International Review of Neurobiology Volume 31

INTERNATIONAL REVIEW OF

Neurobiology VOLUME 31

Editorial Board W. Ross ADEY JUIMJS

AXELROD

SEYMOUR KETY KEITHKILLAM

Ross BALDESSARINI

CONANKORNETSKY

SIRROGERBANNISTER

ABELLAJTHA

FLOYDBLOOM

BORISI .FRFDEV

DANIELBOVET

PAULMANDEL

PHILLIPBRADLEY

HUMPHRY OSMOND

YURIBUROV

RODOLFOPAOLETTI

Josd DELCADO

SOLOMON SNYDER

SIRJ O H N ECCLES

STEPHEN SZARA

JOEL

ELKES

SIRJOHN VANE

H. J. EYSENCK

MARATVAKI'ANIAN

KJELLFUXE

STEPHENWAXMAN

B o HOLMSTEDT

RICHARDWYATT

PAULJANSSEN

INTERNATIONAL REVIEW OF

Neurobiology Wedby

JOHN R. SMYTHIES Department of Neuropsychiatry Institute of Neurology National Hospital London England

RONALD J. BRADLEY Department of Psychiatry and The Neuropsychiatry Research Program The Medical Center The University afAlobama at Birminghom Birmingham, Alabama

VOLUME 31

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CONTENTS Animal Models of Parkinsonism Using Selective Neurotoxins: Clinical and Basic Implications

MICHAELJ . ZICMONDA N D EDWARDM . STRICKER I. I1 . 111. IV . V. VI . VII . VIII .

Introduction .................................................... What Is the Relation between Neuropathology and Sympt Why Are Such Large Lesions Required before Symptoms Does Age of Injury Influence the Deficits? ......................... How Does Stress Influence Symptoms? . How Can Drugs Replace Neurons? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Implications for Future Research . . . . . . . . . . . . . . . . . . . Summary and Conclusions .. ................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 9 15 30 33 40 43 57 60

Regulation of Choline Acetyltransferase

PAULM . SALVATERRA AND JAMES E . VAUGHN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Methods Used to Study ChAT Expression ......................... 111. Immunocytochemical Location of ChAT in the CNS . . . . . . . . . . . . . . . . IV . Development of Cholinergic Neurons ............................. V . Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81 84 100 123 132 134

Neurobiology of Zinc and Zinc-Containing Neurons

CHRISTOPHER J . FREDERICKSON ........................

I . Introduction . I1 .

n ......................

111.

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

IV . Zinc and Brain Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Zinc and Membranes . . . . . . . . . . . . . . . . . . VI . VII . VIII . Functional Significance of Vesicular Zinc ........................... 1x. Zinc and CNS Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . Summary and Conclusions . . . . . ... ........... References . . . . . .................................... V

146 149 159 164 175 177 196 204 214 220 224

CONTENTS

vi

Dopamine Receptor Subtypes and Arousal

ENNIOONGINI AND VINCENZO G. LONGO I.

Introduction, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..... .............. 111. Central Dopamine Rec ........................... IV. D-2 Receptors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ................................ ........................... 11. Arousal: A Definition,

V11. Conclusion References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239 240 24 1 244 246 24Y 25 1 253

Regulation of Brain Atrial Natriuretic Peptide and Angiotensin Receptors: Quantitative Autoradiographic Studies JUAN

M.

SAAWDKA,

EERO( : A S Y K ~ N ,.JoR(;E S. GUTKINI), AND ADILJ . NAZAKALI

.......... .............. ................................ 111. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V1. Keferenres. . . . . . . . . . . . . . ...... ............... 1. Introrlurtion

257 260 265

28 1 290 29 1

Schizophrenia, Affective Psychoses, and Other Disorders Treated with Neuroleptic Drugs: The Enigma of Tardive Dyskinesia, Its Neurobiological Determinants, and the Conflict of Paradigms JOHN

L. WADDINGTON

............. I. Introduction . . . . . . . . . . . . Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................... .. 111. Incidence ....................................... .. IV. Natural H V. Morbidity and Mortality. ......................................... ............ VI. Vulnerability Factors.. . . . V I I . Pathophysiological Mechar v111. Synthesis: The Conflict of References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.

298 30 1 310 31 1 314 315 337 344 346

CONTENTS

vii

Nerve Blood Flow and Oxygen Delivery in Normal, Diabetic, and Ischemic Neuropathy

PHILLIPA. Low, TERRENCE D. LAGERLUND, AND PHILIPG. MCMANIS I. Special Anatomy of Nerve Microvasculature. . . . . . . . . . . . . . . . . . . . . . . . Regulation of Blood Flow Nerve Blood Flow Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diabetic Neuropathy.. . . Ischemic Neuropathy . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . Edematous Neuropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

356 360 362 378 382 396 409 424 433

INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTSOF RECENTVOLUMES ............................

439 455

11. Special Physiology of Nerve Microvasculature 111. Oxygen Delivery . . . . . . . . . . . . . . , . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . .

IV. V. VI. VII. VIII.

This Page Intentionally Left Blank

ANIMAL MODELS OF PARKINSONISM USING SELECTIVE NEUROTOXlNS: CLINICAL AND BASIC IMPLICATIONS By Michael J. tigmond and Edward M. Stricker Depaltmenfr of Behavioral Neuroscience and Psychiatry

and the Center for Neuroscience University of Pittsburgh Pittsburgh, Pennsylvania 15260

I. Introduction A. Overview of the Disease B. Pharmacotherapy C. Recent Animal Models for the Study of Parkinsonism D. Some Unanswered Questions 11. What Is the Relation between Neuropathology and Symptoms? A. The Role of the Nigrostriatal Bundle B. Other Monoaminergic Projections C. Secondary Responses to NSB Lesions 111. Why Are Such Large Lesions Required before Symptoms Emerge? A. Role of Residual DA Neurons in Maintaining Function B. Characteristics of Dopaminergic Systems C. Rapid Compensations after Subtotal Injury D. Recovery of Function after Large NSB Lesions IV. Does Age of Injury Influence the Deficits? A. Attention Deficit Disorder as a Consequence of Neonatal NSB Injury B. Effects of Early Damage to Dopaminergic Neurons in Experimental Animals V. How Does Stress Influence Symptoms? A. Paradoxical Kinesia B. Stress-Induced Impairments C. Implications for Cannon's Fight-or-Flight Theory VI. How Can Drugs Replace Neurons? A. Atropine B. [.-DOPA VII. Implications for Future Research A. Diagnosis B. Treatment C. Prevention VIII. Summary and Conclusions A. How Good Are the Models? B. What Have We Learned? References

1 IN'I'ERNATIONAL REVIEW OF NEURORIOLOGY, VOL. 31

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

MICHAEL J. ZIGMOlVD A N D EDWARD M. STKICKER

I. Introduction

I n the history of science and medicine, basic research and clinical pr-nctice play synergistic roles. Within neuroscience, nowhere is this more in evidence than in the case of Parkinson's disease. For example, our understanding of the cause of this disorder as well as the principal modes of its treatment were derived from animal studies, whereas efforts to understand parkinsonisni have led to nia.jor insights into the basic neurobiology of catecholaminergic systems, with irriplications extending well beyond the disease itself. This review deals with two decades of interplay between the laboratory and the clinic in analyzing the biological bases of parkinsonism arid formulating a rational approach to its treatment. First, we describe the syndrome, its accompanying neuropathology, and the current modes o f treatment. Second, we review t w o animal models of the disorder. Third, we describe the progress that has been made with these models toward answering some of the niajor questions regarding the disease. Finally, we discuss some of the implications of these results, focusing on avenues of future research.' A. OVERVIEW

OF THE

DISEASE

1. Symptoms

Parkinson's disease is a progressive neurodegenerative disorder of the basal ganglia that ultimately robs the afflicted individual of the ability to initiate any voluntary movement. It is observed in approximately 1 % of the population over 55, the approximate age at which the disease usually is first diagnosed; it affects men and women equally; and it occurs worldwide. The disease commonly is detected as a mild resting tremor of one or more limbs, muscular rigidity, postural abnormalities, arid brad ykinesia. 'These abnormalities tend to be accompanied by other symptoms, including decreased food intake and concomitant weight loss, characteristic autonomic dysfunctions, depression, and a general slowing of intellectual processes. The severity of the symptoms is exacerbated by physical or emotional trauma, although there also are reports of a temporary stress-induced improvement in the behavior of

'

The literature review on which this article is based was completed in k c e m b e r 1987. A few additional citations were added while the manuscript was in press.

ANIMAL MODELS OF PARKINSONISM

3

otherwise akinetic patients. Both neurological and psychiatric symptoms usually worsen slowly but inexorably and lead to an end-state of complete akinesia, often accompanied by dementia, within 10- 15 years of the initial diagnosis (see reviews in Selby, 1968; Birkmayer and Riederer, 1983; Jankovic and Calne, 1987).

2. NeurofmtholoRy Postmortem examination of parkinsonian brains reveals a number of neurochemical and histological abnormalities (see reviews in Hornykiewicz and Kish, 1987; Javoy-Agid et al., 1986; Jellinger, 1987; Zigmond et al., 1987). Most striking is the degenerative loss of the dopaminergic neurons of the nigrostriatal bundle (NSB). This is manifest as a loss of pigmented cells in the substantia nigra and of dopamine (DA) in the caudate and putamen of the corpus striatum. Even patients with relatively mild symptoms usually have striatal DA depletions of 70-80%, and severe akinesia commonly is associated with a loss of 95% or more of this transmitter. Typically, parkinsonism is accompanied by the loss of other monoaminergic neurons as well. Some degeneration of DA-containing neurons of the mesocortical, mesolimbic, and hypothalamic systems usually is observed. In addition, there is loss of norepinephrine (NE)-containing projections of the locus coeruleus and of the autonomic nervous system, and apparent degeneration of central serotonin (EiHT)-containing projections. Changes in nonmonoaminergic systems also have been identified, including a reduction in the concentration of enkephalins, substance P, bombesin, cholecystokinin-8, and neurotensin, and a decrease in the activities of choline acetyltransferase and glutamic acid decarboxylase.

B. PHARMACOTHERAPY The predominant form of therapy for parkinsonism involves the use of drugs (see reviews in Lang, 1984; Bianchine, 1985; Calne, in press). Initially, pharmacotherapy involved the belladonna alkaloids, of which atropine was the most common. These drugs usually are effective in alleviating the symptoms of rigidity and many of the autonomic dysfunctions that commonly accompany the disorder. However, they have little effect on akinesia and may elicit or exacerbate dementia. In the late 1960s, L-dihydroxyphenylalanine (L-DOPA)emerged as an alternative to atropine in the treatment of Parkinson’s disease, and this amino acid precursor of DA rapidly became the pharmacotherapy

4

MICHAEL. .I. ZIGMOND AND EDWAKD M . STKICKER

of choice. When given in conjunction with an inhibitor of peripheral decarboxylation, L-DOPA provides a considerable reduction of neurological symptoms in about a third of all patients, and it affords at least moderate relief in another third. Unfortunately, L-DOPA treatments d o not remain beneficial. There is a gradual reduction in their effectiveness beginning 3-5 years after the onsct of treatment, and most symptoms return to pretreatment levels by 8 years after the onset of treatment. C. RECENTANIMAL MODELSFOR

THE

STUDYOF PARKINSONISM

T h e initial observation that animals became akinetic when treated systemically with reserpine (Carlsson et nl., 1975) soon led to the discovery that parkinsonism is accompanied by a loss of DA in striatum. Since then, animal models have played an important role in studies of this disorder (see reviews in Schultz, 1982; Zigmond and Stricker, 1984). T w o selective neurotoxins have been of great value in this regard, 6-hydroxydopamine (6HDA) and 1-methyl-4-phenyl-1,2,5,6tetrahydropyridine (MPTP) (see reviews in Kostrzewa and Jacobowitz, 1974; Breese, 1975; Jonsson, 1980; Langston and Irwin, 1986; Kopin and Markey, 1988). 1. 6-Hydroxydopamine

The effects of GHDA (Fig. 1) were described first in studies of the autonomic nervous system, in which it was observed that the drug produced a depletion of N E that lasted several months (Porter et al., 1963) and was accompanied by the selective degeneration of noradrenergic terminals (Thoenen and Tranzer, 1968). Although GHDA administered systernically failed to cross the blood-brain barrier, the drug could be used to damage catecholamine-containing neurons in brain selectively by administering it directly into brain parenchyma or the cerebrospinal fluid (Ungerstedt, 1968; Bloom et nl., 1969; Uretsky arid Iversen, 1970). 'I'his effect was accompanied by the loss of DA, N E , epinephrine, and various biochemical arid histochernical indices of catecholaminergic neurons, including catecholamine metabolites, tetrahydrobiopterin, an uitro high-affinity catecholamine uptake, and tyrosine hydroxylase (TH) activity (Iversen and Uretsky, 19'70; Bullard et al., 1978; Levine et al., 1981; Reader and Gauthier, 1984). These and most other studies involving 6HDA have used rats as experimental subjects, although relevant observations also have been made with other species, including mice (Mandel and Randall, 1985), cats (Beleslin et al., 198 I), dogs (Van Woel-t et al., 1972), and monkeys (Maas et al., 1972; Redmond et al., 1973).

5

ANIMAL MODELS OF PARKINSONISM

OH HonCHz-CHz-NH2

H o ~ i H - c ~ 2 N-H,

HO DOPA M I N E

HO NOREPINEPHRINE

Hoa~~ N H~

HO

OH

6 - H Y D ROXY DO PAM I N E

FIG. 1, The structure of 6-hydroxydopamine (6HDA) and the endogenous catecholamines, dopamine and norepinephrine.

Subsequent studies indicated that GHDA could produce these central effects without permanently reducing the concentrations of other neurotransmitters in brain, including 5HT, acetylcholine (ACh), and y-aminobutyric acid (GABA) (Jacks et al., 1972; Kostrzewa and Jacobowitz, 1974). Histological studies further support the specificity of this neurotoxin (Bloom et al., 1969; Fibiger et al., 1972; Hedreen and Chalmers, 1972; Agid et al., 1973b; Hiikfelt and Ungerstedt, 1973; Simon et al., 1974; Lidbrink and Jonsson, 1975). Moreover, when coupled with an inhibitor of high-affinity NE transport, such as desipramine, GHDA can be used to deplete tissue of DA without affecting NE (Breese and Traylor, 1970). Like any drug, however, the specificity of 6HDA is not absolute; there have been reports of nonspecific damage after administration of large doses of the toxin (Poirier et al., 1972; Poirier, 1975; Butcher et al., 1974; Butcher, 1975), and even moderate doses may affect 5HT as well as the catecholamines (Reader and Gauthier, 1984). Consequently, the specificity of the drug must be determined in each new condition in which it is used. The selective effects of GHDA apparently result because it is a structural analog of the catecholamines and therefore is concentrated by the high-affinity transport system present in catecholarninergic neurons, especially their terminals. Because it also is highly electroactive, it oxidizes rapidly to form several cytotoxic compounds, including hydrogen peroxide, which destroy the terminal from within (Heikkila and Cohen, 1972a; Sachs and Jonsson, 1975) (Fig. 2). The successful use of 6HDA led to the development of other selective neurotoxins (see reviews in Breese, 1975; Jonsson, 1980). These include 6-hydroxydopa, which crosses the blood-brain barrier and is converted within the CNS to GHDA by aromatic amino acid

6

MICHAEI..]. ZIGMONI) AND EDWAKI) M , STRI(:KEK

6 - hydroxydopomine

A

6-hydroxydoparnine quinone

B

6-HDA

DMI

FIG. 2. (A) A iiiodel for the mechanism of action of 6-hydroxydopamine (GHDA). In neutral, aqueous solution, GHDA is oxidized to GHDA quinone, H@,, and several other cytotoxic products (B). When present in extracellular fluid in sufficiently low concentrations, this step is preceded by a selective high-affinity uptake into norepinephrine (NE) and dopamine (DA) neurons, resulting in the accumulation of these metabolites in aminergic terminals. If the resulting concentration of metabolites exceeds the buffering capacity of the cytoplasm, degeneration will occur. Increased potency can he obtained by pretreating animals with M A 0 inhibitors, such as pargyline, while increased specificity can be obtained by pretreatment with drugs that block one of the high-affinity transport systems such a s desipramine (DMI). (A) From Heikkila and Cohen, 197'La.

7

ANIMAL MODELS O F PARKINSONISM

decarboxylase (Jacobowitz and Kostrzewa, 1971 ; Sachs and Jonsson, 1972); N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), which selectively damages noradrenergic terminals (Ross and Renyi, 1976; Jaim-Etcheverry and Zieher, 1980; Jonsson et al., 1981); and 5,7dihydroxytryptamine, which can be used to destroy serotonergic neurons selectively (Baumgarten and Lachenmayer, 1972; Baumgarten et al., 1973).

2. MPTP MPTP (Fig. 3) was discovered after the sudden development of parkinsonism in young adults soon after they had unknowingly selfadministered the substance (Davis et al., 1979; Langston et al., 1983). Subsequently, it was observed that when given systemically to monkeys, MPTP produced a selective cell loss in substantia nigra and depletion of DA and other markers of dopaminergic terminals in striatum (Burns et al., 1983,1986;Langston et al., 1983).MPTP also can be used to produce hemiparkinsonism by administering the drug unilaterally into the internal carotid (Brooks et al., 1987). Thus far, primates have proven to be the animal class that is most

MPPP y

3

y

3

8 \

M PTP

MPP+

FIG. 3. The structures of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP); its active metabolite, 1-methyl-4-phenylpyridinium (MPP+); and I-methyl-4-phenyl-4propionoxyl piperidine (MPPP), the meperidine analog during whose synthesis MPTP can be formed.

8

MICHAEL]. ZICMOND A N D EDWARD M. STKICKER

sensitive to MPI'P. However, higher doses of the drug can produce NSB damage in some but not all strains of mice (Hallman et al., 198413, 1985; Heikkila et al., 1984a, 1985a), as well as in dogs (Parisi and Burns, 1985), cats (Schneider et al., 1986), and frogs (Barbeau et al., 1986). In contrast, the drug is relatively ineffective in rats (Soyce et al., 1984; Chiueh et ul., 1984; Sahgal et al., 1984) and guinea pigs (Chiueh et al., l984), although its active metabolite destroys DA neurons when injected into rat brain (Heikkila et al., 1985b). MPTP is one of a large class of piperidine compounds that are neurotoxic (Bradbury et al., 1985; Wilkening et ul., 1986; Youngster et al., 1987; Heikkila et al., 1985b). The first step in the mechanism of action of the drug appears to be its deamination by monoamine oxidase (MA0)-B in glial cells (Chiba et al., 1984; Heikkila et al., 198413; Langston et al., 1984b), resulting in the formation of l-methyl-4-phenylpyridinium (MPP') (Markey et al., 1984; Mytilineou et al., 1985; Sayre et al., 1986). MPP' then is selectively accumulated in DA-containing nerve terminals by way of the high-affinity DA transport system (Chiba et al., 1985; Javitch et al., 1985). Once present within DA nerve terminals, MPP' is thought to act either by generating hydrogen peroxide and free radicals (in a manner analogous to that of 6HDA) (Perry et al., 1985; Kopin et d,, 1986), or by interfering with mitochondria1 respiration (Nicklas et al., 1985; Poirier and Barbeau, 1985; Kamsay et al., 1986) (Fig. 4). In primates MPTP ultimately leads to DA cell death. In mice, however, cell bodies may be preserved and striatal DA content may gradually recover, at least in younger animals (Hallman et al., 1985; Melamed et al., 1985; Ricaurte et al., 1985, 1986). Moreover, extensive reinnervation of mouse striaturn by DA-containing terminal axons can be visualized several weeks after MPTP treatment (Mizukawa et al., 1988). Thus, although MPTP is able to reduce DA content in mouse brain, this can occur without the permanent loss of NSB neurons. D. SOMEUNANSWERED QUESTIONS

During the past several years, investigators have utilized animal models to examine many questions of relevance to parkinsonism. In this review we focus on five such issues. First, what is the relation between the neurological deficits seen in the disease and the complex neuropathology that ultimately is observed upon postmortem examination? Second, why do symptoms not emerge until NSB degeneration is nearly complete? Third, are the symptoms that result from degeneration of the

ANIMAL MODELS OF PARKINSOXISM

9

Ast roc y te

FIG. 4. A model for the mechanism of action of MPTP. MPTP is oxidized in astrocytes by MAO-B to l-methyl-4-phenyl-2,3-dihydropyridinium (MPDPI). This intermediate then undergoes further oxidation either within the glial cell or in extracellular fluid to form the cytotoxic conipound MPP'. MPP' is accumulated via high-affinity transport in dopaminergic neurons, where it causes degeneration, possibly as a result of its inhibition of mitochondria1 respiration. (From Heikkila et al., 1987).

NSB affected by the age at which the degeneration occurs? Fourth, what are the mechanisms by which stress affects parkinsonian symptoms? Fifth, what is the mechanism by which drugs act in the treatment of parkinsonism, and what insights d o these mechanisms provide for the design of alternative modes of therapy?

