. C., 220 Breit, A., 107 Breurer, H., 110 Brindley, G. S., 49 Broman, T., 74, 168 Brown, D. V. L., 156 Brown, K. T., 244 Brustad, T., 91,97-99, 103 BueII, M. Y.,36 Bullough, W. S., 107 Bulrner, D., 94, 118 Bunge, M. B., 270 Bunge, R. P., 270

Bailey, P., 105 Bairati, A., 22 Bakay, L., 129,155-183 Ballantine, H. T., 179 Ballard, A., 49 Barlow, C. F., 126, 165, 168 Barnes, J. M., 176-178 Barrnett, R. J., 69 Barron, K. D., 39 Becker, B., 86 Becker, H., 168 Beecher, H. K., 157, 168 Behar, A., 91, 97-99, 103 Bekaert, J., 131 Bendixen, H. H., 160, 169, 171, 179 Bennett, H. S., 6, 55, 64 Bensch, K., 69 Bercel, N. A., 166 Berl, S., 257, 258 Bessman, S. P., 222, 223 Bingham, W. G., 135, 147 Bingley, M. S., 240 Birks, R. I., 140, 273, 274 Bland, J. 0. W., 237 Blaw, M., 38 Boatman, J. B., 166 Bornstein, M. B., 36, 270, 271 Bourne, G. H., 256 Bowsher, D., 86 Boyd, D., 163 Boyle, J. P., 131, 205, 206

Cajal, S. Ram6n y, 12-34, 49, 202, 245, 249 Campbell, A. C. P., 111 Canti, R. G., 237 Caputo, C., 63 Carlsson, A., 258 Casal, S. T., 253, 258 Casamajor, L., 109 Causey, G., 270 Chamberlain, R., 166 Chambers, R. A., 168 Chang, J. J., 237 Chang, M. W., 36 Cheng, S. C., 257, 258 Cherry, R. B., 260 Chetverikov, D. A., 168 Chiarandini, D. J., 207, 208 Clasen, R. A., 135, 147, 156, 163 Clemente, C. D., 105 Cogan, D. G., 40 Cohen, R. B., 36 Collewijn, H., 184-195 Colmant, H. J., 38 Conel, J. L., 268 Conn, H. J., 185 Conway, E. J., 131, 196,205,206 Cook, E. R., 220 Cooke, P. M., 163 Coombs, J. S., 140 Corson, R., 166 Costero, I., 268 Coxon, R. V., 129, 147 Cremer, J. E., 174

*

Italics indicate the pages on which the paper of the author in these proceedings is printed.

287

288

AUTHOR INDEX

Creutzfeldt, 0. D., 261 Crowell, J., 185 Cumhgs, J. N., 165, 176-178 Cummins, J., 243, 256 Curran, P. F., 196,201-207, 214 Curtis, D. R., 10 Cusick, J. T., 129 Darling, L., 268 Darrow, M. A., 185 Davson, H., 49,70,124-134, 147, 148, 155, 196, 219 Dayes, L. A., 144, 147, 149 De Castro, F., 6, 20 De Estable, R., 108, 117 Demeester, G., 131 Dempsey, E. W., 49, 53, 196 De Robertis, E. D. P., 1-11, 44, 49, 53, 55, 63, 111, 135, 140, 144, 148, 151, 155, 156, 192, 196-218,243,270,284-286 Diaz Borges, J. M., 252, 253, 256, 258 Dick, D. A. T., 212 Dobbing, J., 148 Domek, N. S., 126 Dose, K., 111 Dossetor, J. B., 144 Draskoci, M., 86 Drujan, B. D., 243-266 Eccles, J. C., 10, 140, 234 Eccles, R. M., 10 Edstrom, R., 86 Egafia, E., 111 Egyhazi, E., 2, 274, 275 Elliott, K. A. C., 135, 140-143, 146, 150, 156, 165,206,222,223 Emmel, V. M., 185 Estable-Puig, J. F., 89,108,117 Evans, J. P., 86,108, 135, 148, 167 Everett, N. E., 165 Farquhar, M. G., 6, 49, 53, 56, 57, 67, 196 Fatehchand, R., 243-266 Fatt, P., 140, 237 Fawcett, D. W., 54,202 Feigin, I., 174 Feldberg, W., 86 Fernandez-MorBn, H., 124 Ferris, P. J., 79, 87 Fisher, R. B., 124 Fleischhauer, K., 86 Fleming, L. M., 36, 39, 40, 243 Frank, K., 234 Franke, F. R., 166 Frankenhaeuser, B., 54 Freygang, Jr., W. H., 237 Friede, R. L., 35-47, 243, 259 Friedman, V., 126