II. What Is the Relation between Neuropathology and Symptoms?

In explaining the neuropathological basis of the disorder, clinical investigators have focused almost exclusively on the degeneration of NSB. This is partly for historical reasons: The loss of cells in substantia nigra and of DA in corpus striatum were among the first biological abnormalities detected in Parkinson's disease. However, there is clinical support for the assumption that NSB degeneration underlies the prominent features of parkinsonism (Hornykiewicz, 1982). First, within the restricted range of DA depletions associated with clinical symptoms, the loss of striatal DA is well correlated with the degree of neurological deficit. Second, in severe parkinsonism, the loss of nigrostriatal cells is

10

MICHAEL J . ZIGMOND AND EDWARD M. SI'RICKER

almost complete and certainly the most extensive of the neuropathological changes that have been described. Third, both striatal DA levels and behavior can be normalized temporarily with L-DOPA. Despite the apparent significance of cell loss in NSB to parkinsonism, however, it is important to remember that postmortem examination of parkinsonian brains reveals a large number of other neurochemical and anatomical changes (see Section I,A,2). Moreover, extensive degeneration of NSB can occur without any detectable symptomatology. Finally, the clinical deficits are complex and extend well beyond the akinesia, rigidity, and tremor first described by Parkinson. For these reasons, much remains to be learned regarding the relation between the neuropathology and symptoms of parkinsonism. Because animal studies utilizing neurotoxins produce more selective lesions than those that occur as part of the disease process, such studies can provide insight into this issue. In this section we review these studies (see also reviews in Robbins and Everitt, 1982; Iversen, 1984; Simon and Le Moal, 1984; Bjorklund and Lindvall, 1986). A. THEROLEOF THE NICKOSTRIATAL BUNDLE 1 . Early Studies with Surgical Lesions Soon after the discovery that parkinsonism was accompanied by degeneration of the DA-containing projections to the caudate and putamen, investigators began to examine the behavioral effects of such lesions in laboratory animals. T h e first such studies involved electrolytic lesions of the NSB in monkeys. These lesions provided support for the link between NSB damage and parkinsonism since they produced tremor, bradykinesia, and rigidity when depletion of striatal DA was extensive (Poirier et al., 1966; Sourkes and Poirier, 1966; Stern, 1966; Goldstein et al., 1973). Moreover, those neurological deficits could be reversed by DA agonists (Larochelle et al., 1971; Goldstein el al., 1975). In retrospect, it is clear that comparable lesions had been made in rats, cats, dogs, and monkeys some years earlier (eg., Anand and Brobeck, 1951; Anand et ul., 1955; Rozkowska and Fonberg, 1970). In those studies, most of which preceded the description of the NSB, the lesion was produced at the level o f the lateral hypothalamus, and the behavioral deficits were interpreted in terms of putative hypothalamic regulatory centers. Subsequently, however, it became clear that most of the effects of such lesions were best understood in terms of damage to axons traversing lateral hypothalamus rather than to hypothalamic tissue. This insight came from three types of observations. First,

ANIMAL MODELS OF PARKINSONISM

11

comparable deficits could be achieved with lesions that were either rostra1 or caudal to the hypothalamus (Gold, 1967; Oltmans and Harvey, 1972; Stricker, 1976). Second, several rostrally directed monoaminergic projections that passed through the lateral hypothalamus were transected by lesions in this area, resulting in extensive loss of monoamines throughout the telencephalon (Heller and Moore, 1965; Ungerstedt, 1968, 197la,d). Third, specific lesions of lateral hypothalamic cells, but not fibers of passage, failed to reproduce the effects of electrolytic lesions in this region (Stricker et al., 1978).

2 . Studies with Neurotoxins More recent studies of the impact of NSB injury have used selective neurotoxins. These agents destroy dopaminergic neurons but have minimal effects on other neuronal elements that inevitably are damaged nonspecifically by surgical lesions. Extensive bilateral damage to the NSB in rats with GHDA leads to severe behavioral dysfunctions within several hours. The brain-damaged animals are akinetic, cataleptic, and show deficits in sensorimotor integration, as well as a loss of motivated behaviors such as feeding and dr.inking (Ungerstedt, 1971d; Cooper et al., 1972; Marshall and Teitelbaum, 1973; Fibiger et al., 1973; Zigmond and Stricker, 1973). GHDA also can be administered unilaterally along the NSB. Because the dopaminergic neurons of the NSB are largely uncrossed (Anden et al., 1966b; Fallon and Moore, 1978; Rice et al., 1987),such lesions lead to contralateral neurological dysfunctions and postural asymmetries (Anden et al., 1966a; Marshall, 1979). By mimicking hemiparkinsonism, this preparation permits a complete unilateral depletion of DA without causing prolonged deficits in ingestive behaviors. This eliminates the need for extensive maintenance of the animals, as is the case when animals become akinetic after bilateral lesions. The unilateral lesion preparation also can be used to great advantage in certain pharmacological experiments, as seen below. Although detailed investigations of complex behaviors have not yet been conducted in MPTP-treated animals, parkinsonian symptoms have been reported in nonhuman primates (Burns et al., 1983; Langston et al., 1984a), cats (Schneider et al., 1986), mice (Hallman et al., 1985), and frogs (Barbeau et al., 1986). The impact of MPTP in nonhuman primates is particularly striking because of the similarity of the motor deficits to those seen in parkinsonian patients. In many studies utilizing those neurotoxins, the loss of DA is rather widespread. Nonetheless, it is destruction of the dopaminergic input to striatum that appears to be responsible for the major neurological

12

MICHAELJ. ZIGMONL) AND EDWARD M. STRICKER

components of the parkinsonian syndrome. There are several reasons for this conclusion. First, injections of6HDA at any point along the NSB will produce comparable neurological deficits whenever striatal DA is depleted severely (Ungerstedt, 1971d). Second, microinjections of apomorphine into striatum cause a reversal of GHDA-induced sensorimotor dysfunction, whereas injections into other brain regions do not (Marshall el al., 1980). Third, although MPTP can act at multiple sites when given to older monkeys (Mitchell et al., 1985; Forno et al., 1986), it appears to be relatively specific to the dopaminergic neurons of the NSB when administered to younger nonhuman primates (Burns et al., 1983; Langston et ul., 1984a). Fourth, large lesions of NSB that elicit behavioral deficits also increase markedly the spontaneous firing rate of striatal cells (Ohye et al., 1970; Schultz and Ungerstedt, 1978). This increase presumably reflects the normal inhibitory influence of DA on the spontaneous activity of these neurons (McLennan and York, 1967; Connor, 1970). In contrast, somewhat smaller DA-depleting lesions that fail to produce behavioral deficits do not alter striatal firing rates (Orr et d., 1986). Finally, the nature of the major neurological impairments in parkinsonism are consistent with the apparent role of striatum in both sensory and motor function (see reviews in Krauthamer, 1975; DeLong and Georgopoulos, 1981; Schneider and Lidsky, 1987). Thus, both the absence of behavioral deficits seen after moderate lesions and the marked behavioral effects of larger lesions appear explainable in terms of the extent to which DA continues to influence striatal function. Additional studies suggest that the critical area of damage responsible for much of the neurotoxin-induced parkinsonian syndrome may be limited to the lateral portion of striatum. Behavioral deficits comparable to those produced by intraventricular or intranigral GHDA can be obtained by injecting GHDA into the lateral but not the medial region of rat striaturn (Snyder et al., 1985). Moreover, transplants of embryonic substantia nigra cells into DA-depleted rats reduce most of the behavioral impairments produced by GHDA, but only when there is new growth into ventrolateral striatum (Dunnett et al., 1981) (see also Section VlI,B,2). These findings are consistent with information concerning the relation between lateral striatum and motor function (e.g., West et al., 1987).

B. OTHER MONOAMINERCIC PROJECTIONS In addition to determining the impact of damage to the DA projections of NSB, investigators have examined the functional effects

ANIMAL MODELS OF PARKINSONISM

13

produced by lesions of other dopaminergic pathways and of central projections utilizing NE and 5HT. 1 . Extrastriatal Dopaminergic Pathways Damage to the mesolimbic DA projections to nucleus accumbens and olfactory tubercle usually is found to impair the integrated motor components of motivated behavior, as contrasted with the impairment of discrete motor responses caused by NSB injury. Thus, animals with a loss of dopaminergic input to these limbic structures are able to move but d o not engage in normal exploratory behavior or in the hoarding behavior normally elicited by stress (Iversen, 1984; Simon and Le Moal, 1984; Kelley and Stinus, 1985). Lesions of the mesocortical projection to medial prefrontal cortex are found to produce a constellation of deficits described as behavioral disinhibition. These impairments include an increase in spontaneous activity, increased responsiveness to external cues, and impaired performance on an alternation task (e.g., Wikmark et al., 1973; Brozoski et al., 1979; Simon et al., 1980). On the basis of these and other studies, investigators have suggested that some of the intellectual and emotional effects of parkinsonism may result from damage to extrastriatal dopaminergic projections. However, this remains a controversial issue because cognitive functions also have been ascribed to the striatum (Oberg and Divac, 1979; Marsden, 1984; Phillips and Carr, 1987).

2 . Nondopaminergzc Pathways Damage to nondopaminergic pathways usually accompanies parkinsonism. Although the possible contribution of such damage to the symptomatology of the disease has not been carefully explored in postmortem studies, investigations with animals indicate that the damage does not play a significant role in the major neurological impairments that are characteristic of the disease. For example, we are unaware of any reports that lesions of serotonergic, cholinergic, or noradrenergic pathways produce severe motor impairments. Damage to some of these nondopaminergic pathways may, however, provide a basis for some of the more subtle abnormalities observed in Parkinson’s disease. T h e best studied of these neurochemical systems are the noradrenergic projections from locus coeruleus. Electrophysiological and neurochemical studies indicate that activity in this system is highly responsive to stress (e.g., Bliss et al., 1968; Thierry et al., 1968; Korf et al., 1973; Aston-Jones and Bloom, 1981; Abercrombie and Jacobs, 1987). Moreover, lesions of the system reduce electrocortical

14

MICHAF.1.J.ZIGMOKU A N D EDWARD M. SI'KICKER

signs of arousal (Jones Pt al., 1973; Lidbrink, 1974; see, however, Jones ~t al., 1977), impair learning (Robbins and Everitt, 1987), decrease accuracy under stressful testing conditions (Carli et al., 1983), and reduce the rates at which nonreinforced learned behavior extinguishes (Mason and Iversen, 1979; but see Pisa and Fibiger, 1983). These and other observations have led some investigators to suggest that the locus coeruleus niay he involved in coordinating the brain's response to emergencies, especially the processes of vigilance and selective attention (see reviews in Amaral and Sinnamon, 1977; Mason, 1980; Aston-Jones, 1985; Iversen, 1984; Robbins et a/., 1985; Jacobs, 1986). If so, then damage to noradrenergic prqjections may play a role in some of the intellectual impairments observed in parkinsonism, including the bradyphrenia (Agid el al., 1984) and the impaired response to stress (Schwab and Zieper, 1965; see also Section V). Like central NE-containing neurons, serotonergic neurons can be shown to increase their activity in response to sensory stimulation (see reviews in Anisman et ul., 1981; Jacobs et al., 1984). However, lesions of this system are reported to produce quite a different syndrome, one that includes increased responsiveness to sensory stimulation (Lints and Harvey, 1969; Baumgarten and Lachenmayer, 1972), hyperactivity (Lorens Pt al,, 1976), aggressive behavior (Baumgarten and Lachenmayer, 1972; Grant et al., 1973; Breese and Cooper, 1975), facilitation of avoidance learning (Breese arid Cooper, 1975), and increased food intake (Lorens et al., 1971; Saller and Stricker, 1976). Such findings provide some support for earlier formulations in which 5 H T was viewed as having functions antagonistic to those of the catecholamines (Brodie and Shore, 1957;Jouvet, 1972; Mabry and Campbell, 1973; Kostowski et al., 1974; see, however, section IV,B). It therefore seems possible that the loss of serotonergic neurons in parkinsonism actually may help to restore balance within the central nervous system rather than contributing to the neurological deficits. In this regard, it may be noteworthy that some of the functional impairments induced by lesions of serotonergic neurons are reduced when the lesions are accompanied by damage to noradrenergic neurons (Saller and Stricker, 1978). TO NSB LESIONS c. SECONDARY RESPONSES

Although many of the neuropathological changes observed in parkinsonism may reflect primary neuronal degeneration, others can be explained as secondary responses to such damage. For example, changes in the concentration of GABA, a transmitter present in the striatonigral

ANIMAL MODELS OF PARKINSONISM

15

projection, occurs in the absence of any detectable loss of striatal cells in parkinsonian brains. Moreover, the accompanying changes in glutamic acid decarboxylase activity that occur in parkinsonism (Berheimer and Hornykiewicz, 1962) are reversed by L-DOPA therapy (Lloyd, 1980). Thus, changes in GABAergic indices presumably are secondary to the loss of dopaminergic input. Studies with selective neurotoxins have demonstrated several neurochemical effects that occur as a secondary consequence of the loss of dopaminergic input. For example, GHDA-induced destruction of NSB causes a decrease in the ACh content of striatum (Grewaal et al., 1974; Agid et al., 1975; Rommelspacher and Kuhar, 1975; MacKenzie et al., in press), and a similar effect can be produced transiently with DA receptor antagonists (Ladinsky et al., 1974; Sethy and Van Woert, 1974; Sherman et al., 1978). Moreover, ACh levels return to normal within days of GHDA treatment (see Section III,D,l). Thus, it may be assumed that the initial loss of ACh does not reflect a degenerative process but is a transient, secondary response to the loss of dopaminergic input and reflects a period of hyperactivity of the disinhibited cholinergic neurons during which ACh stores are reduced. This interpretation is supported by the observation that MPTP administered to mice produces a brief period during which striatal muscarinic receptors are reduced in concentration, as would be expected from an increase in ACh release. This effect can be reversed by L-DOPA (Ogawa et al., 1987). Other presumably secondary responses to NSB degeneration include an increase in striatal glutamic acid decarboxylase activity (Vincent et al., 1978), an increase in 5 H T metabolism (Blondaux et al., 1973), and a transient rise followed by a fall in somatostatin in striatum and other regions (Ogawa et al., 1987; see also Costa et al., 1978; Hanson et al., 1981).

111. Why Are Such Large Lesions Required before Symptoms Emerge?

A. ROLEOF RESIDUAL DA NEURONS IN MAINTAINING FUNCTION As noted previously, postmortem analysis of parkinsonian brains indicates that even minor symptoms are associated with extensive loss of DA in caudate and putamen. Similarly, the gross behavioral deficits produced by GHDA do not occur unless NSB degeneration is almost complete (Zigmond and Stricker, 1973; Stricker and Zigmond, 1974), and comparable results have been observed with MPTP (Burns et al., 1983; Forno et al., 1984; Chiueh et al., 1985; Barbeau et al., 1987). These

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MICHAEL J. ZIGMOND AND EDWARD M . STRICKER

findings presumably explain the failure of several earlier investigators to note significant behavioral dysfunctions in animals with more moderate DA depletions (e.g., Bloom et al., 1969; Breese and Traylor, 1970; Uretsky and lversen, 1970). One possible explanation for the absence of detectable symptoms after extensive NSB degeneration is that functions normally subserved by DA are transferred to another, nondopaminergic system. However, this hypothesis seems unlikely. Rats whose behavior appears to be normal despite GHDA-induced lesions of NSB show severe functional deficits when dopaminergic transmission is compromised further by acute treatment with such drugs as a-methyltyrosine and spiroperidol. In fact, the DA-depleted, brain-damaged animals are more susceptible to the disruptive effects of these' drugs than are intact animals (Schoenfeld and Zigmond, 1973; Zigmond and Stricker, 1973; Breese et al., 1973, 1974; Heffner et al., 1977; Marshall, 1979).Thus, we have proposed that the residual dopaminergic neurons are able to assume many of the functions that normally are mediated by the full complement of NSB neurons (Zigmond and Stricker, 1974; Stricker and Zigmond, 1976). Furthermore, we have suggested that this alteration does not simply reflect redundancy within the NSB but is a result of active, compensatory events that occur within striatum, including neurochemical adaptations in the residual dopaminergic afferents to this structure.

B. CHARACTERISTICS OF DOPAMINERCIC SYSTEMS For the impact of NSB injury to be offset by adaptive changes within the remaining DA neurons and denervated striatal cells, there would have to be a mechanism for detecting the injury and initiating rapid compensatory changes. Moreover, DA released from one nerve terminal would have to be capable of substituting for transmitter normally released from another terminal. In this section, we review briefly investigations indicating that dopaminergic transmission within the NSB does have such properties. We then will consider the evidence in support of lesion-induced compensations at the synaptic level. 1. Homeostasis at Dopamanergac Synapses Activity in dopaminergic NSB neurons is regulated homeostatically (Fig. 5). This occurs at both the cellular and the systems levels and involves adaptive changes in the synthesis and release of DA and in the response of the postsynaptic neuron to the transmitter. (For a more extensive review of these issues see Zigmond and Stricker, 1985; Stricker and Zigmond, 1986.)

ANIMAL MODELS OF PARKINSOKISM

\\

17

-5

FIG.5. Pathways of synaptic homeostasis. The influence of a monoaminergic neuron (shaded) on its target can be regulated both by modulation of transmitter release (1-7) and by amplification of the signal provided by that release (8). This involves a large variety of cell surface receptors, including receptors that respond to the monoamine transmitter itself as well as receptors responding to other chemical signals. The principal pathways for regulating transmitter release are (1) direct action of recurrent collaterals onto the soma; (2) indirect action of recurrent collaterals mediated via influence on presynaptic afferents; (3) direct action of the transmitter (T) on presynaptic terminal; (4)alterations in rate of transmitter reuptake; (5) humoral signals generated by the target; ( 6 ) neural signals providing short-loop negative feedback from the target; and (7) neural signals providing long-loop negative feedback from the target. In addition, (8)the extent to which the signal is amplified can be modulated by short-term modification of the sensitivity ofthe target, by long-term changes in number of receptors, and by other means. (From Stricker and Zigmond, 1986.)

DA release is regulated in part through homeostatic control over impulse flow. This is achieved both by short feedback loops that permit these neurons to regulate themselves (Groves et al., 1975; Wilson et al., 1977) and by longer, multisynaptic feedback loops involving target cells (Bunney et al., 1973). Release also is regulated at the presynaptic terminal, where DA can stimulate autoreceptors and influence subsequent release of transmitter (Starke et al., 1978). In addition, other humoral signals can act on the terminal to modulate DA release (Taube et al., 1977; Hedqvist, 1981; Lehmann and Langer, 1982). Despite these feedback loops, transmitter release does change under certain conditions. However, by adjusting the sensitivity of the postsynaptic cell to its afferent input, the ultimate objective of synaptic homeostasis still can be maintained. These alterations in sensitivity may result in part from rapid, transient changes in target cell responsiveness

18

MICHAELJ. ZICMOND A N D EDWARD M . STRICKER

such as have been described in the peripheral noradrenergic system (Mukherjee et al., 1974; Kebabian et al., 1975). In addition, more gradual and long-lasting changes occur in the number of transmitter receptors (Burt el al., 1977; Muller and Seeman, 1977). In addition to elaborate mechanisms that exist to maintain the constancy of transmitter release from monoaminergic neurons, complex schemes also have evolved that maintain adequate stores of monoamines should their rate of usage change. This appears to be accomplished primarily through the coupling of DA synthesis to release, which allows changes in synthesis to occur within seconds of a rise in impulse flow. The rapid replacement of released transmitter with newly synthesized material permits monoamine levels to remain constant despite high levels of release and catabolism. Because dopaminergic nerve terminals are situated some distance from their mesencephalic cell bodies (the principal site of protein synthesis), rapid changes in DA synthesis must involve enzymes that already exist in the terminals. Indeed, short-term modulation of DA synthesis seems to occur as a result of posttranslational, covalent modification of tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in DA biosynthesis (Zivkovic and Guidotti, 1974; Murrin et al., 1976).

2. Nonspecificity at Dopaminergic Synapses The dopaminergic NSB has other properties that differentiate it from classical neurons. Among these are its anatomical characteristics (see reviews in Bjorklund and Lindvall, 1986; Moore and Bloom, 1978). For example, like other monoaminergic systems, the NSB is composed of a small number of neurons whose cell bodies are located in a cluster within the brain stem and whose axons are thin, unmyelinated, and highly branched. There is a general topographic relation between the location of the cell bodies and their terminations (Fallon and Moore, 1978). However, each neuron gives rise to a lengthy, highly branched terminal axon, which in the rat contains about 250,000 synaptic varicosities en passage over a total axonal length of approximately 30 cm (Anden et al., 1966c; Moore and Bloom, 1978; Bjorklund and Lindvall, 1986). This geometry must inevitably lead to a relatively large field of influence for a given DA neuron. This field may be even larger than that indicated by such considerations, given observations suggesting that conventional synapses may occupy only a small amount of the terminal membrane of DA neuron (Descarries et al., 1980; Pickel et al., 1981; Pickel, 1986) and that interactions can occur between NSB neurons and other striatal elements in the absence of apparent synaptic contacts (e.g., Lehmann and Langer, 1982). Another factor is the reciprocal striatoni-

ANIMAL MODELS OF PARKINSONISM

19

gral projection. This pathway, which provides an important component of the homeostatic system regulating NSB activity, appears less precise in its organization than is the NSB itself (T. W. Berger, personal communication). If so, then the area within striatum that provides input to the substantia nigra is larger than the area that receives nigral afferents. T h e electrophysiological characteristics of the NSB also warrant consideration. These neurons fire at a low and stable rate (approximately 4-5 Hz) (Steinfels et al., 1983; Grace and Bunney, 1984), and they conduct these impulses slowly (approximately 1 m/sec) (Guyenet and Aghajanian, 1978). Moreover, although DA can inhibit the firing rate of striatal neurons (McLennan and York, 1967; Connor, 1970), it also can act to influence other afferent input (Bergstrom and Walters, 1984; Rolls et al., 1984; Schneider et al., 1984; Abercrombie and Jacobs, 1985; Chiodo and Berger, 1986). Finally, these postsynaptic changes probably are mediated through second messenger systems (Kebabian et al., 1972), which presumably contributes to their long postsynaptic delay and duration of action (Connor, 1970).

3. Functional Implications T h e tendency of monoaminergic neurons to maintain a relatively constant level of activity is in marked contrast to the conventional model of the nervous system as a highly responsive communications network. Indeed, monoaminergic systems appear to operate on an entirely different principle. Rather than reflecting alterations in input with high fidelity, monoaminergic systems such as the NSB are designed to resist changes. This characteristic is consistent with the relatively diffuse anatomical connections made by these systems and with the electrophysiological evidence that they can facilitate transmission along the circuits that they influence. Thus, it would appear that dopaminergic neurons are designed to modulate synaptic transmission that is initiated by other pathways (Fig. 6). These properties of DA systems probably account for the behavioral tolerance that develops to repeated administration of DA antagonists (Moore, 1968; Pirch and Rech, 1968; Hynes et al., 1978). In addition, the same characteristics may permit systems such as the NSB to operate somewhat independently of the number of synapses that are available. Elsewhere, we have argued that this latter feature would allow the NSB to function before innervation was complete and thus might underlie the rapid maturation of motor function in developing animals (Zigmond and Stricker, 1985; see also Coyle and Campochiaro, 1976; and Wallace and Zigmond, 1989). Moreover, we have suggested that the failure of

‘LO

MICHAEL J. ZIGMOND AND EDWARD M. STRICKER

A

FIG. 6. Modulation of synaptic transmission by DA. Transmission at a conventional synapse, characteristic of primary sensory or motor pathways, is shown. (A) In the absence of neuromodulation, the primary transmitter elicits a relatively small postsynaptic response and thus a small impact on the firing rate of the next neuron. (B) However, in the presence of a neuromodulator such as DA, the impact of the transmitter is amplified. (At some sites DA may exert the opposite influence, diminishing transmission.)

homeostasis at dopaminergic synapses may be related to many of the functional deficits that emerge during aging (Zigmond and Stricker, 1985; see Section III,D,6). In the next section, we explore the evidence that these same characteristics also are responsible for the absence of gross neurological deficits after subtotal degeneration of NSB. C.

RAPID

COMPENSATIONS AFTER SUBTOTAL INJURY

1. A Model for Rapid Compensations

We believe that several events occur immediately after partial destruction of NSB that serve to reduce the functional consequences of the injury. According to our formulation, some of the events reflect the same rapid compensatory responses that occur during acute treatment with DA antagonists: increases in the firing rate of residual DA neurons,

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21

increases in DA release, and increases in DA synthesis. Others reflect changes that are specific to the loss of terminals. In this section, we present a model for rapid compensation (Fig. 7), and in the sections that follow, describe the supporting evidence. It is likely that a certain amount of redundancy exists within the NSB and that some damage can be tolerated without the need for any adaptive response. I n addition, the degeneration of some DA terminals will decrease the rate at which DA released by intact terminals is inactivated by neighboring terminals and thereby lead to an extracellular accumulation of DA without provoking an active, compensatory process. With somewhat larger lesions, however, there is an increase in the number of DA neurons that are firing at any given time. This occurs in conjunction with an increase in the amount of DA released per pulse from a given terminal, an increase in DA synthesis, and a decrease in the local inactivation of DA due to saturation of the high affinity DA transport system. T h e net result of these events will be an increased concentration of DA in the synaptic cleft and a concomitant increase in the overflow of DA into the extracellular space. Moreover, once within extracellular fluid, DA should diffuse greater distances from the local synaptic space because of the terminal degeneration and resulting loss of high affinity DA uptake sites. This would serve to reestablish dopaminergic control over denervated striatal targets. 2. Increased NSB Activity T h e destruction of DA terminals in striatum by intraventricular 6HDA is associated with a loss of cells in substantia nigra as quantified histochemically and electrophysiologically (Onn et al., 1986; Hollerman et al., 1986). Moreover, those cells that remain show little if any compensatory increase in their firing rates except when striatal DA loss is extreme (>95%) (Hollerman and Grace, 1988). These phenomena may be contrasted with the effects of 6HDA-induced damage to central noradrenergic projections from locus coeruleus. In that system, in which cell loss does not occur as long as some minimal number of terminals remain, there is a three-fold increase in the average firing rate of' spontaneously active units (Chiodo et al., 1983). Despite the absence of much compensation as assessed electrophysiologically, there is a clear rise in DA turnover in the remaining terminals. This can be measured by examining the rate of formation or disappearance of radiolabeled DA in striatum (Uretsky et al., 1971 ; Agid et al., 1973a), the ratio of DA metabolites to DA content (Hefti et al., 1980; Zigmond et al., 1984; Altar et al., 1987), or the extracellular concentration of DA and its metabolites (Robinson and Whishaw, 1987; Zhang et

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MICHAEL J. ZIGMOND A N D EDWARD M. STRICKER

A. NORMAL

c. LARGER LESION

9. MODERATE LESION

D. EXTREME

LESION

FIG. 7. A model for compensatory changes after NSB lesions. (A) Under normal conditions synaptic transmission may occur with relatively little interaction with neighboring synapses. In addition, some synapses may he inactive. (B) Moderate lesions may have little or no functional impact for one of several reasons. First, some transmitter released at one synapse may act at a denervated site (shown). In addition, there may have been some redundancy in the intact system, or previously silent synapses may become active (not shown). (C) A larger lesion may require an increase in the synthesis and release of DA to keep postsynaptic function under dopaminergic control. This may result in an increase in the field of influence of the residual DA neurons (shown) or an increase in the postsynaptic response of innervated sites (not shown). (D) After still larger lesions, rapid compensatory processes may he inadequate to restore function immediately. As a result (1) there will be an initial period of failure. However, (2) a delayed Compensation and recovery of function may occur. A gradual increase in the availability of tyrosine hydroxylase may increase the capacity for DA synthesis and release still further, thereby enlarging the field of influence of the few neurons that remain. Moreover, an increase in the number of receptors at distant targets may permit inhibitory control to be restored with a relatively low concentration of DA.

ANIMAL MODELS OF PARKINSONISM

23

al., 1988; Bonatz et al., 1989; see also Abercrombie and Zigmond, in press). Comparable findings have been made in monkeys and mice given MPTP (Burns et al., 1986; Duvoisin et al., 1986), in monkeys with surgical lesions of NSB (Sharman et al., 1967), and in postmortem analyses of parkinsonian brains (Bernheimer and Hornykiewicz, 1965; Bernheimer et al., 1973). T h e increase in DA turnover is accompanied by an increase in DA synthesis, which can be measured either as an increase in the formation of DA from tyrosine or an increase in L-DOPA accumulation after inhibition of aromatic amino acid decarboxylase (Altar et al., 1987; Hefti et al., 1980). An increase in tyrosine hydroxylase activity also may be detectable (Zigmond et al., 1984; Onn et al., 1986; Lloyd et al., 1975a; see also Acheson and Zigmond, 1981), although this does not always appear to be the case (Hefti et al., 1980; R. E. Heikkila, personal communication). T h e observation of an increase in the synthesis and release of DA in the absence of an increase in firing rate raises the possibility of an important modulatory influence on DA release that is exerted at the terminal level. Recently, evidence for such a phenomenon has been accumulating. In fact, there now is reason to believe that transmitter release can be initiuted at the terminal level through a process that does not require mediation by action potentials (Abercrombie et al., 1989a; Cheramy et al., 1986; Lonart and Zigmond, 1989). These findings may cause us to reevaluate our understanding of the relation between electrophysiological and biochemical indices of neuronal activity. 3. Increased DA Overflow p e r Pulse 6HDA reduces the net release of DA from striatal slices, both under basal conditions and in response to electrical field stimulation. However, DA efflux is reduced much less than is DA content. Consequently, fractional DA efflux, a measure of efflux from residual terminals, is increased considerably (Stachowiak et al., 1987; Snyder et al., 1986). Fractional efflux also can be increased by the addition of nomifensine, an inhibitor of DA uptake, suggesting that the increased fractional efflux occurring after NSB lesions is due in part to a decrease in the rate at which DA is removed from extracellular fluid (Stachowiak et al., 1987; Snyder at al., 1986). This is consistent with the observation that the distance over which DA can diffuse is influenced by the density of DA terminals in the region (Kelly and Wightman, 1987) and implies that the field of influence of residual DA neurons is increased by NSB injury. An analogous phenomenon has been shown directly in studies of the impact

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MICHAEL J. ZIGMOND AND EDWARD M . STRIGKER

of partial denervation on the sympathetically mediated rhythm of N-acetyltransferase in pineal (Zigmond et al., 1981, 1985). However, the presence of nomifensine does not abolish the difference in fractional DA efflux between intact and lesioned slices (Snyder and Zigmond, 1987). Thus, there appears to be an increase in the amount of DA released by the nerve in response to depolarization. T h e mechanism of this latter effect is not yet known. However, we have observed that DA overflow is enhanced by the DA receptor antagonist sulpiride in control tissue but not in slices prepared from 6HDAlesioned rats, suggesting that the lesions might have increased DA efflux per terminal by reducing DA autoinhibition (Snyder et ul., 1986).

OF FUNCTION AFTER LARGE NSB LESIONS D. RECOVERY

A degree of recovery of function after brain damage is the rule, not the exception, and the biological basis of this phenomenon has fascinated neuroscientists at least since its description by Flourens in 1824 (see reviews in Rosner, 1974; Laurence and Stein, 1978; Marshall, 1984). Recovery from NSB injury first was described 35 years ago. In those studies, it was observed that most rats given electrolytic lesions of the lateral hypothalamus gradually reattained the ability to eat and drink when they were maintained for a lengthy period of time by intragastric intubation of nutrients (‘Teitelbaum and Stellar, 1954; see also Section II,A,l). Since then, the pattern of recovery after NSB lesions has been described in great detail, both after surgical lesions (Teitelbaum and Epstein, 1962; Marshall and Teitelbaum, 1974) and after treatment with 6HDA (Ungerstedt, 1971d; Zigmond and Stricker, 1973; Marshall et al., 1974; Ljungberg and Ungerstedt, 1976a). Recovery from MPTP treatment also has been reported (Langston, 1985; Eidelberg et al., 1986; Kopin and Markey, 1988). As noted previously, animals with large DA-depleting brain lesions are initially akinetic, do not eat or drink, and fail to respond to diverse sensory stimuli. Gradually, however, the animals begin to show improved motor performance, consume highly palatable foods, and orient to sensory stimuli. Full recovery can occur within a few weeks, although several months may be required. 1. A n Expanded Model: More Gradual Compensutions We believe that recovery of function after large lesions of NSB, like the absence of behavioral deficits after more moderate lesions, reflects compensatory changes within those elements of the system that are

ANIMAL MODELS OF PARKINSONISM

25

spared (see Fig. 7). We have proposed that two changes in particular form the basis of this gradual recovery: an induction of tyrosine hydroxylase synthesis and an increase in the responsiveness of striatal neurons to DA. T h e increase in tyrosine hydroxylase levels would be expected to elevate the maximal rate at which DA release could occur without depleting transmitter stores. Moreover, it might obviate the need for maintaining the enzyme in a tonically activated state, thereby preserving the capacity for tyrosine hydroxylase activation as a phasic response to emergencies (see Section V,B). T h e increase in DA receptors would enhance the impact of extracellular DA, thus extending the anatomical range over which the residual, hyperactive DA terminals could influence striatal targets. T h e evidence in support of this extension of our recovery model, together with some of the its functional implications, is described in the following sections. 2. A Role for Residual DA Recovery of function without regeneration of the injured NSB neurons originally was interpreted to indicate that this projection had little o r no role in controlling behavior. However, there are several reasons for believing that this is not the case and that recovery of behavioral function requires the reestablishment of dopaminergic control over striatal cell function. First, large depletions of DA that produce behavioral deficits are associated with a marked increase in the spontaneous firing rates of Type I1 striatal cells (Orr et al., 1987). Yet, 4-6 weeks after such lesions, these cells show normal activity in animals that recover their behavioral function, whereas the cells continue to show high spontaneous firing rates in animals that d o not recover behaviorally (Nisenbaum et al., 1986; see also Schultz and Ungerstedt, 1978). Moreover, when rats that had recovered from the behavioral and electrophysiological effects of GHDA are given either a second intraventricular injection of GHDA or a systemic injection of haloperidol, striatal firing rates again are elevated and behavioral deficits are reinstated (Breese et al., 1973, 1974; Zigmond and Stricker, 1973; Nisenbaum et al., 1986). A second way in which the interactions between dopaminergic afferents to striatum and their targets have been monitored is by examining the inhibitory influence of DA on ACh release from cholinergic interneurons. DA has been shown to decrease ACh release both in vivo (Stadler et al., 1973; Guyenet et al., 1975) and in vitro (Vizi et al., 1977; Starke et al., 1978). Immediately after NSB lesions, there is a fall in striatal ACh content and an increase in ACh release (see Section 11,C).

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MICHAELJ. ZIGMOND A N D EDWARD M. STRICKER

However, such effects may not be evident several days o r weeks later (Kim, 1973; Grewaal et al., 1974; Agid et al., 1975). Moreover, striatal ACh levels still can be reduced and ACh release increased by the acute exposure of GHDA-lesioned rats to DA antagonists (Guyenet et al., 1975; MacKenzie et al., in press). Collectively, these behavioral, electrophysiological, and biochemical observations suggest a continuing role for DA in the maintenance of striatal function even when the number of NSB afferents has been reduced greatly. 3. Evidence ,for Gradual Compensatory Changes There is evidence in support of gradual compensatory responses to NSB injury. First, in addition to the short-term activation of existing tyrosine hydroxylase molecules, an induction of tyrosine hydroxylase also can occur, leading to a gradual increase in the number of available tyrosine hydroxylase molecules. This process has been studied most completely in the peripheral nervous system after damage to sympathetic noradrenergic neurons (Fluharty et al., 1987; Stachowiak et al., 1986a),but comparable events have been observed in the NSB (Zigmond et al., 1984). Presumably, this increase in tyrosine hydroxylase would further elevate the capacity of the residual DA neurons to synthesize and release their transmitter. Indeed, such time-dependent increases in DA turnover have been reported after NSB damage (Chiueh, 1988; see also Acheson et al., 1980). A second change is an increase in the responsiveness of striatal targets to DA. Areas deprived of their normal complement of dopaminergic afferents ultimately become more sensitive to DA. The initial phase of this response appears to result from the loss of DA terminals and a consequent decrease in the availability of high-affinity transport sites that inactivate DA (Ungerstedt, 197lc; Schoenfeld and Uretsky, 1972, 1973; Zigmond and Stricker, 1980; see also Section 111,C). However, when the lesion is sufficiently large, supersensitivity to DA continues to develop long after degeneration is complete. Moreover, animals also gradually become supersensitive to DA agonists, such as apomorphine, that are not inactivated by high-affinity uptake (Ungerstedt, 197lc; Schoenfeld and Uretsky, 1973; Zigmond and Stricker, 1980). This latter phenomenon is accompanied by increases in the electrophysiological and neurochemical responsiveness of striatal cells to DA agonists (Feltz and DeChamplain, 1972; Fibiger and Grewaal, 1974; Ungerstedt et al., 1975), the number but not the affinity of postsynaptic DA receptors of the D2 subtype (Creese et al., 1977; Creese and Snyder, 1979; Nagy et al., 1978; Neve et al., 1982; MacKenzie and Zigmond, 1984), and the sensitivity of adenylate cyclase to DA (Mishra et al., 1974;

ANIMAL MODELS OF PARKINSONISM

27

Zigmond and Stricker, 1980). Increases in the number of DA receptors and in DA-sensitive cyclase also have been reported in parkinsonian brains (Lee et al., 1978; Nagatsu et al., 1978; Rinne, 1982; Guttman and Seeman, 1985, 1987; Raisman et al., 1985), although other investigators have failed to observe such effects (Reisine et al., 1977; Riederer et al., 1978; Pimoule et al., 1985). 4. Functional Implications of Gradual Compensation

These compensatory changes after NSB lesions have several further implications. First, unlike the compensations described in Section III,C, these processes require days or longer to develop, as they involve bc th protein synthesis and the transport of new materials to their sites of action. Thus, to the extent that more rapid adaptations are successful in restoring synaptic transmission, the stimulus for tyrosine hydroxylase induction and DA receptor proliferation may not occur. Consistent with this prediction are the observations that increased DA turnover can occur in the absence of an increase in tyrosine hydroxylase after moderate NSB lesions and that increases in postsynaptic supersensitivity do not occur until at least 90% of the NSB has been destroyed. The model now predicts, however, that while these long-term processes are developing in response to very large lesions, there will be a period of functional impairment during which rapid compensations are inadequate to restore NSB control over striatal targets. This latter period of gradual neurochemical change may underlie the prolonged neurological deficits that occur after large lesions. Finally, as with the short-term model described earlier, this conceptualization of the adaptive response to NSB injury depends on the presence of some minimal number of DA neurons. Thus, one would predict that no recovery would occur after total destruction of this pathway. This, in fact, has been our experience when 6HDA-induced lesions are made in adult animals. 5. Generality of the Model The characteristics of NSB described above (Section II1,B) hold for virtually all neuronal systems that utilize biogenic amines as their transmitters. Thus, it is not surprising that many of the rapid and more gradual compensatory changes that occur in response to partial NSB injury also occur in other neuronal systems (see reviews in Zigmond and Stricker, 1985; Stricker and Zigmond, 1986). Increases in NE turnover have been reported after injury to the locus coeruleus induced by 6HDA (Jonsson et al., 1979; Acheson et al., 1980; Acheson and Zigmond, 1981) and by DSP4 (Logue et al., 1983;

28

MICIIAEL J. ZIGMOND AND EDWARD M . STRICKER

Hallman and Jonsson, 1984; Hallman et al., 1984a). 6HDA also has been reported to stimulate epinephrine turnover in brainstem nuclei (Burgess et al., 1980), and GHDA-induced damage to the sympathetic postganglionic system results in increases in tyrosine hydroxylase activity in the remaining noradrenergic terminals as well as the adrenal chromaffin cells (Mueller et al., 1969; Brimijoin and Molinoff, 1971; Fluharty Pt al., 1985a,b; Stachowiak et al., 1986a). Moreover, such lesions have been shown to cause a number of changes that should serve to increase responsiveness to catecholamine, including loss of NE uptake sites, increased noradrenergic binding sites, and increased NE-sensitive adenylate cyclase (Jonsson and Sachs, 1972; Chiu, 1978; Yamada et al., 1980; Fluharty et al., 1987). Although other systems have been examined less thoroughly, there is evidence for compensatory changes after partial damage to noncatecholaminergic systems as well. For example, such changes may occur in the serotonergic projections of the raphe nuclei. Neurotoxin-induced damage to these neurons increases 5HT turnover and/or tryptophan hydroxylase activity in residual 5 H T terminals in spinal cord (Gerson et al., 1974) and brain (Victor et at., 1974; Baumgarten et al., 1977; Bjiirklund and Wiklund, 1980; Harvey and Gal, 1974; Stachowkdk et al., 1986b; see, however, Hyyppa et al., 1973; Lytle et al., 1974). Evidence also exists for compensations in cholinergic neurons. Blockade of striatal DA receptors can increase high-affinity choline uptake, and thus ACh synthesis, in that structure (Atweh et al., 1975; Kuczenski et al., 1977; Pedata et al., 1980; see, however, Sherman et al., 1978). Moreover, after the initial decrease in cortical high-affinity choline uptake that occurs in response to lesions of the nucleus basalis magnocellularis, uptake gradually increases to control levels (Pedata et al., 1982). Thus, it seems likely that many of the conclusions drawn in this review about NSB injury also are applicable to situations in which other transmitter systems have been damaged. Later, we will discuss one such example, the partial destruction of the sympathoadrenal system, (see Section V,C) in some detail. 6 . Why Do Parkinsonian Patients Not Recover? Although animals that sustain large NSB lesions often recover from their initial neurological deficits, Parkinsonian patients usually do not improve but instead show a gradual worsening of symptoms. ‘I’here are several possible explanations for this lack of‘parallelism. First, unlike the neurotoxin-induced lesions, which usually are produced abruptly, Parkinson’s disease appears to result from a gradual and progressive

ANIMAL MODELS OF PARKINSONISM

29

degenerative process. Consequently, once symptoms emerge, degeneration may proceed at a faster rate than that of the compensatory events. This hypothesis would predict that patients whose symptoms were the result of acute NSB damage also would show some recovery of function when the damage was not too great. In fact, spontaneous clinical improvement has been observed after the initial effects of MPTP (Langston, 1985). Parkinsonian patients also may fail to improve because the disease represents the effects of a subthreshold loss of DA superimposed on the natural deterioration of NSB function that occurs with aging. Parkinsonian-like movement disorders do occur in senescent humans and rats, and these have been attributed in part to age-related changes in basal ganglia (see reviews in Critchley, 1956; Finch et al., 1981). In support of this hypothesis are the observed therapeutic effects in aged rats of DA agonists (Marshall and Berrios, 1979) or nigral transplants (Gage et al., 1983; see also Section VII,B,2), although DA agonists have been reported to be ineffective in aged patients (Newman et al., 1985). Deterioration of NSB function during senescence may be associated with the age-related loss of DA neurons (McGeer et al., 1971) or receptors (Govoni et al., 1978; Severson rnd Finch, 1980). Alternatively, it may result from a failure of the homeostatic mechanisms that previously had served to compensate for the disorder (Zigmond and Stricker, 1985). In either case, the hypothesis implies that it may not be a progressive increase in the pathology that is responsible for the emergence of permanent neurological deficits, but a progressive decrease in the capacity for compensation owing to the aging process. However, this hypothesis suggests that animals sustaining neurotoxin-induced lesions at a later age would show less recovery than younger animals, a prediction not supported by available data (Marshall et al., 1983). A third explanation for the failure of parkinsonian patients to recover spontaneously is that the cellular deficits are more general than in the animal models using selective neurotoxins and that some aspects of the clinical pathology ultimately interfere with the compensatory processes. For example, the neuropathology of parkinsonism may involve a loss in the capacity for DA receptor upregulation, an hypothesis consistent with several reports (Reisine et al., 1977; Quik et al., 1979; Riederer et al., 1978). Moreover, a decline in the activity of choline acetyltransferase has been observed in the striatum of patients with advanced Parkinson's disease (Lloyd et al., 1975b; Reisine et al., 1977), which could reflect the loss of cholinergic neurons as a result of transsynaptic atrophy of DA-sensitive targets.

30

MICHAEL]. ZIGMONU A N D EDWARD M. STRICKER

IV. Does Age of Injury Influence the Deficits?

A. ATTENTION DEFICIT DISORDER AS NSB INJURY

A

CONSEQUENCE OF NEONATAL

During the encephalitis epidemic of 1917-1928, the virus that often led to parkinsonism in adults produced a very different syndrome in children, a “postencephalitic behavior disorder” (Hohman, 1922) that bears a close resemblance to the disorder later termed minimal bruin dysfunction and, more recently, attention deficit disorder, Attention deficit disorder first emerges in childhood and consists of behavioral, intellectual, and physical abnormalities. Such patients frequently are hyperactive and impulsive, especially under stressful conditions. They have a shortened attention span; an insensitivity to environmental cues; a difficulty in handling co’mplex tasks, especially those that require multiple sensory modalities; and learning disabilities (for reviews see Wender, 1971, 1975; Cantwell, 1975; Weiss and Hechtman, 1979; Hunt et al., 1982). At least 50% of such children have soft neurological signs including motor incoordination, mild choreiform movements, and impaired performance on tasks requiring sensorimotor integration. Although many of these patients show behavioral signs of hyperactivity, the syndrome often can be treated successfully with drugs such as amphetamine and methylphenidate (Bradley, 1937; Millichap and Boldrey, 1967; Rapoport, 1983). These observations are consistent with evidence of excessive slow wave electroencephalographic activity, reduced reaction time, and decreased levels of homovanillic acid in cerebrospinal fluid (Shaywitz et al., 1977; Hunt et al., 1982). Such findings, together with the original epidemiological evidence of a link to parkinsonism, have led to the suggestion that attention deficit disorder may represent a manifestation of dopaminergic hypofunction in children (Wender, 197 1, 1975; Snyder, 1973; Shaywitz et al., 1977; Zigmond and Stricker, 1977). In this regard, it is noteworthy that recent data suggest the presence of attention deficits in adult parkinsonian patients (Girotti et al., 1987).