F r o m , G. H., 261 Frost, R. W., 130 Fuortes, M. G. F., 234 Furshpan, E. J., 237 Furukawa, T., 237 Galmbos, R., 2,244, 267-277 Garcia Argiz, C. A,, 219-224 Garzoli, R. F., 54 Geiger, A., 110 Geoghegan, H., 196 Gerard, R. W., 201 Geren, G. B., 270 Gerschenfeld, H. M., 1,4,10,44,49,53, 63,111, 135, 140, 144, 148, 151, 155, 156, 192, 196, 243,270 Giacobini, E., 44,54,256 Gimknez, M., 249,250 Glees, P., 12, 187 GIobus, J. H., 185 Goldberg, M. A., 126, 168 Goldstein, A. L., 111 Goldstein, N. P., 164 Golgi, C., 12-34 Golubtsova, A. V., 111 Gonatas, N. K., 108, 167, 168, 177, 178 Gonzalez Aguilar, F., 2, 10 Gordon, S., 117 Gouras, P., 244 Granit, R., 49 Grant, F. C., 166 Gray, E. G., 6, 64 Green, D. E., 259 Grillo, M. A., 117 Grontoft, O., 168 Groth, D., 256 Gulati, D. R., 144, 146 GUM, C. G., 168 Hager, H., 74, 107 Haigh, A. L., 164 Hall, J. C., 111 Hamberger, A., 2, 45, 48 Hamuro, Y., 108, 111 Haque, I., U1, 156, 160-162, 164, 165 Haranath, P. S. R., 86 Harris, B., 148, 150, 192 Harris, E. J., 204, 210 Harrison, R. G., 29 Hartmann, J. F., 49, 53, 124, 196 Hashimoto, Y., 49 Hass, G. M., 135, 147, 156 Hassin, G. B., 130 Hastings, A. B., 49, 62, 127 Hauser, H. M., 164 Hawk, P.B., 94 Hawkins, A., 44 Haymaker, W., 89-114, 117, 256

AUTHOR INDEX

Hayner, R., 135 Heisey, S. R., 219 Held, H., 19 Hellman, L., 166 Henry, J., 91, 97-99, 103 Hernhndez, J. A., 252, 256 Herned, H. S., 204 Hess, A., 64 Hess, H. H., 36 Heywood, P. M., 168 Hicks, S. P., 151 Hild, W., 15, 192, 193, 205, 234, 237, 249, 268, 270 Hill, A. V., 203 Hillarp, N. A,, 258 Hills, C. P., 168, 172 Hines, M., 256 Hirano, S., 221, 223 Hirschberger, W., 105, 107 His, W., 13 Hodgkin, A. L., 54 Hoffmann, J. F., 214 Hofmann, H. F., 132 Holst, E. A., 105 Hooper, N. K., 129 Horstmann, E., 4, 54, 124, 196 Horstmann, V., 44 Hortega, P. del Rio, 12-34 Hubbard, R. S., 220 Hueter, T. F., 179 Hurst, E. W., 151 Huxley, H. E., 274 Hydtn, H., 2, 44, 48, 243, 256, 274, 275 Hyman, C., 166 Ibrahim, M. Z. M., 38, 42 Inoma, S., 244 Ishii, S., 135 Jacobs, M. H., 204 Jacobson, S., 270 Jahn, T. L., 258, 260 Jankovic, B. D., 271 Janssen, P., 91, 97-99, 103, 107 Jasper, H., 146, 150, 156 Javid, M., 147 Jen, M. K., 36 Jordan, E. F., 219 Kalsbeck, J. E., 176178 Kamiya, N., 240 Kapp, H., 261 Kapphan, J. I., 36 Kaps, G., 164 Katz, B., 273, 274 Katzman, R., 44, 140, 166, 168, 176-178, 192 Kelly, W. A,, 135