B. EFFECTS OF EARLY DAMAGE TO DOPAMINERCIC NEURONSI N EXPERIMENTAL ANIMALS

1 . Behavioral Efects The effects of NSB lesions made in neonatal rats are in striking contrast to those made in adults. Although electrolytic lesions of lateral hypothalamus or substantia nigra can produce a broad range of neuro-

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31

logical deficits in rat pups (Almli and Golden, 1974; Lytle and Campbell, 1975; Almli and Fisher, 1977), none of these marked behavioral dysfunctions are observed when the lesion is limited to the DA projections (Breese and Traylor, 1972; Lytle et al., 1972; Shaywitz et al., 1976; Bruno et al., 1984). For example, rat pups given intraventricular GHDA at 3 days of age continue to suckle and grow; they have little difficulty in weaning 2-3 weeks later despite permanent striatal DA depletions of 99% (Bruno et al., 1984). We find a similar lack of neurological deficits in animals given near-total DA-depleting brain lesions at 10 or 15 days of age. Indeed, it is only in rats that are at least 35 days old at the time of GHDA treatment that severe behavioral dysfunctions are observed (Bruno et al., 1987). Although younger animals sustaining NSB lesions show no parkinsonian symptoms, their behavior is not completely normal. Instead, the animals are much more active than intact animals of a comparable age (Pappas et al., 1975; Shaywitz et al., 1976; Sorenson et al., 1977; Stoof et al., 1978; Erinoff et al., 1979) and are deficient in certain learning tasks (Thieme et al., 1980). Such observations strengthen the proposed link between attention deficit disorder and parkinsonism. In further support of this hypothesis, some investigators have observed that both the motoric and cognitive deficits produced by neonatal GHDA can be reversed with DA agonists (Shaywitz et al., 1976, 1978; Sorenson et al., 1977), although others have failed to see such effects (Pappas et al., 1980; Thieme et al., 1980).

2. Neurobiological Effects Brain injury sustained in developing animals frequently produces different behavioral deficits than does comparable injury in adulthood. Often, early lesions cause much less functional disruption; in other cases the nature of the disruption is qualitatively different (Kennard, 1936; Teuber, 1971; Goldman, 1974; Schneider, 1979; Goldberger and Murray, 1985). Among the several different hypotheses that have been proposed to account for such findings are the following: 1. T h e developing brain may be more capable of growth and the formation of new anatomical connections (see Section VII,B,P). Such connections might serve either to compensate for the injury o r to cause disruptive responses. 2. T h e individual developing neuron may be more or less sensitive to injury. This may be particularly true in the case of monoaminergic neurons, which gradually develop an extensive collateralization that may protect the soma from degeneration in response to the loss of a portion of the terminal field.

32

MI(:HAEL J . ZICMOND A N D EDWARD M . SI'RICKER

3. A given projection within the developing nervous system may contain excess neurons that normally die but are sustained in response to injury to that o r a related projection. 4. The injured brain may make use of existing pathways to perform the same task in a different way. Although the basis for the age-dependent behavioral effects of NSB lesions is unknown, there are several differences between the neurobiological consequences of such lesions given to neonates and those observed after adult lesions. T h e most striking difference is that rats treated with GHDA 3 days postpartum show a four-fold increase in the 5 H T content of the rostral striatum, whereas rats given the lesions as adults show no such change (Stachowiak et al., 1984). T h e increase in 5 H T levels in the neonatal rats is paralleled by an equivalent rise in 5 H T uptake into striatal synaptosomes (Stachowiak et al., 1984),an increase in the density of 5HT-positive terminals and an increase in the labeling of 5HT-positive raphe cells after the injection of a retrograde tracer into rostral striatum (Berger et al., 1985; Luthman et al., 1987; Snyder et al., 1986). It is noteworthy that neonatal treatment with 6HDA also has been shown to increase the serotonergic innervation of neocortex (Blue and Molliver, 1987). (In that study, GHDA was given systematically and without pretreatment, resulting in the loss of N E as well as DA.) Thus, the presence of catecholamines normally may either exert a general inhibitory influence on the growth of 5HT neurons or promote an early pruning of serotonergic terminals. Pups given intracisternal GHDA at 5 days of age also show signs of abnormalities in tachykinin synthesis as adults (Sivam et al., 1987). The relative abundance of preprotachykinin mRNA is decreased markedly in the striatum of these animals, as are the concentrations of substance P and neurokinin A. Substance P also is reduced in substantia nigra. In contrast, little or no change in these substances is observed when striatal DA depletions of comparable size are produced in adult rats. Neonatally lesioned rats also exhibit different behavioral responses to L-DOPA than do rats given GHDA-induced lesions as adults. When given L-DOPA, intact rats show an increase in locomotor activity, and, when the doses are large, such stereotypic behaviors as grooming, sniffing, and gnawing. Animals given DA-depleting brain lesions as adults show a similar behavioral syndrome in response to L-DOPA, although lower doses of the drug are required to produce the same effects (Schoenfeld and Uretsky, 1973; Zigmond and Stricker, 1980). In contrast, animals given the brain lesions as neonates exhibit a preponderance of self-mutilating behaviors in response to L-DOPA (Breese

ANIMAL MODELS OF PARKINSONISM

33

et al., 1984, 1987a). The effect has been attributed to an increase in responsiveness to D- 1 agonists, although no change in D- 1 binding sites has been observed (Breese et al., 198713).

3. Relation between Behavioral and Biologacal Responses

Several possible explanatior .s for the behavioral sparing that occurs in immature animals given extensive NSB lesions can be discarded based on recent studies. For example, unlike rats given brain lesions in adulthood, behavioral function in the young animals does not appear to depend on residual dopaminergic neurons; indeed, they show little or no sensitivity to the akinesia-inducing effects of DA receptor-blocking agents (Bruno et al., 1985). Moreover, unlike rats lesioned as adults, animals sustaining NSB lesions as neonates show no residual dopaminergic inhibition of ACh release (Jackson et al., 1988b), and striatal cells have a persistently elevated firing rate (Onn et al., 1987). Endogenous 5HT, like DA, exerts an inhibitory influence on the release of striatal ACh (Euvrard et al., 1977; Vizi et al., 1981; Jackson et al., 1988a). Thus, one possible explanation for the unusual ability of neonatal rats to be spared from severe dysfunctions after NSB lesions is that the elevated 5 H T takes over the functions normally mediated by DA. However, this does not appear to be the case. Although direct and indirect serotonergic agonists decrease ACh release in striatal slices prepared from animals with NSB lesions sustained as adults, no such response is observed in animals given the lesions as neonates (Jackson et al., 1988b). Moreover, the gross behavioral deficits produced in animals given 6HDA as adults are not reproduced even when animals given 6HDA as neonates are additionally treated with 5,7-dihydroxytryptamine to destroy 5HT-containing neurons (Bruno et al., 1987). Thus, additional experimentation will be required to elucidate the mechanisms that underlie the capacity of young animals to withstand NSB injury. V. How Does Stress Influence Symptoms?

In laboratory animals, the degree of neurological dysfunction that accompanies adult NSB lesions is determined largely by three variables: lesion size, the time that has elapsed since the lesion, and the degree of emotional, environmental, and physiological stress that is being experienced. Stress can induce either of two seemingly opposite phenomena in brain-damaged animals, both of which have their counterparts in human patients with Parkinson’s disease: paradoxical kinesia and stress-

34

MlCHAEL J. ZIGMOND A N D EDWARD M. STRICKER

induced akinesia. In this section, we discuss the mechanisms that might underlie these two phenomena. A. PARADOXICAL KINESIA During the initial akinetic phase of the response to GHDA, an acute stress, such as being placed in a deep tub of water, can temporarily improve the performance of the brain-damaged rats (Levitt and Teitelbaum, 1975; Marshall et al., 1976). T h e ability of an intense stimulus to produce a transient remission in parkinsonian patients and NSBlesioned animals has its parallel in earlier studies of the behavioral effects of DA antagonists such as chlorpromazine; indeed, these drugs were termed neuroleptics because they induced a parkinsonian-like bradykinesia in patients. In describing this condition, early investigators found that the neuroleptic-induced sedation could be overcome when patients were exposed to a sufficiently intense stimulus (Laborit et al., 1952). Analogous findings were made with rats, which would fail to respond to a tone signaling that foot shock was imminent but would escape from the shock itself (Courvoisier et al., 1953; Verhave et al., 1959; Posluns, 1962). The studies with neuroleptics in patients and experimental animals foreshadowed more recent investigations on the relation of sensory responsiveness to NSB injury. Some components of the syndrome produced by NSB or basal ganglia lesions also can result from partial sensory deafferentation (Zeigler and Karten, 1974; Zeigler, 1987; see, however, Stricker et al., 1975b). Moreover, the pattern of recovery from large NSB lesions can be interpreted in part in terms of a decreasing dependence on activation from the sensory stimulation provided by the environment (Stricker and Zigmond, 1984). Thus, brain-damaged rats will eat sweetened mash before dry chow and drink sugar water before tap water (Teitelbaum and Epstein, 1962; Zigmond and Stricker, 1973). However, the gross behavioral dysfunctions seen after large NSB lesions cannot be explained simply in terms of sensory or motor impairment, and more complex sensorimotor integrations are envisioned (Marshall et al., 1971; Turner, 1973; Marshall et al., 1980). There is a physiological and anatomical basis for the interaction between sensory input and NSB activity. For example, in substantia nigra, sensory stimulation increases the activity of neurons in zona reticulata (Schwarz et al., 1984; Joseph and Boussaoud, 1985) and 'in presumed dopaminergic cells in zona compacta (Barasi, 1979; Harper et al., 1979; Chiodo et al., 1980; Grace et al., 1980; Steinfels et al., 1983). Moreover, increasing evidence points to a role for striaturn, a structure

ANIMAL MODELS OF PARKINSONISM

35

known to receive extensive projections from sensory cortex (Kunzle, 1977; Malach and Graybiel, 1986), in sensory processing (see reviews in Krauthamer, 1975; Schneider and Lidsky, 1987). Given the strong reciprocal connections between substantia nigra and striatum, it would be surprising if sensory input to these two regions was not interrelated. Support for this presumption comes from the recent observation that NSB stimulation can enhance the striatal response to sensory input (West and Michael, 1987). These observations suggest that in paradoxical kinesia, the response of patients and NSB-lesioned animals to intense stimulation may reflect a greatly increased threshold for sensory activation. It has been presumed that this temporary improvement in performance reflects a transient release of DA from the few residual terminals (see Section V,B,2). The capacity of DA agonists to improve stress-induced deficits in performance is consistent with this formulation (Ljungberg and Ungerstedt, 1976b; Marshall and Ungerstedt, 1976; Snyder et al., 1985). Alternatively, it is possible that a sufficiently intense stressor is able to elicit a behavioral response without the involvement of DA. In support of this hypothesis, we have observed recently that akinetic rats with near total DA-depleting brain lesions nevertheless will begin to swim when placed in a tub of water; they will do so even when they have been pretreated so as to block both D1 and D2 DA receptors (Keefe et al., 1989a). Thus, paradoxical kinesia may involve other, nondopaminergic afferents to striatum or may occur without dopaminergic modulation when sensory stimulation is sufficiently intense. B. STRESS-INDUCED IMPAIRMENTS 1. Specific or Nonspecific? Stress also can impair the performance of brain-damaged rats. Indeed, despite the normal appearance of the animals under basal laboratory conditions, 6HDA-lesioned rats often show striking functional impairments when exposed to intense challenges (Breese et al., 1973; Fibiger et al., 1973; Marshall and Teitelbaum, 1973; Stricker and Zigmond, 1974; Snyder et al., 1985). These effects occurred both in animals that had sustained large DA depletions and had recovered from their initial deficits, and in animals with more moderate lesions that never had shown gross impairments. These residual deficits include the absence of expected feeding arid drinking responses during such acute challenges as insulin-induced hypoglycemia or colloid-induced hypovolemia (Epstein and Teitelbaum, 1967; Stricker and Wolf, 1967). Studies with this animal model have suggested that these dysfunc-

36

M1CHAEL.J. ZICMOND AND EDWARD M. STRICKER

tions represent a general inability of the brain-damaged animals to deal with any intense challenge, including hypoglycemia, cellular dehydration, cold, and pain. Thus, when NSB-lesioned animals with no gross behavioral dysfunctions are exposed to such severe stress, they soon deconipensate and resemble animals immediately after large DAdepleting lesions: that is, they show signs of akinesia, catalepsy, and sensory neglect (Stricker et al., 1979; Snyder et al., 1985). However, 6HDA-lesioned animals that are unable to respond appropriately to an intense stimulus often will respond normally to a stimulus of the same quality but lower intensity. For example, although the brain-damaged animals do not increase their food intake when given a single large dose of insulin, they do become appropriately hyperphagic when chronic hypoglycemia is induced with small daily doses of a long-acting form of insulin (Stricker et al., 1975a; Rowland and Stricker, 1982). 2. Stress and Dopamine Release There is considerable reason to believe that stress increases DA release in striatum. First, stress-induced akinesia can be observed in animals that had recovered from the initial deficits produced by 6HDA or surgical lesions of the NSB (Teitelbaum and Epstein, 1962; Zigmond and Stricker, 1972; Stricker and Zigmond, 1974; Snyder et al., 1985). The 6HDA can be administered intraventricularly, along the NSB, o r directly into the lateral striatum. Rats with smaller DA depletions that had not shown initial neurological dysfunctions also will become impaired when the stressor is sufficiently intense or the animals are pretreated with neuroleptic drugs that compromise dopaminergic function (Snyder at al., 1985). Second, animals that have recovered from the initial effects of unilateral NSB lesions will turn away from the innervated side when either stressed or given amphetamine (Ungerstedt, 197lb). Finally, stress-induced akinesia can be reversed by treatment with DA agonists such as L-DOPA (Snyder et al., 1985). ‘Thus, like the initial deficits, stress-induced impairments in neurological function appear to result from the loss of DA. However, although a variety of stressful challenges can be shown to increase DA turnover in frontal cortex and limbic regions, most studies report no increase at all in DA turnover in striatum (e.g., Thierry et al., 1976; Fadda et aZ,,1978; Lavielle et al., 1978; Tissari et al., 1979; see, however, Dunn and File, 1983). Furthermore, whereas NSB neurons are more active during arousal than at any other time during the sleepwake cycle (Steinfels et al., 1983) and may be further increased by sensory stimuli (see Section V,A), there is no evidence for a sustained elevation in activity during more prolonged stress (Strecker and Jacobs, 1985).

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37

How might these two groups of apparently conflicting data be reconciled? We believe that regional differences in the responsiveness of DA neurons to stress d o not reflect either the extent to which cell groups are activated or the relative importance of different regions in the response to stress. Instead, we propose that stress-induced release of DA occurs in cortical, limbic, and striatal regions and that differences in circuitry (including local feedback loops) and in the rate of DA reuptake determine the amplitude and duration of response. In particular, we believe that the relatively small response of NSB neurons is a consequence of the highly developed capacity for homeostatic control within the system and the extremely high density of DA terminals, the principal sites for DA inactivation (see Section III,B, 1). T h e evidence in support of this hypothesis comes from studies of in uiuo DA release. We have monitored in uiuo dopaminergic activity in striatum using both voltammetry (Keller et al., 1983) and microdialysis perfusion (Abercrombie et al., 1989b; Salamone et al., 1989). With our voltammetric electrode we observed a DA-like signal that could be blocked by either a-methyltyrosine or y-butyrolactone and increased by amphetamine. T h e signal also was increased by various external stimuli, although not always in the same way. For example, intense exteroceptive stimuli, such as tail shock or cold water, produced a large and abrupt rise in the signal that decayed rapidly, even under conditions in which exposure to the stimulus was maintained. Milder stimuli such as food after a 24-hr fast, water after a period of dehydration, or a novel olfactory or visual stimulus elicited a smaller, more gradual, and more prolonged electrochemical response. In contrast, homeostatic challenges such as hypoglycemia, hypotension, and cellular dehydration were ineffective in producing a change in apparent dopaminergic activity in striatum (Keller et al., 1983). In addition, we have monitored dopaminergic activity in perfused rat striatum by a microdialysis probe. This approach provides an identification of the compounds under investigation that is less ambiguous than voltammetric techniques, although the temporal resolution is considerably lower. Using dialysis, we observed an increase in DA overflow during 30 min of intermittent tail shock in rats. This was accompanied by a somewhat smaller, more delayed, and more prolonged increase in 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) (Abercrombie et al., 1989b). No such changes were observed in response to insulin-induced hypoglycemia (R. Keller, M. Zigmond, and E. Stricker, unpublished observations). Collectively, these observations suggest that NSB neurons are activated by many stressors, although not by interoceptive ones. Moreover, they suggest that the period of time during which increased dopaminer-

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MICHAEL J. ZIGMOND A N D EDWARD M. SI’RICKEK

gic activity can be maintained is determined in part by the rate at which extracellular DA concentration rises. Rapid increases are terminated quickly, whereas gradual increases can be sustained over a longer period of time. Such behavior is characteristic of a homeostatic feedback system. Given these findings, it is not surprising that changes in DA activity have been difficult to detect using conventional measures of DA release based on postmortem analysis of tissue. This is particularly true of studies involving the quantification of tissue levels of DA or its metabolites, for which the baseline values are high.

3. Stress and Dopamine Release a f e r NSB Lesions T h e inability of preclinical parkinsonian patients and laboratory animals with NSB lesions to tolerate stressors may follow directly from the increase in the activity of the remaining elements of the damaged system. Presentation of a stressor that normally increases dopaminergic activity may exceed the limits ofthe compensated system in one or more of several ways. For example, the neurons may be incapable of a further increase in firing. In support of this hypothesis, it has been observed that acute administration of neuroleptics can precipitate a depolarizationinduced blockade of subsequent neuronal activity in 6HDA-lesioned rats (Hollerman and Grace, 1988), whereas this phenomenon normally occurs only after several weeks of drug treatment (Bunney and Grace, 1978). In addition, compensated neurons may be incapable of maintaining their elevated response to depolarization when firing rates get too high. This hypothesis is supported by the observation that DA release from slices prepared from the striatum of control rats is proportional to stimulus frequencies up to 20 Hz or more, whereas slices from GHDAlesioned animals are responsive in a much more restricted range (Snyder et al., 1986; Stachowiak el al., 1987). Such an explanation does not appear to hold for visceral stressors that do not produce behavioral activation, such as hypovolemia and glucoprivation, because such homeostatic imbalances fail to release DA in striatum (Keller et al., 1983). Indeed, DA release actually is inhibited by some of these stimuli (Zigmond et al., 1986). Moreover, recently we have observed that an exteroceptive stressor such as tail shock can increase extracellular DA in rat striatum even after NSB lesions (Keefe et al., 1989b). Thus, further experimentation is required before stressinduced deficits can be substituted. 4. Therapy f o r Stress-Induced Akinesia One of the problems faced by a parkinsonian patient is the sudden onset of akinesia, or freezing. The cause of this symptom is unknown,

ANIMAL MODELS OF PARKINSONISM

39

but it often is precipitated by stress and thus may be related to the phenomenon under discussion. As noted above, dysfunctions induced by stress can be reversed in GHDA-treated rats (Ljungberg and Ungerstedt, 1976b; Marshall and Ungerstedt, 1976; Snyder et al., 1985). In contrast, several investigators have reported that L-DOPA fails to improve freezing in parkinsonian patients and even may exacerbate this problem (Barbeau, 1976; Ambani and Van Woert, 1973; Narabayashi and Nakamura, 1981). Recently, threo-3,4-dihydroxyphenylserine (DOPS) has been used successfully in the treatment of this problem (Narabayashi et al., 1984a). Unlike L-DOPA,which is decarboxylated to form DA and has little or no impact on the NE content of brain (see Section VI,B), DOPS is decarboxylated in brain to form NE (Bartholini et al., 1971; Suzuki et al., 1984). Thus, the clinical efficacy of this drug suggests the possibility that under certain conditions noradrenergic projections can substitute for the NSB. This, in turn, may be related to the observation that paradoxical kinesia may involve a nondopaminergic mechanism (see Section VA. C. IMPLICATIONS FOR CANNON’S FIGHT-OR-FLIGHT THEORY

Parallels between central and peripheral catecholamines have been noted by several investigators (e.g., Zigmond and Stricker, 1974; Stricker and Zigmond, 1976; Amaral and Sinnamon, 1977). These include utilization of the same neurotransmitters, an anatomical organization that involves relatively few clusters of neurons having a diffuse field of influence, and responsiveness to stressors (see also Sections III,B and V). Thus, it seems appropriate to ask whether the insights that have been obtained from studies of injury to central aminergic systems also might have implications for studies of damage to the sympathetic nervous system. T h e first systematic examination of the functional impact of damage to a monoaminergic system was contained in Cannon’s studies of the peripheral sympathetic nervous system. He noted that extensive removal of the sympathetic ganglionic chain in cats had few, if any, apparent physiological consequences under basal laboratory conditions. On the other hand, such lesions did disrupt the physiological responses to acute homeostatic challenges. From these findings he concluded that the sympathetic nervous system was involved primarily in the response of animals to fight-or-flight situations (Cannon et al., 1929; Cannon, 1932).