289

Kendrick, T. R., 166 Kerr, S. E., 94 Kies, M. W., 270 Kiyota, K., 164 Klatzo, I., 38, 39, 73-88, 89, 91, 92, 94, 97-99, 103,107,117,135,141,144,148,156,163-165 Kleeman, C. R., 129,131-133,147,219 Knapp, F. M., 166 Knoller, M., 36, 39 Koch, A., 44,49, 166, 192 Koelle, G. B., 272 Koenig, H., 39 Kolliker, A,, 13 Kosugi, Y.,244 Kozak, P., 38 Krebs, H. A., 110 Kreutzberg, G., 38 Kruger, L., 89, 117 Kuffler, S. W., 193, 249, 250 Kulenkampff, H., 44 Kuss, B., 110 Kuwabara, T., 40 Lajtha, A., 219, 221, 223 Lange, P. W., 2, 44, 48 Langfitt, T. W., 147 Lasansky, A., 6, 48-72 Lascano, E. F., 185, 186 Laskowski, E. J., 75, 135, 144, 148, 156, 163 164 Laufer, M., 243,245,250-252,255,256-259 Laughlin, J. S., 105 Lawrence, Jr., W., 166 Leaf, A., 140, 143, 196 Leavitt, S., 156 Leblond, C. P., 107 Leduc, E. H., 164 Lee, J. C., 86, 165 Lehninger, A. L., 55 Lehrer, G. M., 271 LenhossBk, M. W., 14 Leslie, W. G., 164 Levin, E., 129,131-133,147,219-224 Levine, S., 108, 167, 168, 177, 178 Libet, B., 201 Lierse, W., 111 Lilly, J. C., 260 Linder, J., 166 Loevinger, R., 89 Loomis, T. A., 220 Lourie, H., 164 Lowry, 0. H., 36 Luft, J. H., 56, 58 Lugaro, E., 25 Lukenbill, A., 162 Lumsden, C. E., 151, 268 Luscombe, M. J., 220 Luse, S. A., 49, 53, 124, 148, 150, 192 Lyman, J., 91, 97-99, 103

290

AUTHOR INDEX

MacCormack, J. I., 196 MacNichol, Jr., E. F., 244 MacPherson, L., 244 MacRobbie, E. A. C., 212 Maeda, S., 55 Magee, K. R., 38 Magee, P. N., 176178 Magnes, J., 110 Magoun, H. W., 268 Malhotra, S. K., 185 Malis, L. J., 89 Manery, J. F., 127 Marazzi, A. S., 258 Marshall, R. O., 220 Martin, F. A., 135, 147 Matsumati, T., 221, 223 Mawas, P., 22 Maxwell, D. S., 54, 117 Maynard, E. A,, 49, 124, 136, 140 McGee Russell, S. M., 117 McIlwain, H., 136 McKenzie, B. F., 164 Menzel, D. B., 89, 94, 117 Merlis, J. K., 219 Meves, H., 4, 54, 124 Mihailovic, L. J., 271 Miledi, R., 273, 274 Milkovic, K., 81 Miquel, J., 38, 39, 73-75, 77, 79, 86, 87, 89-114, 117, 148, 164, 165, 256 Mitarai, G., 243-245, 248, 250-252, 255, 256, 258,259 Moiseyenko, E. V., 111 Moses, C., 166 Mossakowski, M. J., 40 Motokawa, K., 244 Mullan, S., 86, 108, 135, 148, 167 Miiller, H., 49 Miiller, W., 38 Murakami, M., 49 Murray, M. R., 36, 268, 270, 271 Nair, V., 112 Nagata, Y.,221, 223 Nageotte, J., 22 Naka, K., 244 Napolitano, L., 54, 202 Nasu, H., 38 Neame, K. D., 223 Negishi, K., 243-266 Nelson, E., 38 Nesbitt, F. B., 132 Newman, B. L., 44, 166, 192 Nicholls, J. G., 193 Niimi, K., 258, 259 Noell, W. K., 54, 70 Nogueira, G. J., 219-224 Novikoff, A. B., 87