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MICHAEL. J. ZIGMOND A N D EDWARD M . STRICKER

We now know, however, that the sympathetic nervous system plays an important role in various aspects of homeostasis under conditions other than severe stress. Thus, for example, a-adrenergic blocking agents precipitate acute arterial hypotension in animals maintained in a laboratory environment providing little stress (Hosutt and Stricker, 1981). What Cannon neglected to take into account in his initial formulation was the possibility that compensatory changes offset the functional impact of the lesions, a possibility suggested by his own experiments. For example, Cannon noted that undamaged elements of the sympathoadrenal system influenced the denervated tissue more effectively after partial sympathectomy than before (Cannon et al., 1926; Cannon and Rosenblueth, 1949). Subsequent investigations have indicated that these functional changes resulted from the loss of presynaptic uptake sites for catecholamine and an increased sensitivity of target cells to transmitter (see review in Trendelenburg, i966). Furthermore, it is clear that destruction of a portion of the sympathetic postganglionic neurons is followed by an increased capacity of the system’s residual elements to synthesize catecholamine (Mueller et al., 1969; Brimijoin and Molinoff, 1971; Fluharty et al., 1987; see also Section 111,D,3). Thus it seems probable that, as a natural consequence of synaptic homeostasis, damage to the sympathetic nervous system is partly offset by increased catecholamine synthesis and release from residual elements of the system and increased sensitivity to catecholamine at the denervated site. As already seen in the case of NSB damage, the benefit of such homeostatic processes is a reduction in functional impairment. The cost, however, is a constriction of the range of stimuli to which the system can respond. Thus, the deficits observed in sympathectomized animals under special testing conditions do not indicate that catecholamines are of importance only in those limited circumstances, but that the compensatory responses to sympathectoniy are limited and cannot fully restore function.

VI. How Can Drugs Replace Neurons?

A.

ATROPINE

As in the case of parkinsonian patients, neurological deficits in NSB-lesioned rats can be alleviated temporarily with muscarinic receptor-blocking agents (Snyder et al., 1985). These drugs originally were prescribed as a treatment for some of the autonomic disturbances

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41

observed in parkinsonism; indeed, to the extent that those disturbances are due to sympathetic hypofunction, parasympathetic blockade would be expected to provide some relief. However, subsequent animal studies suggest that the autonomic nervous system probably is not the main site of action for these compounds in the treatment of parkinsonism. As described above (Section III,D, l ) , dopaminergic NSB fibers normally inhibit ACh-containing interneurons in striatum, and degeneration of NSB can lead to a hyperactivity of those cells. By blocking muscarinic receptors in striatum, the alkaloids should reduce the impact of' excess ACh release. In addition, many antimuscarinic agents also inhibit high-affinity DA transport (Coyle and Snyder, 1969), a factor that also may contribute to their therapeutic actions. B. L-DOPA 1. The Site of D A Synthesis from DOPA L-DOPA, which reduces the neurological dysfunctions observed in Parkinson's disease, also is effective in animals with DA-depleting brain lesions produced by MPTP and 6HDA (Van Woert et al., 1972; Ljungberg and Ungerstedt, 1976b; Marshall and Ungerstedt, 1976; Snyder et al., 1985; Burns et al., 1983; Langston et al., 1984c; Ogawa et al., 1985). These behavioral effects of L-DOPA are abolished by inhibition of DOPA decarboxylase (Bartholini et ad., 1969; Schoenfeld and Uretsky, 1973). Thus, the therapeutic efficacy of L-DOPA appears to result from an enhanced availability of DA. However, the increase in brain DA content produced by L-DOPAis not abolished by NSB injury (Ng et al., 1971; Lytle et al., 1972; Snyder and Zigmond, 1987; Hefti et al., 1980). This is consistent with the apparent presence of decarboxylase immunoreactivity in various nondopaminergic sites in brain, including cerebral capillaries (Bertler et al., 1966; Langlier et al., 1972) and 5HT-containing neurons (Duvoisin and Mytilineou, 1972; Ng et al., 1972; Hokfelt et al., 1973; Jaeger et al., 1984), as well as the appearance of significant decarboxylase activity in neurons intrinsic to striatum (Melamed et al., 1980a).These findings raise the possibility that although DA is essential to the action of L-DOPA, dopaminergic neurons are not. 2. The Release of D A Formed from L-DOPA: Impact of NSB Lesions L-DOPA also can increase striatal DA release, both in uiuo and in uitro (Hefti and Melamed, 1981; Ng et al., 1970; Tyce and Rorie, 1985; Misu et al., 1986; Keller et at., 1988a). This increase can be seen under basal conditions and in response to depolarization (Snyder and Zigmond,

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MICHAEL J. ZIGMOND AND EDWARD M. STRICKER

1987; Keller et al., 1988a) and presumably underlies L-DOPA’Stherapeutic efficacy. Although much of the DA formed and released from exogenous L-DOPA derives from dopaminergic neurons, a significant component appears to originate from nondopaminergic sites. In the intact rat this extradopaminergic pool may contribute only 20% of the DA released in the presence of L-DOPA; however, after large NSB lesions, this pool can represent the great majority of that DA (Snyder and Zigmond, 1987; see also Melamed et al., 1980a). Studies utilizing an in vzvo microdialysis probe suggest that the capacity of L-DOPA to increase extracellular DA actually is enhanced by 6-HDA-induced lesions (Bonatz et al., 1989). This seemingly paradoxical finding is not due to an increase in DA release but to a decrease in the rate of DA inactivation, an indirect consequence of the loss of high affinity uptake sites for DA that accompanies NSB degeneration (E. D. Abercrombie, A. Bonatz, and M. J. Zigmond, unpublished observations). Thus, NSB damage appears to have two impacts on the dopaminergic response to L-DOPA. First, it reduces the rate at which DA, formed from L-DOPA,is removed from the extracellular space and thus greatly increases the resulting extracellular concentration of DA. Second, it increases the relative contribution of nondopaminergic sites to extracellular DA. Further experimentation is needed to determine the precise location of the nondopaminergic sites of DA release, the mechanism by which the release occurs, and the importance of such release to the therapeutic effects of L-DOPA.

3. Significance for Pharmacotherapy In attempting to explain the decline in therapeutic efficacy of L-DOPA, it seems appropriate to focus on two factors: the long-term impact of L-DOPA and the progressive degeneration of the NSB. It has been noted that chronic exposure to large doses of L-DOPA reduces the number of DA receptors (e.g., Reisine et uE., 1977; Rinne et al., 1981; Lee et al., 1978; Raisman et al., 1985; see, however, Guttman and Seeman, 1987). In addition, chronic L-DOPA treatment can reduce the capacity of brain to decarboxylate L-DOPA to form DA (Melamed et al., 1983, 1987). These observations suggest that adaptations within striatum occur during long-term L-DOPA treatments that gradually reduce the efficacy of these treatments, and have led to the proposal that L-DOPA treatment be withheld for as long as possible (Yahr, 1976; Lesser et al., 1979; Melamed, 1986). In support of delaying L-DOPA treatment, it has been noted that

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43

responsiveness to the therapy sometimes is seen to increase in patients removed from drug treatment for several weeks (Direnfeld et al., 1978; Weiner et al., 1980), although the extent of the improvement caused by such drug holidays has been questioned (Kofman, 1984; Kaye and Feldman, 1986). Moreover, some investigators report that the period of time during which L-DOPA is effective is independent of the degree of neurological deficit at the outset of treatment (Barbeau, 1969; Yahr, 1977). T h e data discussed here suggest that the gradual degeneration of NSB also may play an important role in limiting the effectiveness of L-DOPA treatments. This factor would not mitigate against early L-DOPA treatment unless L-DOPA promoted NSB degeneration, a possibility that in fact has been raised (Cohen, 1983). It does, however, further strengthen the conclusion that L-DOPA ultimately will become ineffective and that further therapy will require another approach, most likely one that does not involve indirect-acting dopaminergic agents. Alternative therapeutic strategies are discussed in the next section.

VII. Implications for Future Research

A. DIAGNOSIS Considerable attention has been paid to the early diagnosis of certain disorders known to derive from genetic abnormalities, such as Huntington’s disease. The benefits of being able to diagnosis an idiopathic disease such as parkinsonism in asymptomatic patients also should not be overlooked. Early diagnosis would permit a more accurate assessment of causal factors, especially important when the factors include the environment to which the patient is then being exposed. Moreover, as methods for halting the progression of the disease are developed, early diagnosis may become an important aspect in its treatment (see Section VI1,C). T h e diagnosis of parkinsonism in early stages of the disease can be difficult because current neurological examinations usually are insensitive to lesion-induced dysfunction until NSB degeneration is extensive. Indeed, even postmortem examination of brain tissue in advanced parkinsonism may be in error because NSB degeneration produces anatomical changes that are not readily detectable using conventional neuropathological approaches (Wolf et al., 1978). Despite these problenis, it has been rather easy to detect subclinical NSB damage in an

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animal model of parkinsonism. As mentioned, although such animals appear normal under basal laboratory conditions, parkinsonian symptoms emerge when they are exposed to a stressor, such as cold or hypoglycemia. Moreover, the animals show a heightened behavioral response to dopaminergic agonists and antagonists, such as L-DOPAand These findings suggest the haloperidol (see Sections II1,C and 111,D). feasibility of initiating a prospective study in human subjects in which performance under stress or after a pharmacological challenge is recorded and is correlated with subscquent neurological status (Zigmond and Striker, I98 1).

B. TREATMENT

1 , Pharmacologzcal Approaches The success of L-DOPA therapy in the treatment of parkinsonism has led to investigations of at least four additional pharmacological approaches to increasing dopaminergic activity (see reviews in Lang, 1984; Bianchine, 1985; Calne, in press) (Fig. 8). a. Substrate Supplementation. Attempts have been made to increase DA synthesis at the rate-limiting hydroxylation step by increasing the availability of the substrates or cofactors for the reaction. Studies with the use of tyrosine in experimental animals have yielded equivocal results. From kinetic studies with cell-free tyrosine hydroxylase, one would predict that the enzyme is nearly saturated with its amino acid precursor and, indeed, that raising tyrosine levels might actually lead to inhibition of enzyme activity (Kaufman, 1986). The hypothesis that adequate tyrosine already is available to dopaminergic neurons is further supported by in vitro and in vivo studies of DA synthesis and release (Carlsson and Lindqvist, 1978; Kapatos and Zigmond, 1977; Snyder and Zigmond, 1987), as well as by the relative lack of efficacy of tyrosine in treating parkinsonian symptoms either in an animal model (Snyder et al., 1985) or in patients (Growdon et al., 1982). There are, however, reports that under some circumstances manipulation of precursor availability can alter DA turnover (Wurtman et al., 1974; Gibson and Wurtman, 1977) and release (Milner and Wurtman, 1984; Melamed et al., 1980b; see review in Wurtman, 1987). Further research will be required to resolve this apparent contradiction. Although kinetic studies of tyrosine hydroxylase raise questions about the usefulness of tyrosine supplementation, they permit more optimism regarding treatment of parkinsonism with tetrahydrobiopterin. This pterin is the presumptive cofactor for the rate-limiting step in

ANIMAL MODELS OF PARKINSONISM

DOPAC

45

I I

FIG. 8. Possible sites for pharmacotherapy in Parkinsonism. Drugs may act (1) to increase dopaminergic activity at DA synapses (see inset) or (2) by increasing the firing rate of DA cells. In addition, a drug could (3) produce a DA-like effect on striatal targets by acting via another input. Finally, (4) a drug could produce the same net effect by acting downstream from the DA-sensitive cell. Inset: An increase in dopaminergic activity could be produced in a variety of ways, including increasing either (A) tyrosine (TYR) hydroxylation or (B) L-DOPA decarboxylation; increasing DA release ( C )by a direct action on a release mechanism, (D) affecting a presynaptic DA receptor, or (E) acting via a heterosynaptic input; decreasing the inactivation of DA via (F) high-affinity DA uptake or (G) catabolism to DOPAC; or stimulating the postsynaptic cell directly, either (H) via the DA receptor on the cell membrane surface or (I) at a more distal site in the receptor complex.

DA synthesis (Kaufman, 1963; Brenneman and Kaufman, 1964; Nagatsu et al., 1964, 1981; Levine et al., 1981), and most estimates of its concentration yield a value that is far below the apparent K , of the enzyme (Kaufman, 1974). These findings have led to studies in which synthetic cofactor has been added, either in vivo or in vztro (Kettler et al., 1974; Patrick and Barchas, 1976; Hirata et al., 1983). However, the maximal effect observed with this approach has been about a two-fold increase in DA synthesis, far less than predicted from studies of the cell-free enzyme. This may indicate that tyrosine hydroxylase is much closer to being saturated by cofactor than had previously been proposed, a possibility supported by recent studies in which tyrosine hydroxylase was examined under more physiological conditions (Kaufman, 1986). Moreover, even if such supplementation were effective in increasing DA synthesis and release, one might expect a number of side effects from

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MICHAEI.,J. ZIGMOND A N D EDWARD M. STRICKER

the administration of tetrahydrobiopterin since it acts as a cofactor in the synthesis of all biogenic amines. For these reasons this approach also may not prove effective in the treatment of parkinsonism, although some preliminary success has been reported when cofactor supplementation was given early in the course of the illness (Birkmayer and Kiederer, 1983; Curtius et al., 1982; Narabayashi et al., 1984b). 6. Indirect Agonihts. An alternative to enhancing dopaminergic activity by increasing tyrosine hydroxylation would be to use drugs that act to stimulate DA release directly. While there has been some indication from animal models that this approach might be effective (Stricker and Zigmond, 1976), neither amphetamine, methylphenidate, and nomifensine have not proven to be of much value in the clinic (Lang, 1984; see also Section VI,B). A possible exception is amantadine, which has been reported to have some therapeutic value. However, although amantadine can increase I)A release, its mechanism of action in the treatment of Parkinson’s disease is unclear and will require further investigation (Heikkila and Cohen, 1972b; Lang, 1984). Another antiparkinsonian treatment suggested recently is the administration of a neuroleptic, which in low doses can block presynaptic DA autoreceptors and thereby increase transmitter release from residual terminals. However, we found that neuroleptics had little or no effect on DA turnover in rats with 6HDA-induced lesions, either in vivo or in vitro (Zigmond and Stricker, 1984; Snyder and Zigmond, 1987), possibly reflecting the fact that the remaining DA neurons are operating near or at their maximal capacity for the synthesis and release of transmitter. Haloperidol, however, has been reported by others to increase DA turnover in NSB-lesioned animals (Hefti et al., 1985a), and thus this approach may warrant further investigation. c. Deprmyl and Inhibition of MAO, Soon after the first attempts to use L-DOPA in the treatment of parkinsonism, MA0 inhibitors were introduced as a possible form of adjunctive therapy. They soon were discontinued because of side effects. However, with the appreciation that at least two forms of MA0 existed in brain, more specific MA0 inhibitors were examined. T h e MAO-B inhibitor deprenyl given in combination with L-DOPA was found to reduce the akinesia and depression associated with parkinsonism (Birkmayer et al., 1975, 1982; Birkmayer, 1978), and subsequent animal studies have further supported the value of the combined treatment (Yahr, 1978). The basis of the effects of deprenyl is not clear, however. DA is a good substrate for MAO-B (Glover et al., 1977), and inhibitors of this enzyme appear to increase DA release, as measured by behavioral and biochemical means (Knoll et al., 1965; And& et al., 1 9 6 6 ~Harsing ; and

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47

Vizi, 1984). Thus, deprenyl may act to inhibit M A 0 and thereby increase the availability of DA formed from endogenous, as well as exogenous, L-DOPA. In addition, deprenyl can be metabolized to amphetamine (Reynolds et al., 1978). Thus, administration of deprenyl would be expected to release DA directly as well as block DA reuptake. MAO-B inhibitors also can increase the concentration of phenylethylamine, another biogenic amine with dopaminergic properties (Yang and Neff, 1973; Reynolds et al., 1978). Finally, it has been suggested that inhibition of M A 0 may actually reduce the degenerative process (see Section VII,C, below). Consistent with the latter hypothesis is the observation that patients treated with deprenyl have a longer life expectancy (Yahr, 1978; Birkmayer et al., 1985). Clinical trials with deprenyl currently are being conducted (Shoulson, in press). d. Direct Agonists. Drugs that act directly on DA receptors might be expected to have an advantage over indirect agonists in that they do not depend on the presence of DA terminals. Early experiments with direct agonists focused on the DA agonist apomorphine (Ernst, 1967), which was shown to reverse neurological deficits in animal models (Ljungberg and Ungerstedt, 1976b; Marshall and Ungerstedt, 1976; Snyder et al., 1985). However, although it proved effective in reducing parkinsonian deficits, the clinical value of apomorphine was limited by the need for parenteral administration, its tendency to cause nausea, and its short duration of action (Schwab et al., 1951; Cotzias et al., 1970). Reversal of neurological deficits also has been obtained in 6HDAtreated rats with the direct administration of DA into striatum via an osmotic minipump (Stromberg et al., 1985). T h e latter approach has not yet been attempted in patients but may be worth considering as an effective if unconventional approach. DA might be expected to have fewer nonspecific effects than other DA agonists. Moreover, it should exhibit a tendency to work primarily at denervated sites, because areas with intact DA terminals would rapidly inactivate the exogenous transmitter. It is most usefully contrasted not with other pharmacological approaches but with the surgical transplantation techniques described in the next section. Another reason for exploring DA agonists as forms of pharmacotherapy emerged with the realization that there existed at least two subtypes of DA receptors, D-1 and D-2, and that apomorphine failed to differentiate between them (Kebabian and Calne, 1979). Subsequently, studies with more selective DA agonists suggested that activation of only the D-2 site was capable of reversing the impact of NSB lesions in animals (Nomoto et al., 1985). This finding increased the importance of ongoing trials with bromocriptine, which was shown to act selectively on

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D-2 receptors, and with other relatively selective D-2 agonists, including lergotrile, lisuride, and mesulergine (see reviews in Lang, 1984, 1987; Lataste, 1984). However, investigations have indicated that D- 1 agonists have behavioral effects, as well (Rosengarten el al., 1983; Molloy and Waddington, 1985; Trugman and Wooten, 1986). Moreover, it has been observed that D-1 agonists can potentiate the behavioral impact of D-2 agonists in DA-depleted animals (Robertson and Robertson, 1986; Groppetti et al., 1986) and that D-1 and D-2 agonists are effective in reversing the behavioral effects of reserpine in mice only when the drugs are given simultaneously (Rabey et al., 1981). These results are consistent with electrophysiological data indicating that D-1 and D-2 receptors may act synergistically in some cases and suggest that a careful examination of the therapeutic value of combinations of D-1 and D-2 agonists may be warranted (Calne and Kebabian, 1987). Yet, despite multiple leads and changing approaches, the clinical trials reported to date have not provided reason for optimism regarding the use of direct agonists. Although such drugs can be useful in patients whose response to L-DOPA has become greatly reduced (Kurlan, 1988), the efficacy of these agents generally is short-lived (Lieberman et al., 1987; Riopelle, 1987). One possible explanation for this is that Pdrkinson's disease eventually leads to neuropathological changes in striatal cells (see Section III,D,5). If so, then it will be important to determine whether these changes are a primary response to the disease, a secondary response to NSB injury, or a response to L-DOPA; in any case, a high priority must be placed on exploring ways to halt the loss of target cells. In the meantime, the search for more effective pharmacological treatments must continue. e. A lternatiue Pharmacologzcal Strategaes. The avenues for developing pharmacological approaches to the treatment of parkinsonism have not been exhausted. Indeed, several new strategies are suggested by the known anatomy and pharmacology of the basal ganglia (see Fig. 8) (see reviews in Graybiel and Ragsdale, 1983; McCeer et al., 1984). First of all, NSB neurons can be influenced by a variety of heterosynaptic inputs to the cells of origin of this pathway. Moreover, DA release is modulated in part through presynaptic input within striatuni, including influences mediated by ACh (Giorguieff et al., 1977), 5HT (Ennis el al., 1981), GABA (Starr, 19'79; see, however, Giorguieff et al., 1978), glutamic acid (Giorguieff-Chesselet et al., 1979a), glycine (Giorguieff-Chesselet et al., 1979b), and neurotensin (De Quidt and Emson, 1983) (see review in Raiteri et al., 1984). Thus, it is possible that pharmacological manipulation of transmission at these sites could be of some benefit.

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49

A second approach would be to duplicate the influence of DA through the manipulation of parallel inputs to striatal cells. For example, the striatum receives innervation from 5HT-containing neurons (Anden et al., 1966b; Lorens and Guldberg, 1974; Azmitia and Segal, 1978),and some of the behavioral and neurobiological effects of DA can be produced by this amine (Waddington and Crow, 1979; Jackson et al., 1988b). Such effects might be enhanced pharmacologically. In addition, 5-15% of NSB neurons are nondopaminergic (Fibiger et al., 1972; Guyenet and Crane, 1981; Van d e Kooy et al., 1981). The nature of the transmitter utilized by these neurons is unknown, although neurotensin has been suggested as a likely candidate (McGeer et al., 1987). A determination of the relation between the dopaminergic and nondopaminergic components of the NSB may be fruitful. As we learn more about the circuitry of the striatum, it also might become possible to restore function by bypassing both the NSB and parallel inputs and influencing the next synapse down the line. Indeed, this presumably is the basis for the therapeutic effects of atropine (see Section V1,A). Some of the transmitters that participate together with ACh in distributing the influence of NSB to other brain regions are Met-enkephalin (Brann and Emson, 1980; Yang et al., 1983), GABA (Araki et al., 1985), substance P (Brownstein et al., 1977), substance K (Maggio and Hunter, 1984), and dynorphin (Vincent et al., 1982).

2. Transplantation It has been known since the early part of the century that embryonic neurons can be made to grow in the brain of an adult host (Dunn, 1916-1917). Such fetal nerve cells can grow neurites, make synaptic connections, and even function in a manner comparable to the normal innervation (see review in Lund, 1978). Brain transplantation has served primarily as a tool for the study of developmental processes in brain. However, investigators have begun to explore the possibility of using brain transplantation to repair damage as well, and one of the conditions for which transplantation offers particular promise as a form of therapy is parkinsonism. DA appears to serve primarily as a neuromodulator in brain, one that acts diffusely and can be replaced in large part by hyperactive residual DA terminals or direct-acting agonists. If so, then fetal DAsecreting cells transplanted into striatum ought to restore function as well. Of course, one would not expect complete elimination of behavioral dysfunctions, because such transplants probably would not receive the same afferent inputs that normally are delivered to cells in substantia nigra. However, in theory transplants could provide several important

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advantages over other approaches. First, it might be possible to raise the density of innervation toward that of normal tissue, thereby providing more compensation than could be expected from residual terminals. Second, DA release from transplanted neurons might be subject to some local regulation, thus providing a more physiological level of stimulation of target cell receptors than could be accomplished pharmacologically. Third, successful surgical intervention might reduce or even obviate the need for pharmacotherdpy. During the past decade, there has been considerable research on the effects of transplanting catecholaminergic cells into striatum after NSB injury (Olson, 1985; Bjorklund et al., 1987; Sladek and Gash, 1984; Azmitia and Bjiirklund, 1987). In the following sections we review that research and its implications for the treatment of parkinsonism. a. Transplant Donor and Hust. ‘Transplantation involves a donor and a host. Transplanted material has included pieces of tissue o r cell suspensions derived from fetal mesencephalon (Bjijrklund and Stenevi, 1979; Perlow et al., 1979), or pieces of adrenal medulla (Freed et al., 1981; Stromberg et al., 1984). Use also has been made of primary cultures of fetal mesencephalic or adrenal chromaffin cells (Gage el al., 1985a; Collier et al., 1987; Kamo et al., 1987) or catecholamine-synthesizing cell lines (Brundin et al., 1985; Hefti et al., 1985b; Hargraves et al., 198’7; Jaeger, 1987; Kordower el al., 1987). Material has been placed both within and adjacent to the striatum of NSB-lesioned animals, using a protocol in which donor and host were the same animal, two different animals of the same species, and/or two animals of different species. T h e viability of transplants depends in part on the nature of graft and of host. With respect to donor material, cell lines examined to date have had a very limited period of survival (Hefti et al., 198513; Hargraves et al., 1987). Moreover, although chromaffin cells from adrenal medulla form neuron-like terminal axons in the anterior chamber of the eye (Olson, 1970), they show little fiber outgrowth when transplanted into striatum (Freed et al., 1981; Strijmberg et al., 1984, 1985). The greatest success has come from transplants of fetal ventral mesencephalon. This material, when taken from a donor of the right age and transplanted under the proper conditions, can survive, grow axons, form extensive plexuses of terminal axons, and raise the overall level of DA in the host striatum (Bjorklund and Stenevi, 1979; Perlow et al., 1979; Freed et al., 1980). As might be expected, the nature of the host also is important in determining transplant viability. Thus, for example, transplantation of fetal mesencephalon into rats appears to be more successful when the hosts are very young (Carder et al., 1987). In addition, survival of

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adrenal medullary cells is high in rats but thus far has been very poor in primates (Morihisa et al., 1987). b. Activity of Transplants. Successful transplants can show electrical activity comparable to that seen in DA cells (Wuerthele et al., 1984) and release catecholamine spontaneously, as indicated by microdialysis and voltammetric analyses of extracellular fluid (Gerhardt et al., 1984; Rose et al., 1985; Zetterstrom et al., 1986). Transplants also respond to indirect agonists (Bjorklund and Stenevi, 1979; Perlow et al., 1979) and may even be influenced by the external environment (Carder et al., 1987; Keller et al., 1988b). Release from transplanted cells often appears to occur under some degree of regulation. This is suggested by the observation that small mesencephalic grafts that raise the density of innervation to 10-20% of normal result in an extracellular level of DA that is about 40% of normal, whereas larger grafts that restore the amount of innervation to control levels also normalize the concentration of DA in extracellular fluid (Zetterstrom et al., 1986; Strecker et al., 1987). Moreover, indices of DA synthesis and turnover, elevated after NSB lesions (see Section III,C), are reduced toward normal after transplantation of mesencephalic tissue (Schmidt et al., 1982, 1983; D. Jackson, R. Carder, R. Lund, and M. Zigmond, unpublished observations). This regulation may be mediated in part by DA autoreceptors, which can be shown to operate in mesencephalic transplants (Wuerthele et al., 1984). In addition, a wide array of synaptic inputs has been described (Bolam tt al., 1987) and these also may play a regulatory role. Once released, catecholamine appears to make its way from the transplant to striatal target sites, since the presence of a transplant decreases behavioral and neurochemical indices of target cell sensitivity to catecholamines (Bjorklund and Stenevi, 1979; Perlow et al., 1979; Freed et al., 1983),decreases the firing rate of striatal targets (Wuerthele et al., 1981), and reestablishes an inhibitory influence over ACh release (Carder et al., in press). Moreover, as discussed in the next section, transplants can reverse the behavioral deficits caused by NSB lesions. In the case of mesencephalic grafts, synaptic contacts have been observed between donor and host cells (Freund et al., 1985; Mahalik et al., 1985) and may serve to mediate these influences. However, the diffusion of transmitter to more distant targets also may play a role in these phenomena. c. Impact of Transplants on Neurologzcal Deficits. Transplants are able to reverse many of the behavioral deficits caused by NSB injury in experimental animals. For example, transplants can improve symptoms of rigidity, bradykinesia, and sensory neglect in rats (Perlow et al., 1979;

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Bjorklund et al., 1980; Dunnett et al., 1983a,b) and monkeys (Bakay et al., 1985; Morihisa et al., 1987; Sladek et al., 1987), although deficits in ingestive behaviors have been resistant to this approach (Dunnett et al., 1983b). The nature of the therapeutic effect of a transplant depends largely on the amount and location of fiber ingrowth. For example, reinnervation of dorsal striatum appears to be necessary to reduce rotation after unilateral NSB lesions, and reinnervation of ventral and lateral striatum is required to reduce contralateral sensory neglect (Dunnett et al., 1983a). There have been several attempts to use adrenal medullary transplants in human parkinsonian patients. Some indications of reduced neurological deficits have been reported (Backlund et al., 1985; Madrazo et at., 1986, 1988; Lindvall et al., 1987; Jiao et al., 1988). However, to date there has been little evidence for long-term improvements and there is concern about the morbidity and mortality associated with the procedure (Sladek and Shoulson, 1988). Moreover, there is reason to believe that even when improvement is observed, it may not be caused by the sustained reinnervation of striatum by donor cells (see Section VII,C,3,b). Thus, such an approach to therapy must be regarded as highly experimental and not yet appropriate for widespread use. Moreover, evidence suggests that if and when transplantation does become an effective procedure, the tissue to be utilized probably will not derive from host adrenal medulla but from mesencephalon taken at a critical stage in embryogenesis or from a cell line that has not yet been examined. Among the issues that remain to be explored are the best tissue and age of donor material, the best time in the course of the disease for transplantation, and the best procedures to promote survival of donor material.

C. PREVENTION

Each of the above approaches accepts the inevitability of NSB degeneration. Recently, however, some investigators have begun to focus on ways to reduce the incidence of the disorder. Two interrelated hypotheses have been suggested: that parkinsonism results from MPTPlike toxins present in the environment or endogenous to the brain (Calne and Langston, 1983; Carlsson, 1987), and that it results from an abnormally high accumulation of reactive forms of' oxygen and free radicals (Perry et al., 1982; Cohen, 1983). I n this section we examine several aspects of the effort to minimize the development of parkinsonism.

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53

1. Environmental Factors Research with neurotoxins, especially MPTP, has helped to stimulate the search for an environmental cause of parkinsonism. Investigations have demonstrated significant correlations between the geographical distribution of presumptive neurotoxins and the prevalence of parkinsonian symptoms (Rajput, 1984; Barbeau et al., 1987; Tanner et al., 1987; Schoenberg, 1987; Spencer et al., 1987). Among the environmental agents that have been implicated are herbicides, plant toxins, and an unidentified toxin that accumulates in well water. The possibility of an endogenous toxin also has been proposed (Ambani et al., 1975; Cohen, 1983; Mann and Yates, 1983; Carlsson, 1987), and the potential toxicity of DA itself has been noted (Maker et al., 1986). However, the search is a difficult one, particularly given the possibility that a long latency exists between exposure to toxin and the onset of symptoms, and to date little progress has been made.

2 . Blockade of the Degenerative Process A second approach has been to attempt to halt the degenerative process. This has been shown to be an effective strategy in the case of MPTP, whose conversion to MPP' and thus its damaging effects on NSB can be blocked by inhibitors of MAO-B (Heikkila et al., 1984b; Langston et al., 1984b; Cohen et al., 1985); moreover, neurotoxicity also can be blocked by inhibitors of high-affinity DA uptake, such as mazindol (Javitch et al., 1985). Of course, NSB degeneration may not result from increased formation of free radicals but from an inadequacy of the systems designed to neutralize them. Consistent with this hypothesis, the principal enzyme utilized to buffer cells against endogenous hydrogen peroxide, catalase, is markedly reduced in substantia nigra, caudate, and putamen of parkinsonian patients (Ambani et al., 1975). Likewise, peroxidase and an antioxidant, reduced glutathione, are deficient in such brains (Ambani et al., 1975; Perry et al., 1982; Kish et al., 1985). In either case, however, M A 0 inhibitors (which should reduce hydrogen peroxide formation as well as block the action of MPTP) and antioxidants, such as vitamin E (tocopherol), should be useful to halt the progression of the disease. Preliminary reports describing the use of MA0 inhibitors support this prediction (see Section VII,B, 1,c) and clinical trials to evaluate the hypothesis more thoroughly are being conducted (Grimes et al., 1987; Shoulson, in press). 3. Regeneration A third approach would be to repair the damage once it has occurred. In one sense, this is what is being attempted with transplan-

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tation (see Section VII,B,2). However, a more direct tack would be to promote either the regeneration of damaged axons or the sprouting of as yet unaffected cells. It generally is assumed that such events cannot occur in the adult mammalian CNS. If so, this would be a unique situation, for sprouting and regeneration of neurons is the rule, not the exception. After the initial disruption of function that accompanies peripheral nerve damage in mammals, as well as other animals, a sequence of events occurs that can lead to complete regeneration and restoration of function. Thus, for example, when 6HDA is given systemically, the loss of sympathetic postganglionic nerve terminals is only temporary and within several weeks innervation returns to normal (DeChamplain, 1971). In addition, in many invertebrates, regeneration occurs after damage to central, as well as peripheral, nerves (e.g., Murray, 1976; Wallace et al., 1977; Wood and Cohen, 1979). Moreover, the developing mammalian CNS is capable of considerable neuronal growth after injury (Lund and Lund, 1971; Schneider, 1979). Why, then, is regeneration in the adult mammalian CNS usually limited to distances of a few micrometers at best (e.g., Cajal, 1928; Raisman, 1969; Tsukahara P t al., 1975; Cotman and Lynch, 1976)? Several explanations have been proposed, including the absence of necessary substrates or growth factors and a lack of the proper temporal organization (see reviews in Lund, 1978; Tsukahara, 1981; NietoSampedro and Cotman, 1985; McGeer et al., 1987; see also Cotman, 1985). Next, we review two areas of investigation deemed particularly promising in the effort to reverse the degenerative process in parkinsonism. a. Substrates: Peripheral 9ridges.” Many years ago it was observed that peripheral nerves transplanted into brain became innervated by their host (Tello, 191I). Investigators have begun to examine the possibiIity of using peripheral tissue to promote regeneration of severed axons in CNS (see review in Aguayo, 1985). Using this approach it has been shown that adult CNS neurons that have been injured can grow considerable distances through a peripheral transplant, although their growth stops abruptly when they reach CNS tissue (David and Aguayo, 1981; So and Aguayo, 1985). Moreover, it has been possible to combine peripheral nerve bridges with fetal transplants. For example, it has been shown that central DA neurons are capable of growing 2 cm or more through a bridge of peripheral sciatic nerve (Aguayo et al., 1984). Such preparations reverse the behavioral effects of GHDA, implying the formation of functional connections (Gage P t al., 1985b). This research raises new avenues for transplantation research, suggests the possi-

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bility of using bridges to reconnect substantia nigra to striatum, and provides additional encouragement to the search for factors that stimulate neuronal outgrowth. 6. Growth Factors. The presence of a target can promote innervation. For example, a transplanted limb becomes innervated by motor neurons (Hamburger, 1939). Such experiments suggest the presence of growth factors, and in 1954 the first such factor was reported, later to be named nerve growth factor (NGF) (Levi-Montalcini and Hamburger, 1954; Cohen et al., 1954). NGF was shown to stimulate the survival and growth of embryonic sympathetic neurons and promote the synthesis of catecholamine-synthesizing enzymes (see reviews in Thoenen and Barde, 1980; Levi-Montalcini and Angeletti, 1968; Yankner and Shooter, 1982). Moreover, when given to neonatal rats, NGF could protect sympathetic neurons from the degenerative effects of systemically applied 6HDA (Aloe et al., 1975). NGF also is present in brain (see review in Whittemore and Seiger, 1987), and early reports raised hopes that the factor might promote the growth o r regeneration of catecholaminergic neurons within CNS, as well as those in the periphery (e.g., Bjorklund and Stenevi, 1971; Tarpy et al., 1975). However, a decade of research now suggests that NGF is not a growth factor for these neurons, either during development or in adulthood. In one study, NGF was not observed to affect central NE after being given intracisternally to intact or 6HDA-lesioned rat pups (Konkol et al., 1978). Other studies have obtained comparable results on central NE and DA systems after giving NGF systemically (Crain and Weigand, 1961), via intracerebral injection (Levi-Montalcini, 1975), or in cell culture (Coyle et al., 1973). There are, however, reasons to anticipate that a NSB-stimulating growth factor will be found. First, while NGF does not affect catecholamines in brain, it does appear to influence central cholinergic nerves (Whitternore and Seiger, 1987). Second, as yet unidentified growthpromoting factors have been reported to appear in response to a variety of CNS lesions (Lindsay, 1979; Norrgren et al., 1980; Crutcher and Collins, 1982; Manthorpe et al., 1983; Nieto-Sampedro et al., 1983; Gage et al., 1984; see review in Nieto-Sampedro and Cotman, 1985). Third, neurons transplanted into brain survive better when the grafting procedure is performed in two stages, the first of which involves creating a cavity (and thus a lesion) (Bjorklund and Stenevi, 1979). Fourth, it has long been known that transplants can exert a trophic action on host brain (e.g., Tello, 1911; Svendgaard et al., 1975; Haun and Cunningham, 1984), and this has been extended to include the ability of adrenal transplants to promote the outgrowth of NSB neurons from the

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host striatum (Bohn et al., 1987). Finally, investigators have identified a molecule, GM1 ganglioside, that appears to be capable of directly stimulating the growth of dopaminergic neurons. Gangliosides. Perhaps the most active area of current investigation involving NSB regeneration pertains to the growth-promoting effects of gangliosides. These complex sphingolipids are a natural constituent of biological membranes. Their hydrophobic end consists of stearic acid and sphingosine and is attached to the outer surface of membranes; their hydrophilic end is composed of carbohydrates such as sialic acid and extends into extracellular fluid. Gangliosides first attracted the attention of neurobiologists when it was discovered that Tay-Sachs disease, a condition in which there is a large increase in brain ganglioside levels, was accompanied by abnormal growth of terminal axons (Purpura and Suzuki, 1976). This effect subsequently was attributed to excess GMI ganglioside (Purpura and Baker, 1977; Roisen et al., 1981). Studies have since suggested that gangliosides may serve to promote neurite outgrowth during development (Morgan and Seifert, 1979), and GMl gariglioside was shown to promote regeneration of a wide variety of transected axons in both the peripheral and central nervous systems (e.g., Ceccarelli et al., 1976; Gorio et al., 1980; Wojcik et al., 1982). In 1984, GM, ganglioside was first shown to promote regeneration of DA neurons after partial transection of the NSB in the adult rat. T h e ganglioside reduced DA cell loss in substantia nigra and increased in uitro high-affinity DA uptake, tyrosine hydroxylase activity, and tyrosine hydroxylase immunoreactivity in striatum. Moreover, the ganglioside treatment decreased DA supersensitivity as measured by the behavioral response to apomorphine and radioligand binding (Sabel et al., 1984; Toffano et al., 1984a,b,c). Although GMI ganglioside originally was reported not to have been effective when NSB lesions were produced by GHDA (Toffano et al., 1984c), other investigators subsequently have found growth-promoting effects of the compound on NE neurons after GHDA administration (Jonsson et al., 1984) and on DA neurons after MPTP treatment (Hadjiconstantinou et al., 1986). There is much to be learned about the effect of ganglioside. The mechanism by which the factor acts is unknown, as is the extent to which it can reverse the severe behavioral deficits associated with large, bilateral NSB injury. Moreover, it is not clear whether the ganglioside can he effective if given well after extensive degeneration has take place, a5 is usually the case when parkinsonism has been diagnosed. Finally, studies have not yet been conducted to determine whether the effect c.

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requires the administration of exogenous ganglioside or can be elicited by promoting the release of ganglioside from endogenous stores.

VIII. Summary and Conclusions

A. How GOODARETHE MODELS? No animal model can be expected to mimic a clinical entity perfectly, and animal models of parkinsonism involving neurotoxins are not an exception to this rule. Indeed, several discrepancies between the disorder and the models deserve note. I n nearly all cases involving neurotoxins, the full brain lesion is produced over a very short time and thus does not reproduce the gradual neurodegenerative process that occurs in parkinsonism. Moreover, patients seldom show a prolonged remission from parkinsonian symptoms, whereas recovery usually occurs in animal models unless NSB destruction is complete. Another distinction is that although the toxin-induced lesions destroy the major pathway involved in the clinical syndrome, they do not produce all of the neuropathology associated with the disease. Finally, the animal model provides only an approximation of the neurological syndrome seen in patients and can never be used to examine the more subtle cognitive and emotional manifestations of the disorder. Despite these concerns, models involving 6HDA and MPTP represent a considerable advantage over previous pharmacological and surgical models of parkinsonism. Lesions can be made that are neurochemically specific; they can be restricted to the brain or even to individual DA projections; and they are permanent. Most important, they result in a collection of neurological impairments that parallel the clinical syndrome to a remarkable degree: (1) Destruction of the NSB in adult animals produces akinesia, rigidity, and sensory neglect. (2) Large lesions are required before these neurological deficits occur. (3) Such deficits d o not occur when comparable lesions are made in very young animals, but instead a quite different syndrome emerges. (4) After exposure to stressors, adult “preclinical” animals with moderate DA depletions show behavioral dysfunctions that are similar to those seen after more extensive lesions. ( 5 ) Neurological deficits are reduced when animals are treated with L-DOPA or apomorphine. (6) Severe deficits also may be reduced temporarily when animals are exposed to an intense, acute challenge.

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B. WHATHAVEWE LEARNED? 1. Animal Models Have Taught Us about Parkinsonism

In the two decades since neurotoxins were introduced for the study of catecholamines, much has been learned from them concerning parkinsonism. First, experimental models utilizing GHDA and MPTP have demonstrated that the cardinal neurological symptoms of parkinsonism can be reproduced by relatively selective damage to the dopaminergic component of the NSB. Moreover, studies with GHDA have shown that NSB lesions lead to secondary changes in diverse brain neurons, thereby suggesting that the comparable changes in these neurons seen in parkinsonism may be secondary to NSB degeneration. Second, it has been shown that the emergence of neurological symptoms only after extensive NSB degeneration is due, at least in part, to pre- and postsynaptic neurochemical changes that occur within the striatum. Specifically, residual DA terminals synthesize and release more DA, and that DA is more effective because it is inactivated less rapidly and acts on more responsive targets. Third, the relation between stress and parkinsonism has been partially clarified. Specifically, it appears that stress normally is accompanied by an increase in striatal DA release, and that the compensatory increase in DA release from residual neurons is accompanied by a reduced tolerance for a variety of challenges. According to this formulation, the functional impairments that emerge during stress reflect the limits of the compensations that have occurred, rather than the limited circumstances under which DA is of functional significance. A fourth area of advance has been the establishment of a possible link between parkinsonism and attention deficit disorder, as suggested by the observation that GHDA administered to neonatal rats does not produce akinesia, but rather hyperactivity. Senescent rats, however, show deficits that resemble those seen in GHDA-lesioned adults. T h e capacity of neonates to withstand the functional effects of near-total destruction of the NSB cannot yet be explained, although it presumably involves some transfer of function from the dopaminergic neurons to other pathways in the brain. Parkinsonian-like deficits that occur with age may be caused by a failure of the synaptic homeostasis that provides the basis for recovery from adult injury. Although this is a lengthy list, there certainly remains much more to learn. We do not know how to diagnose the disease in its preclinical stage; in the best of cases we can treat it symptomatically and then only for a limited period of time; we do not know what causes it; and we can

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neither prevent it nor reverse it. One can be confident, however, that the remarkable progress observed in the past two decades will continue and that animal models of parkinsonism will play an important role in that progress.

2. Parkinsonism Has Taught Us about Basic Neurobiology At the interface between basic and clinical science, information flows in both directions. T h e case examined in this chapter is no exception, and basic neuroscience has profited greatly from the attention it has given to parkinsonism. For example, Parkinson’s disease has served to focus enormous attention on the basal ganglia in general, and on NSB is particular. In consequence, we may know more about the neurobiology of striatum than of any other region of brain. Among the many specific areas of investigation that have been stimulated in this regard are (1) interactions between DA and ACh (resulting directly from the use of atropine in the treatment of parkinsonism), (2) the possibility that DA may act as a neuromodulator with a wide field of influence (resulting in part from the successful use of L-DOPA),and (3) the involvement of the basal ganglia in sensory, motor, and cognitive functions (resulting from the multifaceted syndrome accompanying NSB degeneration). Attempts to develop new forms of pharmacotherapy have stimulated research on DA receptor subtypes, which has provided new drugs for research on dopaminergic systems. The development of additional neurotoxins has been stimulated by the successful use of 6HDA and MPTP in animal models of parkinsonism, and toxins now are available that act specifically on other transmitter systems, including ACh, NE, and 5HT. Moreover, the successful use of L-DOPA in the treatment of parkinsonism has stimulated basic research on the relation between amino acid availability and transmitter synthesis and release. The observation that the symptoms of Parkinson’s disease do not emerge until NSB degeneration is almost complete has led to studies on neuroplasticity within DA systems and, by analogy, within other biogenic amine systems. It also has led to questions concerning development and aging, two natural states of dopaminergic hypoinnervation. Moreover, the observation that parkinsonian patients are impaired during severe challenges has forced a reexamination of the relation between DA and stress. Furthermore, studies of transplantation have raised fundamental questions regarding the growth and development of neurons within the CNS. These studies have stimulated attempts to develop new DAsynthesizing cell lines that will be of value to neurobiologists investiga-

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ting a wide variety of problems and to the search for growth-promoting factors. Finally, and perhaps most important, studies of Parkinson’s disease have demonstrated that interactions among and between basic and clinical scientists can be fruitful and that neurologists, neurosurgeons, psychiatrists, neuroanatomists, neurochemists, electrophysiologists, and molecular biologists can learn together what they cannot learn apart.