Ochs, S., 184 Ogawa, K., 36 Ogawa, T., 244 Oikawa, I., 244 Oksche, A., 44, 108, 111, 202 OLeary, J. L., 164 Olssen, O., 74 Olszewski, J., 44, 86, 165 Oppelt, W. W., 129 Oser, B. L., 94 Osterberg, K., 38, 39, 42 Otenasek, R., 73-75, 77, 87, 92, 107, 148, 164, 165 Ottoson, D., 252 Owen, B. B., 204 Palade, G. E., 6, 56, 57, 67 Paladino, G., 24, 25 Palay, S. L., 6, 111, 117 Palladin, A. V., 110 Pandolfi, S., 163 Pappas, G. D., 268 Pappenheimer, J. R., 219 Pappius, H. M., 135-154,165,206 Parchwitz, H. K., 110 Parker, I. T., 168 Parsons, D. S., 137, 142 Patlak, C. S., 49, 129 Pease, D. C., 49, 54, 56, 124, 136, 140 Penfield, W., 34 Perez, J. C., 256 Peters, A,, 52 Peters, G., 38 Peterson, E. R., 36, 268, 270, 271 Pethica, B. A., 64 Pigon, A., 2, 44, 48, 243, 274 Piraux, A., 75, 135, 144, 148, 156, 163, 164 Pol&, M., 12-34 Pollay, M., 129, 219 Polyak, S. L., 49, 59 Pomerat, C. M., 237, 268 Ponder, E., 212 Portela, A., 256 Pope, A., 36, 109 Popoff, N., 174 Potter, D. D., 193, 249, 250 Prado, J. M., 29, 30 Prochorova, M. I., 110 Prokop, 5. D., 79, 87 Prost, J., 268 Prouty, R. R., 135, 147 Psychoyos, S., 110 Purkinje, J., 15, 19 Purpura, D. P., 268 Quadbeck, G., 168 Quastel, J. H., 174, 256 Raimondi, A. J., 86, 108, 135, 148, 163, 167

AUT H O R I N D E X

Rall, D. P., 49, 86, 129 Ranck, Jr., J.B., 44, 166, 186, 192 Reddy, V., 256 Reed, D. J., 129, 147 Revel, J. P., 54, 202 Rhodin, J., 54 Richardson, J. C., 168 Richardson, Jr., R. E., 105 Ris, H., 270 Roberts, N. R., 36 Robertson, W, F.. 24 Robins, E., 36 Robinson, J. R., 4, 143 Roizin, L., 35 Romanul, F., 36 Rose, A. S., 35 Rose, J. E., 89 Rosemberg, T., 214 Rosenbluth, J., 53, 55 Ross, L. L., 270, 271 Roth, L. J., 112, 126, 165, 168 Rougemont, J., 132 Rowbotham, G. F., 164 Rubinstein, L. J., 38, 39, 40, 42, 86 Rushton, W. A. H., 244, 252 Russell, D. S., 237 Sabatini, D. D., 69 Safronova, M. I., 111 SchadB, J. P., 165, 184-195 Schafer, E. A., 269 Schaper, A., 13 Schatzmann, H. J., 214 Scheibel, A. B., 28 Scheibel, M. E., 28 Scheinker, M., 178 Schiffer, D., 36, 38, 40 Schloerb, P. R., 127 Scholte, W., 105 Scholz, W., 105 Schoolar, J. C., 165 Schultz, R. L., 49, 124, 136, 140 Schuster, E. M., 54 Schwerin, P., 222, 223 Scott, B. L., 54 Seegal, B. C., 271 Segar, W. E., 162 Selverstone, B., 157, 168 Selvin de Testa, A,, 243-266 Sheldon, W., 86 Shimizu, N., 55, 108, 111 Shmidt, Z. M., 168 Sickel, W., 256 Sidman, R., 55 Simmons, B., 165 Sjostrand, F. S., 49, 55, 243, 260 Smart, I., 107 Smirnov, A. A., 168