Acknowledgments

We thank Elizabeth D. Abercrombie, Theodore W. Berger, Anthony A. Grace, Barry G. Hoffer, and John D. Salamone for helpful discussions concerning portions of this review, and Terri L. Komar and Cheryl L. Serafin for assistance in the preparation of the manuscript. Support has been provided in part by USPHS grants MH-29670, NS-19608, MH-00058, MH-18273, and MH-30915.

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REGULATION OF CHOLINE ACETYLTRANSFERASE By Paul M. Salvoterra and James E. Vaughn Division of Neurosciences Beckman Research Institute of the Ciiy of Hope Duarte, California 91010

I. Introduction A. Purpose B. Historical Background C. ChAT as an Example of Neuron-Specific Gene Expression 11. Methods Used to Study ChAT Expression A. Biochemical Approaches B. Genetic Approaches C. Pharmacological Approaches D. Immunological Approaches 111. Immunocytochemical Location of ChAT in the CNS A. General Description B. Regional Distribution of C h A T C. Functional Implications of ChAT Immunocytochemistry in Vertebrate CNS D. Immunocytochemistry of ChAT in Invertebrate Nervous Systems IV. Development of Cholinergic Neurons A. Descriptive Studies B. Experimental Studies V. Future Directions References

1. Introduction

A. PURPOSE Newer cellular and molecular approaches are being applied to study old problems in nervous system organization, function, and development. In most cases these modern approaches are the driving force behind an explosion of new information. Unfortunately, the formulation of new concepts that can increase our appreciation and understanding of how the brain works in health and disease often lags significantly behind the collection of new data. One molecular component present in the nervous systems of all animals is the neurotransmitter biosynthetic enzyme choline acetyltransferase (ChAT) (EC 2.3.1.6). ChAT has reINTERNA I'IONAL REVIEW OF NEUROBIOLOGY, VOL. 31

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Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cently been studied in a variety of new ways that should allow us to propose a deeper and more rational account of chemical neurotransmission in general. Even more importantly, the features that regulate ChAT expression are beginning to yield to experimental attack and have the potential to serve as model paradigms for understanding the regulation and function of other nervous system-specific molecules. Alterations in the operation of these regulatory control points may be intimately involved in a number of neurodegenerative diseases. The newer descriptive and regulatory studies of' ChAT are often published in diverse sources and come from widely separated disciplines. Because of this they usually are not integrated into the comprehensive context that is essential for understanding their significance. T h e purpose of this review is to try to integrate a broad cross section of studies related to ChAT and cholinergic biology into a more comprehensive neurobiological context. We hope this effort will allow us to start to think more about their larger meaning. We have chosen to review the field from the primary perspectives of neuromorphology and biochemistry since these biases not only reflect our background but in our opinion provide some key insights into the role of ChAT in the brain. From these points of view, therefore, we cover recent results of biochemical, immunological, molecular biological, arid immunocytochemical studies of ChAT and its regulation.

B. HISTORICAL BACKGROUND Historically, acetylcholine was the first neurotransmitter to be described (Loewi, 1921). T h e importance of ChAT for catalyzing acetylcholine biosynthesis was also recognized nearly five decades ago (Nachmansohn and Machado, 1943). As a consequence of this long history, a wealth of information has been accumulated with regard to ChAT and cholinergic neurobiology. Often this information is seemingly contradictory with other studies, and part of our job in writing this review is to attempt to resolve some of these apparent conflicts. T h e reaction catalyzed by ChAT is outlined in Fig. 1. The formal name for ChAT is acetyl-CoA:choline-O-acetyltransferase,and the reaction involves the transfer of an acetyl group from acetyl Coenzyme A to choline. Both substrates for ChAT are fairly ubiquitous compounds present in many different cell types including neurons and nonneuronal cells. Both can participate in a variety of reactions that are not nervous system-specific including lipid biosynthesis, formation of membrane components, and many other reactions of general metabolic importance such as carbohydrate metabolism.

REGULATION OF CHOLINE ACETYLTRANSFERASE

choline

acetylCoA

83

acetylcholine

FIG. 1 . Reaction catalyzed by ChAT.

T h e phylogenetic distribution of ChAT in the nervous systems of all animals indicates a long and distinguished evolutionary history for the production of the protein and the utility of cell-cell communication via acetylcholine. T h e reader is referred to two reviews emphasizing much of the early biochemical work related to ChAT for additional details (Mautner, 1986; Salvaterra, 1987). In addition, a number of earlier, specialized and more comprehensive reviews related to ChAT can also be cited as useful for their excellent discussions of many of the controversial issues in this field and how our ideas about ChAT have evolved over the years (Mautner, 1977; McGeer et al., 1984b; Rossier, 1977b). C. ChAT

AS AN

EXAMPLE OF NEURON-SPECIFIC GENEEXPRESSION

It has become a standard paradigm in recent years to produce monoclonal antibodies o r cDNA clones that identify nervous systemspecific gene products, usually of unidentified function, but with interesting cellular distributions and/or other criteria that recommend these particular molecules for intensive study. Many of these studies have helped shape our current ideas about the exquisite chemical specificity of various cell types within the nervous system. We wish to emphasize in this review that ChAT shares many of the properties of some of the most important molecules identified by the above approaches. ChAT is basically a nervous system-specific gene product. If we can determine what factors are responsible for ChAT expression, we will most certainly learn lessons that can be applied to more general conclusions about nervous system-specific gene expression. ChAT expression is also neuron-specific. Since nonneuronal cell types in the nervous system may share a common lineage with neurons, we may learn something about the major developmental decisions precursor cells make when choosing a particular lineage pathway by studying the regulation of ChAT expression. In addition, ChAT expression is confined to a subset of neurons; thus, the regulatory features of its expression are certain to provide clues about what factors are essential for the selection, maintenance, and plasticity of specific chemical phenotypes for well-defined

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PAUL M. SALVArERRA A N D JAMES E. VAUGHN

neuronal subsets. The major advantage in studying ChAT in the preceding context is that we also know its function. There is now substantial evidence that the major consequence of a particular neuron expressing ChAT will be for that cell to make and use acetylcholine as its primary neurotransmitter. Other uses of acetylcholine for modulatory functions have also been proposed (Mautner, 1986; Rossier, 1977b). In several neurodegenerative disorders, a more or less specific loss of neurons sharing a common neurotransmitter phenotype has been a neuropathological hallmark. The classic example of this type of disorder is Parkinson’s disease. Once this disease was understood as a loss of cells that produce the neurotransmitter dopamine, it was possible to develop pharmacological agents that could alleviate the deficiency. It has even been possible to resupply the brains of patients suffering from parkinsonism with a new supply of dopamine by implanting adrenal cells in their brains (Madrazo et al., 1987). In Alzeheimer’s disease and amyotrophic lateral sclerosis, acetycholine is a specific neurotransmitter phenotype that appears to be destroyed (Bowen et al., 1976; Perry et al., 1977b). Unfortunately, no pharmocologic agents have yet been identified that can relieve the symptoms or slow the progressive degenerative nature of these diseases. If the neurobiology of neurotransmitter phenotype selection and maintenance can be understood at the level of regulation of cell type-specific gene expression, we may be able to propose new therapeutic rationales for treatment or even cures for these tragic diseases. ChAT studies in animals have progressed to a stage at which we can begin to address many of the fundamental questions of neurotransmitter expression and maintenance at the cellular and molecular level. The answers to these questions may serve as valuable intellectual models for designing new therapeutic approaches to a variety of neurodegenerative disorders.

II. Methods Used to Study ChAT Expression

ChAT activity, like that of any enzyme, can be regulated by a number of molecular mechanisms at many different levels. A variety of these potential control points have been investigated using most available techniques and intellectual approaches. In this section, we consider the main outlines of these approaches, including short-term regulation of enzyme activity, regulation of ChAT gene transcription and translation, and intracellular compartmentalization. Another active and productive area of recent research has been the immunocytochemical distribution of ChAT, and we also discuss this information in detail.

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A. BIOCHEMICAL APPROACHES 1. Short-Term Regulation of ChAT Activity T h e two substrates used for the catalytic production of ACh by ChAT are acetyl Coenzyme A and choline. Both of these substrates are ubiquitously distributed in many types of cells and can be used for a variety of other biochemical pathways such as fatty acid biosynthesis, phospholipid production, and intermediates in carbohydrate metabolism. If these substrates encounter active cellular ChAT and ACh synthesis results, they are no longer available for other cellular metabolic pathways. Biosynthesis of ACh by ChAT is thus a committed step in a cellular metabolic pathway, and this step is likely to be regulated at the level of active enzyme in order to ensure that the proper levels of substrates are available for either neurotransmitter synthesis or other uses. In general, the levels of ACh and ChAT seem to be codistributed in nervous systems (Cheney et al., 1976; Hildebrand et al., 1974; Rossier, 1977b; Salvaterra and Foders, 1979), suggesting that the biosynthesis of neurotransmitter is the main function of ChAT in nervous tissue. It should be mentioned, however, that a number of nonneuronal sources for ChAT have been described, including bacteria (Alpert et ad., 1966; White and Cavallito, 1970), plants (Barlow and Dixon, 1973), primate placenta (Hersh et al., l978b; Morris, 1966), and spermatozoa (Bishop et al., 1976). The significance of these nonneuronal sources of ChAT remains unknown, although several interesting proposals have been advanced suggesting a nonsynaptic intercellular communication role for ACh (see, e.g., Mautner, 1986; Rossier, 1977b). During cholinergic synaptic transmission, the ionic composition of a synaptic terminal changes in a dynamic and transient manner as a consequence of the opening or closing of ionic channels in the synaptic membrane (Fatt and Katz, 1951; McCaman and Ono, 1982; Phillis, 1976).In uitro biochemical assays of ChAT activity can be influenced by a number of physiologically important ions (Hersh, 1979; Hersh and Peet, 1978; Rossier, 1977a). Enzyme activity in uiuo may thus be responsive to these ionic changes. The specificity of these in uitro effects on ChAT activity is rather broad and their consequences for an in uiuo cellular regulatory mechanism are difficult to evaluate (Mautner, 1977; Rossier, 1977a). In addition to the ionic responsiveness of ChAT enzyme activity, kinetic studies of ChAT have indicated that the enzyme is subject to strong product inhibition (Morris el al., 1971; Ramasastry and Henderson, 1972). Since the precise concentrations of choline and acetyl CoA are unknown in synaptic terminals, it is impossible to assess the relative importance of product inhibition as an in uiuo regulatory mechanism for

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PAUL M. SALVAI'EKKA AND.IAMES E. V A U G H N

ChAT activity. In any case, however, we know that for the enzyme to be fully functional in vivo it must be in the proper ionic and chemical cellular milieu. Changes in this ionic and chemical milieu can also regulate the enzyme activity in a rapid and precise way. Perhaps a more important feature for intracellular regulation of ChAT involves the spatial distribution of the enzyme within a cholinergic neuron. T h e precise intracellular distribution of ChAT is not yet known with a high degree of certainty. The enzyme protein apparently is made in the cell body of the cholinergic neuron and conveyed to synaptic sites of action in the slowly transported pool of proteins (Frizell et al., 1970; Kasa et al., 1973; Tutek, 1975). All of the studies dealing with ChAT transport have relied on detection of enzyme activity for measuring transported ChAT. If the enzyme is synthesized and transported as a nonenzymatically active precursor it would not have been detected by these studies. Once ChAT arrives at the synaptic endings, its distribution and precise mode of action remains somewhat uncertain. The enzyme activity appears to be largely aqueous-soluble (Fonnum, 1968; Fonnum and Malthe-Sorenssen, 1973), although a small fraction of enzyme activity appears to be associated with particulate structures (Mautner, 1986; Rossier, 1977b). It has been suggested that the particulateassociated ChAT has distinct kinetic properties that may have regulatory importance (Badamchian and Carroll, 1985; Benishin and Carroll, 1982, 1984). It has been argued that some ChAT protein may even be distributed on the extracellular face of synaptic plasma membranes since anti-ChAT antibodies can be used to affinity-purify cholinergic synaptosomes (Docherty et al., 1987). An important question in resolving the regulatory mechanisms of neurotransmitter biosynthesis relates to the subcellular distribution and availability of the substrates. Acetyl CoA is produced by normal metabolic reactions in the mitochondria1 compartment of the cell. It is generally agreed that most intracellular choline is taken up from the extracellular fluid by a high-affinity transport system present in cholinergic nerve endings (Kuhar and Murrin, 1978; Yamamura and Snyder, 1973). I n fact it is often stated that the high-affinity choline transport system, rather than ChAT activity, is the rate-determining step in ACh biosynthesis. The regulation of ACh biosynthesis by various metabolic processes (other than direct regulation of ChAT activity) has been the subject of numerous studies and a particularly comprehensive discussion of this work can be found in TuCek (1985). Much of the synaptic ACh appears to be packaged inside synaptic vesicles that do not contain ChAT and are impermeable to ACh. In an

REGULATION OF CHOLINE ACETYLTRANSFERASE

87

elegant series of studies using Torpedo synaptic vesicles, Parsons and co-workers, as well as a number of other investigators, have demonstrated an ACh uptake process in the synaptic vesicle membrane (Anderson et al., 1981, 1982, 1983a,b; Angel and Michaelson, 1981; Diebler, 1982; Koenigsberger and Parsons, 1980; Rothlein and Parsons, 1982; Toll and Howard, 1980). The uptake of ACh into vesicles is against a concentration gradient and is apparently coupled to a protontranslocating ATPase activity. ChAT could thus function in the cytoplasm to synthesize ACh that in turn would be concentrated into the vesicle compartment of the nerve endings by a separate process. Unfortunately, vesicular transport of ACh has not yet been described for other types of synaptic vesicles, such as those of mammalian brain. The coupling of ACh biosynthesis and vesicular packaging of ACh is still an open question and may be an important potential regulatory point for ChAT activity. 2. Protein Biochemistry A large part of our ignorance with respect to ChAT can be directly attributed to the low abundance of the protein. As a consequence of the minute quantities of the enzyme available from even the best sources, such as Drosophila nervous system, purification of the protein has often required considerable effort. Substantial progress has been made in purifying the enzyme from a number of sources including the nervous systems of Drosophila (Slemmon et al., 1982a), locust (Lutz et al., 1988), nematodes (Rand and Russell, 1985), squid head ganglia (Husain and Mautner, 1973), Torpedo (Brandon and Wu, 1978), chickens (Johnson and Epstein, 1986), rats (Dietz and Salvaterra, 1980; Malthe-Splrenssen et al., 1978; Ryan and McClure, 1979), pigs (Eckenstein et al., 19811, cows (Cozzari and Hartman, 1983; Ryan and McClure, 1979), and human brains and placentas (Hersh et al., 1978b; Peng et al., 1980; Roskowski et al., 1975). The results of various purification protocols have led to substantial controversy over the exact molecular nature of ChAT (see, e.g., Mautner, 1986; Salvaterra, 1987). Most work has resulted in the description of ChAT as a single-subunit globular protein that has no pronounced axial asymmetry and furthermore exists in several different isoelectric forms. T h e molecular mass of ChAT from a variety of species has been estimated to be from 67-75 kDa. In studying the Drosophila protein, we have made several surprising observations relating enzyme activity to SDS gel bands. The enzyme appears to be especially susceptible to proteolytic processing and/or proteolysis during purification. A single peak of activity after gel filtration or sucrose gradient centrifugation (M,. 67,000) results in a

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P A U L M. SALVATERRA A N D JAMES E. V A U GHN

more complicated SDS gel pattern for the polypeptides present in completely purified enzyme preparations (Slemmon et al., 1982a,b). At least three polypeptides are present in completely purified Drosophilu ChAT preparations with molecular masses of 67, 54, and 13 kDa. T h e two smaller peptides appear to be derived from the larger protein by limited proteolysis, although we have not been able to show this conversion directly in vitro. Similar observations have also been reported for the bovine enzyme (Hersh et al., 1984) and locust protein (Lutz et al., 1988). Even more surprising, when we used high-affinity polyclonal antibodies to detect ChAT polypeptides directly in fresh fly head homogenates by Western blotting, we primarily saw a 75-kDa immunoreactive band (Mufioz-Maines et al., 1987). When the same antibodies were used to stain Western blots of even partially purified ChAT, they recognized primarily the lower molecular weight polypeptides present in completely purified ChAT preparations (Muiioz-Maines et al., 1987). Since even the largest ChAT polypeptide we have observed is enzymatically active, proteolytic processing of an inactive precursor to form active enzyme does not seem to be a major regulatory mechanism. Limited proteolysis, however, may regulate the enzyme in other ways. For example, proteolytic processing could result in the modulation of enzyme activity o r even, perhaps, act as a signal for translocating ChAT to different subcellular compartments. T h e turnover rate of ChAT protein in cells would be valuable information for assessing the regulatory dynamics of any signals involved in controlling the expression of the ChAT gene. One estimate has been published from data collected after in vivo pulse labeling and protein purification of the rat brain enzyme (Wenthold and Mahler, 1975). In addition, in vitro studies can be used to estimate the minimal limits of ChAT turnover using protein synthesis inhibitors in cell cultures (Brenneman and Warren, 1983; Kirshner et al., 1986). ChAT seems to be in neither an especially fast nor an especially slow turnover pool from the results of these studies. It would be interesting to know if the turnover rate of ChAT protein varies as a function of the developmental age of the cholinergic neuron, especially during the time when embryonic neurons are making the choice of their neurotransmitter phenotype. In addition, any changes in ChAT turnover rates in older cholinergic neurons may provide insight into the mechanisms involved in neurodegenerative disorders specific to cholinergic neurons. 3. Molecular Biology The techniques of molecular biology are an especially welcome addition to more traditional biochemical approaches used to study

REGULATION OF CHOLINE ACETYLTRANSFERASE

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ChAT. With respect to the biochemical characterization of the enzyme, molecular techniques have now made it possible to obtain the precise molecular structure of ChAT including the complete inferred amino acid sequence. With respect to regulation of ChAT activity in the nervous system, it is always valuable to look directly at the gene being regulated as well as the primary transcript of the gene. The cDNA cloning of both Drosophila (Itoh et al., 1986) and porcine (Berrard et al., 1987) ChAT has been reported. Although neither is a full-length cDNA clone, both have been shown to contain sufficient coding information to produce a fully active enzyme when in vitro-synthesized RNA is injected into Xenopw oocytes (Berrard et al., 1987; McCaman et al., 1988). T h e mRNA size reported for Drosophila ChAT is approximately 5 kbases (Itoh et al., 1986), whereas that for porcine ChAT has been estimated to be about 7 kbases (Berrard et al., 1987). In both cases, the mRNA is substantially larger than that which would be necessary to code for an enzyme the size of ChAT (67-75 kDa). The position of the protein coding region for porcine ChAT cDNA relative to the mRNA sequence has not been reported. We have obtained several overlapping cDNA clones that indicate that the Drosophila ChAT coding sequence is situated approximately 0.8 kbases from the 5' end of the mRNA. There is, thus, a rather large 5' and 3' untranslated region for ChAT mRNA that could play a potential regulatory role. Although no data has been reported for alternatively spliced forms of ChAT mRNA, other neurotransmitter biosynthetic enzymes are thought to undergo this potential regulatory process (Grima et al., 1987). In Drosophila there appears to be only a single mRNA species present at all stages of development at which ChAT mRNA can be detected (L. Carbini and P. M. Salvaterra, unpublished observations). One especially unusual feature of the Drosophila ChAT cDNA clone is the absence of any in-frame, upstream traditional AUG (methionine) codon used for initiating protein translation. The exact position of initiation of protein translation is uncertain, but when RNA derived from Drosophilu ChAT cDNA is translated by Xenopus oocytes, rabbit reticulocyte lysates, o r in several different Escherichia coli expression vectors, a fully active protein is produced with apparently the same size and PI as native Drosphila ChAT (McCaman et al., 1988; H. Sugihara and P. M. Salvaterra, unpublished observations). T h e use of a novel translation initiation codon by Drosophila ChAT may be important for regulating enzyme production in uivo. Porcine ChAT, in contrast, has an AUG start codon that can be unambiguously assigned by comparing the cDNA sequence with the N-terminal amino acid sequence of the purified protein (Berrard et al., 1987; Braun et al., 1987).

YO

PAUL M. SALVATERRA ANDJAMES E. VAUGHN

A comparison of the deduced amino acid sequences for Drosophila and porcine ChAT reveals extensive homology between the two proteins (Berrard et al., 1987; Itoh et al., 1986). ‘The Drosophila ChAT sequence shown in Berrard et al. (1987) contains several typographical errors. Figure 2 shows an alignment of these two amino acid sequences using a corrected Drosophila ChAT sequence. Even though there is global homology between the two proteins, the regions of highest homology appear in the C-terminal third of both proteins. These highly conserved sequences may represent conservation of active site residues essential for enzyme activity or, alternatively, they may be important for other regulatory functions of ChAT. T h e level of homology between Drosophila and pig ChAT is less extensive at the nucleic acid level. An interesting feature we have noted for the deduced Drosophila ChAT amino acid sequence is its homology to acetylcholinesterase, the degradative enzyme for ACh inactivation (Mori et al., 1987). It is apparent that both ChAT and acetylcholinesterase may have evolved from a common ancestral gene. Perhaps these two genes, while clearly under independent regulatory control, may share some common regulatory features. Of course, testing such a possibility must await the identification of the regulatory parts of these genes. In addition, a series of short segmentally analogous peptides are present in both Drosophila ChAT and a rat neuronal nicotinic acetycholine receptor subunit (Mori et al., 1987), and this may indicate the convergent evolution of important functional domains. We have also determined the pattern of ChAT-specific mKNA levels at different stages of Drosophila development. As a fly progresses from early embryonic stages through three larval instars and a pupal stage and finally emerges as an adult, the levels of ChAT mRNA undergo stage-dependent changes. T h e levels of ChAT activity undergo the same pattern of change, but at later developmental times. Our results indicate that the steady-state levels of mRNA correlate quite well with the levels of ChAT activity (L. Carbini and P. M. Salvaterra, unpublished observations), perhaps indicating an important role for transcriptional regulation for ChAT expression during development. It will be important to establish the relative turnover rates of ChAT mRNA and protein FIG.2. Proposed alignment of the amino acid sequences of Drosophzku ChAT (Itoh et al., 1986) and porcine ChAT (Berrard et al., 1987). The respective sequences are indicated by a D or P along the left margin preceding the amino acid residue number. Amino acid residues, indicated by standard one-letter codes, are also numbered along the right of each sequence. Identical amino acids are indicated by a (*) between the sequences; conservative substitutions are indicated by a (I). The horizontal dashes within each sequence represent gaps inserted in order to optimize the amino acid homology.