29 1

Smith, B., 38,40 Smith, D. E., 36, 7 3 9 8 Snezhko, A. D., 111 Sonneblick, B. P., 111 Sosa, D., 179 Spaziani, E., 49, 125, 148, 196 Spector, R. G., 168, 172 Spyropoulos, C. S., 234 Steinwall, O., 74, 86 Stewart, P., 256 Stewart-Wallace, A. M., 147, 167 Stoner, H. B., 176-178 Story, J., 38 Streicher, E., 81, 177 Stubbs, J. D., 133 Sugano, H., 112 Summerson, W. H., 94 Sutton, C. H., 40, 42 Svaetichin, G., 243-266 Wen, H. J., 164 Tachibana, S., 194 Tasaki, I., 192, 193, 205, 234-242, 249 Tasaki, K., 244 Taylor, J. L., 130 Taylor, R. M., 110 Terry, R. D., 167, 176, 177 Terzuolo, C. A., 237 Thesleff, S., 273 Thompson, C. M., 240 Timiras, P., 49 Tobias, C. A., 89, 91, 94, 97-99, 103, 107, 117 Tomita, J., 49 Tomita, T., 244 Tong, C., 244 Torack, R. M., 167, 176, 177 Tosaka, T., 250, 258 Tosteson, D. C., 193, 214 Toth, J., 221, 223 Tower, D. B., 258 Tschirgi, R. D., 86, 111, 130 Tsukada, Y.,221,223 Ungar, G., 110 Ussing, H. H., 212 Valentin, J., 15 Vallecalle, E., 243, 245, 250-252, 255, 256, 258, 259 Van Dyke, D. C., 107 Van Gelder, N. M., 222, 223 Van Goor, H., 54 Van Harreveld, A., 129, 165, 184, 185, 193, 194 Van Houton, W. H., 38,44,243 Van Rossum, G. D. V., 137,142 Vardanis, A,, 174 Varon, S., 136 Veralli, M., 110

292

AUTHOR INDEX

Verlot, M., 157, 168 Vesco, C., 36,38,40 Villegas, G. M., 53,55,64,245,249 Villegas, J., 243, 245, 250-252, 255, 256, 258, 259 Villegas, L., 63, 249, 250 Villegas, R., 53, 63, 64, 245, 249, 250 Voeller, K., 268 Waelsch, H., 222, 223, 257, 258 Waksman, B. H., 270,271 Wald, F., 4, 6, 44, 49, 50, 55, 69, 61, 63-66, 68, 70, 135, 140, 144, 148, 150, 155, 156, 192, 196-21 8 Watanabe, I., 250, 258, 259 Wattenberg, L., 38, 39, 42 Weed, L. H., 60 Weinberg, S., 174 Weinstein, W. J., 164 Weiss, L., 64 Weiss, P., 268 Welch, H., 126 Welch, K., 86

W e d , E. J., 167 Wesemann, I., 110 White, J. C., 157, 168 Wildbrant, W., 214 Williams, G. R., 168 Wilson, C. E., 44, 176, 178 Wislocki, G. B., 49, 5 5 , 196 WiSniewski, H., 73-88 Witkovsky, P., 256-258 Woldeck, B., 258 Wolfgram, F., 35 Woodbury, D. M., 49, 129, 147 Wright, G. P., 270 Wyckoff, R. W. G., 49, 124, 196 Yokota, T., 250, 258 Yonezawa, T., 36, 271 Young, D. A. B., 124 Young, J. Z., 49, 124, 196 Zadunaisky, J. A., 4, 49, 63, 135, 140, 144, 148, 150, 155, 156, 192, 196-218 Zimmermann, H. M., 36, 108, 167, 178

293

Subject Index Amino acids, transport, in CNS, 219-223 and ventriculo-cisternal perfusion, 219-223 Astrocytes, and extracellular fluid, composition, 65, 70 degeneration, and cerebral edema, 156 enzymatic activity, 36-39 and edematous changes, 37, 38 and sodium transport, 42, 43 and Wilson’s disease, 42 histochemistry, 36-45 hypertrophy and enzymatic changes, 39-43 and glycolysis, 39 and lactic dehydrogenase, 39, 40 regional types, 40, 41 triggering factors, 42, 43 pathological conditions, 38 Astroglia, and cerebral edema, 155, 156, 227 function, water-ion compartment, 140 and glycolysis, inhibition, 110-1 12 irradiation, metabolic disorders, 111,112 and mitotic activity, 102-107 a-particle radiation, data, 91 reactions, to ionizing radiation, 89-1 12 X-radiation, data, 89 Astroglial cells, and blood-brain barrier, 2 differentiation, 16-19 function, 2 4 , 20-22 and oligodendroglia, 2, 3 morphology, 16-22 Autoradiographic observations, and vascular permeability studies, 78-81 Biogenic amines, and glial metabolism, 258 Blood-brain barrier, and astroglial cells, 2 and brain irradiation, 91, 92, 95 and cerebral edema, 155 diffusion, of 1311, 128, 129 and extracellular fluid, concentration, 126 and extracellular space, measurement, 127,128 filtration, conditions, 129, 130 function, and cerebral edema, 155, 164 Brain, extracellular fluid, secretion, 130

extracellular space, 124-133 and blood-brain barrier, 127-129 measurement, 124-127 function, and glia-neuronal interaction, 243263,283 irradiated -, and blood-brain barrier, 91, 92, 95 chemical analysis, 94 glycogen accumulation, 117-122 mechanism, 11C112 glycogen content, 97,99-105, 107-1 11 histological changes, 92, 94-105 Brain barriers, permeability, and fluorescent markers, 73, 85 and radioactive markers, 73, 85 Brain edema, and extracellular space, 4, 6 protein movement, dynamic aspects, 78, 79, 85, 86 Brain tissue, antigenic properties, 27C272 astrocytes, and retention of salts and water, 196-217 edema, definition, 156 experimental -, and hypertonic urea, 147, 149 in-vivo studies, 144-149 fluid, distribution, 148, 150, 151, 227 in human pathology, 227-231 and intracranial tumours, 146 and myelin sheath, 229,232 electrolytes, clearance rate, 166 extracellular fluid, and tracer sodium, 202-208 fluid uptake, conditions, 135-1 53 in-vivo studies, 144-151 glial cells, function, and antigenic properties, 272 and myelination, 270-272 and synaptic events, 272-274 glucose distribution, and membrane permeability, 140 G-strophantin, and sodium reflux, 214-217 and immersion fluid, osmometer, 214 morphology, and cell activity, 267-269 osmotic changes, and effective volume, 212-214 quantitative studies, 198-202

294

SUBJECT I N D E X

and potassium permeability, 208-212 and radiosodium, exchange, 207-208 and Ringer’s solution, electron micrography, 197-202 slices, action potentials, 234237 and cellular activity, 268, 269 electric response, 234237 electrical impedance, 239 and glial cells, electric responses, 237-241 electrophysiology, 249, 250 and inulin space, 205,206, 225, 226 and neuron, excitability, 237 photomicrography, 138, 139 protein space, 138, 140 sodium permeability, 207, 208 sucrose, equilibration, 138, 140 swelling, osmotically i n d u d -, 149-1 51 thermal conductivity, 164 thiocyanate, equilibrium, 138, 140 trauma, water and electrolyte content, 145, 146 uremic dog, and hemodialysis, 149-1 51 and water, distribution, 135-153 Central nervous system, and amino acids, transport, 219-223 demyelination, and allergy, 270-272 demyelinative disorders, studies, 271 fluid-tissue barrier, and amino acids, transport, 219 function, and glial cells, 243-263 and neurons, 243-263 glial metabolism, and biogenic amines, 258 perfusion fixation, 2, 27 serum proteins, penetration, 73-87, 232 vascular permeability, dynamic aspects, 75, 76 studies, 74-77,84,85 Cerebral cortex astrocytes, and anoxia, 188-190, 193 and hypothermia, 190, 191 and spreading depression, 191, 192, 194 staining method, 186 fluid compartments, and distribution of tracer substances, 136, 137 freezing conditions, 185 inulin space, 137, 138, 141 oligodendrocytes, and anoxia, 189, 190 and hypothermia, 190, 191 and spreading depression, 191, 192 staining methods, 186, 187 slices, and fluid compartments, 135, 137, 280

fluid uptake, and impaired metabolism, 143, 144 and osmotic gradient, 142, 143 incubation, and osmotic equilibrium, 142144 potassium, uptake, 142 swelling, in isotonic salt solutions, 135-137 and thiocyanate, 148, 149 and ‘tracer’ space, 136, 137 types of swelling, 140-144 Cerebral edema, albumin concentration, and astrocytes, enzyme systems, 165 and asphyxia, physiological components, 168-172 and astrocytes, degeneration, 156 enzymatic activity, 37, 38 and blood-brain barrier, function, 155 permeability, and astrocytes, 164 theories, 155 and cerebral metabolism, 155 cold-induced, and astrocytes, 164 and blood-brain barrier, 166, 167 development, studies, 135 electrolytes, and albumin movement, 155-180 clearance rate, 165 electron microscopy, and extracellular space, 167 electrophoresis, and albumin concentration, 164 experimental -, and astrocytes, 155, 156 and electrolyte content, 158, 165 and electrolytes, port of entry, 163, 167 and hypertonic urea, 147, 149 in-vivo studies, 144-149 and isotopes, 163, 166 radioautography, 157, 159, 160-164 vital staining, 157-159, 163, 168, 171, 177 fluid, distribution, 148, 150, 151 and gray matter, pathological changes, 179 and hypoxia, 168 hypoxic -, histological changes, 172, 173 and radioactive albumin, deposition, 172 inflammatory -, experiments, 167, 168 and intraaanial tumours, 146 and local freezing, studies, 156, 157 mechanism, and pathogenesis, 178, 179 and neurons, electrical impedance, 165 Yroteins, content, 164 electrophoretic pattern, 164 fluorescein-labeled, 165

SUBJECT INDEX

toxic -, experiments, and histological changes, 176 and triethyl tin, 173-178 and blood-brain barrier, 177 and white matter, distribution of water, 176 predilection, 158, 164-168 selective vulnerability, 178 selectivity, explanation, 179 and ultrasonic radiation, 179 Cerebral tissue, glial space, and fluid uptake, 144, 151 Cerebrospinal fluid, and amino acids, transport, 219,220 and extracellular fluid, relationship, 130-133 injected creatinine, concentration, 127 secretion, and choroid plexuses, 132, 133 and plasma concentration, 131-133 'sink-action', 125-127 Chloride space, and extracellular space, 127 Choroid plexus, passage of proteins, mechanisms, 80-84 Creatinine, injected -, concentration, in CSF, 127 Donnan equilibrium, and extracellular fluid, asphyxia, 193 Electroretinogram, and ferrocyanide, distribution, 60-62, 65 Extracellular fluid, brain, asphyxia, and Donnan equilibrium, 193 Gibbs-Donnan equilibrium, 131 and K-concentration, 132, 133 secretion, 130-132 and glial cells, 132 and tracer sodium, 202-208 and cerebrospinaI fluid, relationship, 130-133 composition, 278 and astrocytes, 65 of neural retina, 70 concentration, and blood-brain barrier, 126 and diffusion coefficient, 126, 127 control, 129 Extracellular space, and blood-CSF barrier, 125, 126 brain, 124-133 edema, 4, 6 function, 4, 6 and cerebral edema, electron microscopy, 167 and chloride space, 127 measurement,

295

and blood-brain barrier, 127, 128 osmotic exchange, 279 and sodium space, 127 and tracer sodium, isotope loss curve, 205-207 Fluorescence microscopy, and vascular permeability studies, 74-77, 81 Glia-index, definition, 44 Glial cells, classical types, 1 and CNS, function, 243 cytochemical techniques, 1 excitability, 236241 function, 1, 267-275 and antigenic properties, 272 and ions diffusion, 278, 279 and myelin formation, 270-272 and synaptic processes, 272,274 immunological specificity, and multiple sclerosis, 271 membrane resistance, determination, 239, 240 and neurons, interaction, 243-263 retina, electric responses, 244-249 morphology, 48-70 and RNA content, 2 and memory storage, 274, 275, 281-283, and sodium content, 140 and synaptic activity, 6, 8-10 Glial space, cerebral tissue, and fluid uptake, 144, 151 Glycogen, accumulation, in irradiated brain, 97, 99-105, 107-111, 117122 mechanisms, 110-1 12 localization in brain, 108-1 11 Inulin space, brain tissue, slices, 205, 206, 225-227 cerebral cortex, 137, 138, 141 slices, 137, 138, 143 fluid uptake, conditions, 141-143 retina, glial cells, 225-227 Lactic dehydrogenase, and astrocytic hypertrophy, 39, 40 Microglial cells, origin, 14 Miiller cells, and distribution of tracer substances, 62-65 electron microscopy, 50-70 granules, studies, 54, 55

296

SUBJECT I N D E X

intercellular junction, 58-62, 69 mitochondria, and enzyme activity, 55 retina, structure and function, 48-70 Miiller cell channels, and diffusion, 59, 60 of ferrocyanide, 62-65 function, 52-54 glial junction, 56-61 and partial diffusion barrier, 67 junctional layer, 55-59 permeability, 66-70 Myelin sheath, and brain tissue, edema, 229, 232 Neuroglia, biology, electron microscopy, 1-1 1 Neuroglial cells, considerations, 12, 13 differentiation, 12, 13 embryological data, 13-15, 28, 29 enzyme histochemistry, 35-45 microscopical observations, 12-33 morphology, 12 osmotic changes, 196-217 physiological role, working hypothesis, 44,45 and spinal ganglia, 30, 31 staining methods, 186-188 and sympathetic ganglia, 29-3 1 volume changes, 184-194 and anoxia, 188, 189 and cardiotonic glucosides, 214-217 and hypothermia, 190, 191 quantitative analysis, 185-192 and spreading depression, 191, 192 Neuron, cell membrane, bioelectrical phenomena, 63, 64 excitability, 234-241 and glial cells, interaction, 243-263 Oligodendroglial cells, enzymatic activity, 35-37 function, 1, 2, 25 of astroglia, 2, 3 and glycolysis, 36 histochemistry, 35, 36 and metabolism, normal -, 44 morphology, 22-24 and myelination, 44 satellite cells, 35 staining techniques, 28 Potassium permeability, and brain tissue, 208-212

Potassium uptake, cerebral cortex, slices, 142 Protein space, and brain tissue, slices, 138, 140 Proteins, serum -, diffusion, role of glia, 86, 87 penetration into CNS, 73-87, 232 vascular permeability, degrees of increased -, 84

Radiation, ionizing -, sensitivity of granular cells, 119 a-particle -, brain, vascular changes, 92, 94-96 techniques, 91-93 Radium sodium exchange, and brain tissue, 207, 208 Retina, color system, spectral response curves, 249 controller cells, and excitation spread, 259-261 function, metabolic dependence, 253-259 neurochemistry studies, 253-259 membrane potential, 253 and temperature, 254, 255 excitability, and controller cells, hydrogen-ion concentration, 253-259, 262 and glial cells, 251 steady potentials, 251-253 function, and glia-neuronal interaction, 282, 283 glial cells, activity, and anoxia, 250,251, 253,255 and neuronal processes, 250, 251 and controller cells, 244-249 electric responses, 244-249 and inulin space, 225, 226 morphology, 48-70 and Miiller cells, structure, 48-70 and presynaptic inhibition, 226 and respiratory enzymes, 256-259 tight junctions, 226 morphology, and microphotography, 245-248

.

Sodium permeability and brain tissue, 207, 208 Sodium space, and extracellular space, 127 Synaptic activity, and glial cells, 6, 8-10

SUBJECT INDEX

Synaptic barrier, morphological basis, 6-10 Thioc yanate, and cerebral cortex tissue, in-vivo, 148, 149 Tracer sodium isotope loss curve, and extracellular space, 205-207 Tracer substances, equilibration, conditions, 136 Trypan blue, and cerebral edema, vital staining, 157-159, 163, 164, 171, 177 Ultrasonic radiation, and cerebral edema, white matter, 179 Urea, hypertonic -,

297

infusion, and experimental brain edema, 147, 149 Vascular permeability, and autoradiographic observations, 78-81 CNS, studies, 74-77, 84, 85 and serum proteins, 84 studies, and fluorescence microscopy, 76-77, 81 Wilson’s disease, and astrocytes, enzymatic activity, 42 X-irradiation, techniques, 90, 91

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