D

1

D 61 P

IPDPKGANVASNEASTSAAGSGPESAALFSKLRSFSIGSGPNSPQRWSNLRGFLTHRLS

60

NITPSDTGWKDSILSIPKKWLSTAESVDEFGFPDTLPKVPVPALDETMADYIRALEPITT

120

MPILEKTPPKMAAKSPSSEEEPGLPKLPVPPLQQTLATYLRCMQHLVP

48

1

*

I *

I

I*I* *l*I I

1***1***1*1

I I

D121

PAQLERTKELIRQFSAPQG1GAR~QYLW)KREARITGPITTGSTRG~~FAFPLPINSN180

P 49

EEQFRRSQAIVQQFGAPGGLGETLQQKLLERQEQTANWVSEYWLNDMYLNNRLALPVNSS

D181

*

*I

1 1 **I**

**

*I*

*

**(I

I**[**

108

PGIGVPAASLQDRPRRAHFAARLLDGIRSHREMLDSGELPLERALAE-KNQPLCMAQYYRL 240

*[I 1

*

I*** *I *I

I*

*

I I***

***** *** *

11

P109

PAVIFARQHFEDTNDQLRFAANLISGVLSYKALLDSHSIPIDC~GQLSGQPLCMKQ~GL 169

D241

LGSCRRPGVKQDSQFLPSRERLNDEDRHVWICRNQMYCLVLQASDRGKLSESEIASQIL

P170

I*

* **

**I

*

I

11 I

* **

**I*

I

*

*

I

) * * * I l l I*I

300

FSSYRLPGHTQDT-LVAQKSSVMPEPEHVIVACCNQFFVLDWINFR-RLSEGDLFTQLR 227

D301 WLSDAPCLPAKPVPVGLLTAEPRS~ARDR~LQEDERNqRLIETSQWLCLDEPL360

11

*I

I

** *

*I****I(I**

* *

I*

*I[**

I[***

*

P228

KIVRMASNEDERLPPIGLLTSDGRSEWAEARTVLVKDSTNRDSLDMIERCICLVCLDAPG

329

D361

AGNFNARGFTGATPTVHRAGDRDETNEMIHGGGSEYNSGNRWFDKTMQLIICTDGTW

420

P288

GM------------------ELSDTNRALQLLHGGGCSKNGANRWYDKSLQFWGRDGTC 329

D421

GLCYEHSCSEGIAWQLLEKIYKKIEEHPD-EDNGLPQHHLPPPERLEWHVGPQ~LAFAQ 480

P330

GWCEHSPFDGIVLVQCTEHLLKHMVKSSKKMVRADSVSELPAPRRLRWKCSPEIQGLLAS 390

D481

ASKSVDKCIDDLDFYWRYQSYGKTFIKSCQVSPDVYIQLALQLAHYKLYGRLVATYESA

540

P391

SAEKLQQIVKNLDFTWKFDDYGKTFIKQQKCSPDAFIQVALQLAFYRLHGRLVPTYESA

450

D541

* *** **** **I*II***

P451

SIRRFHEGRVDNIRSATPEALHFVKAITDHASAMPDSEKLLL~DAIR-----------498

D601

RELFRCALARQTEVMVRISWAMASTSRCWPARGQYRGHRRDARAVQRRVLQQCSQCNLLS

I

I

*I

II

***

II

I**

*

I** I** *I1 * I )

1 I*** **I I I

*II***I**ll*

Ill****

I I

I

*******

***

**I*

11

** * I

***

*

I*II*

*I* ****I*****

I**J*****

STRRFLHGRVDCIRAASTEALEWAKAMCQGEGANLPLESDREDEEES~VKFSIYSKDHL 600

* **

I

***

*

I*

I *I

I *

I *

II

I1

*I

I**

660

P499

- - - - - - - AQTQYTVMAITGMAIDNHLLGLRELA--REVCKELPEMFTDETYLMSNRFVLS

549

D661

TSQLACSTDSFMGYGPVTPRGYGCSYNPHPEQIVFCVSAF

720

***I1 I I

* **** * *** *** ** *I**I*

* )* I**J(II**IIII*

P550 TSQVPTTMEMFCCYGPWPNGYGACYNPQPESILFCISSFHGCKETSSTKFAKAVEESFI 609 D721 P610

IMRDLLQN

*I *

EMKGLCSLSQSGMGKPLATKEKVTRPSQVHQP

728 641

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PAUL M. SALVATERRA AND JAMES E. VAUGHN

before this point can be firmly established. It will also be necessary to study the distribution of ChAT mRNA by in situ hybridization techniques to cornpare the relative distributions of mRNA to that seen for ChAT protein by immunocytochemical techniques. Such studies are now underway in our laboratories for Drosophzla ChAT (Barber et al., 1989).

B. GENETIC APPROACHES ChAT is one of the few molecules of neurobiological interest that has been studied in well-characterized genetic systems. The advantages of a genetic approach to neurobiology in general and cholinergic systems in particular has been emphasized in an excellent review (Hall and Greenspan, 1979). Drosophilu contains a single gene for ChAT (cha) that has been mapped cytogenetically to the right arm of the third chromosome using segmental aneuploids (Greenspan, 1980). This locus for ChAT has also been confirmed by in situ hybridization to polytene chromosomes using a ChAT cDNA clone (Itoh et ul., 1986). A variety of mutant alleles for ChAT have been isolated and described including presumptive nulls and conditional temperature-sensitive mutants (Greenspan, 1980; Hall et al., 1979). The availability of temperaturesensitive alleles for ChAT has made it possible to perturb enzyme activity specifically in a variety of experimental situations. The results of some of these experiments have identified important new phenotypes resulting from decreased Ch A T activity, including altered electroretinogram traces, motor behavior, and even subtle alterations in courtship behavior (Greenspan, 1980). Temperature-sensitive ChAT mutants have also been used in one study to present evidence for the participation of specific cholinergic synapses in a Drosophila motor pathway (Gorczyca and Hall, 1984). The significance of ChAT activity for the regulation of ACh levels has been a controversial point (TuEek, 1985). Partly, this is a result of the unavailability of' inhibitors with adequate in uivo specificity for ChAT. Using Drosopltila temperature-sensitive ChAT mutants, we have been able to perturb enzyme activity in classical temperature shift experiments. Our results show a strong linear correlation between in vivo ChAT activity and acetylcholine levels when enzyme activity is perturbed over a 50-fold range (Salvaterra and McCaman, 1985). These results seem to argue for a rather direct role of ChAT activity in regulating ACh levels in Drosophila. Interestingly, ChAT activity does not seem to be essential for early

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neurogenesis in Drosophilu. Animals that have a complete homozygous deficiency for the cha locus and are thus unable to produce any enzyme develop apparently normally throughout early embryogenesis. This complete ChAT deficiency does, however, result in the failure of animals to survive into early larval stages (Greenspan, 1980; Gorczyca and Hall, 1987). In one study, the importance of normal ACh levels for maintenance of the structural integrity of the nervous system has been emphasized (Chase and Kankel, 1988). Using temperature-sensitive ChAT alleles, these investigators observed a significant degeneration of the CNS in adult animals that had been exposed to restrictive temperatures for a variable period at an earlier developmental stage. T h e ChAT gene has also been studied in the genetically well-defined soil nematode Cuenorhubditis eleguns (Rand and Russell, 1984, 1985). The cha gene has been mapped to linkage group IV, very close to the position of the unc-17 gene, whose mutant allele leads to uncoordinated movement. Mutant alleles for ChAT have been described that lead to substantial decreases in enzyme activity in homozygous animals and, interestingly, also result in uncoordinated movement, slow growth, and resistance to acetylcholinesterase inhibition. Complementation analysis of the chu and unc- 17 loci indicate a rather complex pattern for these two genes (Rand and Russell, 1984). Interestingly, cha mutants of C. eleguns lead to a decrease in the number of cholinergic synapses, perhaps indicating a role for ACh in development and/or maintenance of structural integrity of the nervous system (Russell and Rand, 1986). Genetic studies of ChAT have already led to a number of surprising conclusions about cholinergic neurobiology. The ability to reduce ChAT activity in uiuo to levels of less than 10% of normal without any overt phenotypic changes may indicate a built-in redundancy of the neural circuits driving the eventually affected behaviors, or perhaps a substantial reservoir of excess neurotransmitter. It should be kept in mind, however, that most temperature-shift experiments are done only under acute conditions of reduced ChAT activity. We do not know the consequences of longer term exposure of animals to lower than normal ChAT activity and presumably ACh levels. It is not surprising that a complete absence of ChAT is lethal, but lethality is observed only after a substantial early development in Drosophila, including much of neurogenesis. The consequences of a complete absence of ChAT activity for early development in other species remains to be determined. Perhaps Drosophilu shows some developmental tolerance to a complete lack of enzyme because in Drosophilu, in contrast to other species, ACh is not used in neurornuscular transmission, an obviously critical function for the organism’s survival.

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C. PHARMACOLOGICAL APPROACHES A classic biochemical approach used to study macromolecular function is the application of specific inhibitors to perturb normal enzyme activity. A wealth of specific inhibitors is available to study most steps in cholinergic neurotransmission. Available reagents include compounds that effectively inhibit different subtypes of ACh receptors, such as snake neurotoxins and various synthetic and natural toxins extracted from plants (eg., curare, atropine, scopolamine, decamethonium, hexamethonium, and quinuclidinyl benzilate). These compounds have allowed the classification of ACh-induced responses into various categories such as nicotinic, muscarinic, and ganglionic, and they also have been adapted for a variety of ligand binding assays to localize and count the various receptors. Hemicolinium-3 has also been shown to interfere with the high-affinity choline uptake system present in cholinergic nerve terminals. The degradative enzyme for ACh, acetylcholinesterase, has been shown to be the primary target for a number of remarkably specific inhibitors used as insecticides and nerve gases. Unfortunately, only a limited number of compounds have been described that inhibit ChAT activity (for review see Mautner, 1986). In general, these reagents lack the requisite specificity to be used in vim with any confidence that only ChAT activity is being inhibited. Napthylvinyl pyridinium ion, a particularly potent inhibitor of ChAT (Mautner, 1977; Ryan and McClure, 1981), has been adapted as an affinity ligand useful for enzyme purification (Cozzari and Hartman, 1980, 1983). In addition this compound can be used to distinguish ChAT activity differentially from that of the mitochondria1 enzyme carnitine acetyltransferase. A number of cholinergic agents including receptor blockers, acetylcholinesterase inhibitors, and stable cholinergic agonists have been shown to decrease the levels of ChAI' activity in primary embryonic Drosophilu cell cultures (Salvaterra et al., 1987). These actions can be interpreted as indicative of a positive feedback loop regulating ChAT expression mediated at some point through ACh action. A number of other pharmacological approaches have been used to identify potentially important steps for regulatory control of ChAT activity. Various second messenger systems have been implicated in influencing the properties of cholinergic cells in general and ChAT activity in particular. The cyclic nucleotide system has been shown to modulate ChAT activity in a number of neuroblastoma and neuronlike cell lines studied in culture. For example, dibutyryl CAMPor forskolin can significantly increase ChAl' activity when added to the culture medium of various types of cells that express ChAT (Prasad and

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Mandel, 1973; Szutowicz et al., 1983; Kirshner et al., 1986). This increase in enzyme activity may involve an increase in synthesis of active enzyme molecules, since it can be blocked by cyclohexamide (Brenneman and Warren, 1983; Kirshner et al., 1986). In addition to cyclic nucleotide modulation of ChAT activity, a number of other pharmacological agents have been shown to affect ChAT activity both in vitro and in vivo. The cellular differentiationinducing compound retinoic acid increases ChAT activity in SH-SY5Y neuroblastoma cells (Adem et al., 1987). Treatment of rats with intraperitoneal injections of 1,25-dihydroxyvitamin Ds also resulted in increased ChAT activity in discrete brain nuclei (Sonnenberg et al., 1986). Estrogen has also been shown both to increase and decrease ChAT activity in a sex-dependent manner when administered to rats (Luine et al., 1986). ChAT activity has also been described as a sexually dimorphic trait in rats, perhaps indicating its regulation by steroid hormones (Loy and Sheldon, 1987). Glucocorticosteroids have been reported to have a negative effect on development of ChAT activity in cultured neurons (Doupe et al., 1985; Black and Patterson, 1980). This effect may be mediated through the ability of corticosteroids to influence expression of adrenergically related neurotransmitter synthetic enzymes positively (Doupe et al., 1985) rather than through any direct negative regulation of ChAT activity. It is interesting to note that the effects of adrenalectomy in rats did not include any increase in ChAT activity in sympathetic ganglia (Hill et al., 1985). Perhaps the in vitro and in vivo effects of corticosteroids are different, o r perhaps the affected cells must be at the proper developmental stage to be influenced by low corticosteroid levels. T h e thyroid hormone T3 (triiodothyronine) has also been shown to increase ChAT activity in cultured CNS neurons (Hefti et al., 1986). In addition, it is known that thyroid deficiency results in reduced brain ChAT activity in experimental animals. Thyroid hormone has also been shown to interact with NGF in a synergistic manner to increase ChAT activity in septa1 neuronal cultures (Hayashi and Patel, 1987). T h e precise mechanism(s) responsible for the ability of any of these agents to modulate ChAT activity remains unknown. I n addition, virtually nothing is known about any synergistic actions o r combinations of these agents. Possible mechanisms could range from a direct stimulation of enzyme activity through posttranslational mechanisms such as changes in protein turnover rates or protein phosphorylation to an increase in ChAT gene transcription, mRNA translation, or changes in RNA turnover rates and could include even very indirect pathways mediated by other cell types (either neurons or nonneuronal cell types).

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Now that specific antibodies and nucleic acid probes are available for measuring ChAT protein and mRNA, the effects of many of these agents need to be reexamined to determine their specific mechanism of action. With regard to modulation of ChAT activity by products in other cell types, there is a considerable amount of information available concerning regulation of ChAT activity by soluble factors or insoluble cell surface molecules produced by other cells, including the normal targets of cholinergic neurons (see below). D. IMMUNOLOGICAL APPROACHES 1. Immunochemical Studies

T h e production of antibodies to ChAT has been one of the more controversial areas of ChAT research in the past (see, e.g., Maunter, 1986; Rossier, 1975; Salvaterra, 1987), largely because of the small amounts of insufficiently characterized protein for use as an antigen. With the availability of monoclonal antibody technology, however, many laboratories have succeeded in producing a number of wellcharacterized ChAT-specific antibodies to only partially purified protein preparations from several different species. Thus far, antibodies are available against Drosophila (Crawford et al., 1982b), locust (Lutz et al., 1988), chicken (Johnson and Epstein, 1986), rat (Crawford et al., 1982a; Ichikawa et al., 1983; Ishida et al., 1983; Levey et al., 1983; Park et al., 1982; Strauss and Nirenberg, 1985), cow (Levey et al., 1981), pig (Eckenstein and Thoenen, 1982), and human (Peng et al., 1980) enzymes. Several of these antibodies cross-react with ChAT from other species, indicating a certain degree of immunological conservation of the protein structure of ChAT over great evolutionary distances (Ichikawa et al., 1983; Johnson and Epstein, 1986; Levey and Wainer, 1982; Yeng et al., 1980; Salvaterra et al., 1986). Many of the antibodies are useful for a number of studies, including immunocytochemical localization of ChAT in tissue sections (see below). Other studies using ChAT monoclonals have shown some to be excellent reagents for recognizing ChAT polypeptides on Western blots (Levey et al., 1981, 1982, 1983; Salvaterra and McCaman, 1985) or as ligands for constructing immunoaffinity columns useful for purifying ChAT (Bruce et al., 1984, 1985; Eckenstein and Thoenen, 1982). We have used anti-Drosophila ChAT monoclonal antibodies to isolate a ChAT cDNA clone from a Drosophila head poly-A(+) RNA library (Itoh et al., 1986). Anti-rat ChAT monoclonal antibodies have been used to map the relative distribution of their epitopes on the surface of the enzyme (Crawford et al., 1982b).

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A number of laboratories have also produced polyclonal antisera specific for ChAT (Bruce et al., 1984, 1985; Cozzari and Hartman, 1980; Eckenstein and Thoenen, 1982; Johnson and Epstein, 1986; Lutz and Tyrer, 1987; Lutz et al., 1988). Polyclonal antibodies often have higher affinity and a broader range of immunoreactive epitopes than the monoclonal antibodies. As mentioned above and stressed in the literature, it is important to assure the monospecificity of polyclonal antibodies when used for certain studies (Rossier, 1975, 1981; Salvaterra, 1987). We have described the production of polyclonal antisera by using a fusion purified from E . cola lysates as antigen in rabbits (Munoz-Maines et al., 1987). These antisera are especially useful for immunostaining Western Blots of Drosophila homogenates.

2. Immunocytochemical Studies a. Rationale and Assumptions. In order to understand many aspects of nervous systems, it is important that experiments provide information at the level of the individual unit of functioning, the neuron. It has been possible for a number of years to study biochemical activity of ChAT, as well as other enzymes, in small, precisely defined samples of the brain. However, one only needs to look at electron micrographs of the CNS to realize that even the most precisely obtained biochemical samples of the CNS are going to be extremely heterogeneous. The diversity of cellular types represented in even a single cubic micrometer of brain makes interpretations of such biochemical assays difficult because of a lack of resolution of individual cells and their processes. A partial solution to this problem became available between the late 1960s and the middle 1970s when molecules involved in the synthesis of neurotransmitter substances were localized within individual neurons (Fuxe et al., 1971; Geffen et al., 1969; Hartman and Udenfriend, 1970) and their synaptic boutons (McLaughlin et al., 1974; Pickel et al., 1976) by immunocytochemistry. This method provides a precise way to label chemical constituents of cells in both light and electron microscopic preparations of nervous tissue. Although antibodies recognize antigenic determinants or epitopes rather than molecules per se, immunocytochemistry can be used to label specific molecules, if the epitopes can be shown to be unique for a given molecule. Immunocytochemical labeling of cell-specific molecules adds greatly to the functional significance of data derived from morphological preparations. In the case of neurotransmitter-synthesizing enzymes such as ChAT, immunocytochemical localization permits the identification of neurons by virtue of the specific substance (e.g., acetylcholine) they use to effect synaptic transmission. This rationale is based on the assumption

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that the localization of a transmitter-synthesizing enzyme within certain neurons means that such cells produce and use a certain substance to mediate synaptic transmission or modulation. Substantial evidence supports this assumption for several transmitter-synthesizing enzymes, including ChAT (see reviews in Kuhar, 1976; McCaman and McCaman, 1976; Storm-Mathisen, 1977). b. Technical Aspects. Although other immunocytochemical techniques, such as irnmunofluorescence, have been used to localize ChAT, the most commonly applied method has been the immunoperoxidase procedure or one of its modifications (Sternberger, 1979; Vaughn et al., 1981). We have relied on several different monoclonal antibodies against ChAT that, taken together, bind with ChAT-specific epitopes in several vertebrate and invertebrate species (see above). We have used these monoclonal antibodies in conjunction with modified peroxidaseantiperoxidase (PAP) protocols that have the following general design:

1. Preparation of tissue: Since ChAT is predominantly a soluble enzyme (Fonnum, 1968; Fonnum and Malthe-SGrenssen, 1973), it is necessary to fix the tissue chemically in order to prevent loss of antigen during subsequent processing in aqueous media. In addition, chemical fixation is presently the best way to achieve the morphological preservation that is necessary for meaningful light and electron microscopic studies. Thus, a fixative must be formulated that produces adequate morphological preservation of nervous tissue, as well as retention of ChAT molecules in the tissue, but does not alter the antigen’s structure in such a way as to abolish antibody-antigen binding. A fixative that has been used successfully for ChAT immunocytochemistry by a number of investigators is a simple forniulation consisting of buffered 4% paraformaldehyde, with small amounts (usually 0.1% or less) of glutaraldehyde being added if tissue is to be analyzed by electron microscopy. Following fixation, specimens are sectioned to provide preparations suitable for microscopic examination and to enhance penetration of immunoreagents. Frozen sections are commonly used for specimens that are to be used exclusively for light microscopy. The freeze-thaw sequence inherent in making frozen sections enhances the permeabilization of cells. However, freeze-thawing has a deleterious effect on tissue preservation. Therefore, sections intended for subsequent processing for electron microscopy are generally cut on a vibratome without prior freezing. 2. Immunocytochemical incubations: Free-floating sections are commonly used in immunocytochemical incubations for ChAT because the surface area for the penetration of immunoreagents is twice that available if sections are fixed to slides prior to incubation. In addition,

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more effective use of expensive antibodies often can be realized by the use of free-floating sections, since more specimens can be processed in smaller quantities of reagents by this method. Basically, a “sandwich” of immunoreagents is built over ChAT molecules in tissue sections by the various incubations that make up the immunocytochemical procedure. The first layer of this sandwich is the antigen-specific step, whereby a complex is formed between ChAT molecules in the tissue section and anti-ChAT primary antibodies in the monoclonal antibody solution. The second layer of the sandwich is formed by complexing a secondary, or bridge, antibody to the anti-ChAT molecules. The bridging antibody is often made in goats against immunoglobulins of the species used to produce the primary antibody. For example, if the primary antibody is an IgG made in mice against ChAT, then the secondary antibody is goat anti-mouse (GAM) IgG antibody. Secondary antibodies that bind exclusively to immunoglobulins of the species used in producing the primary antibody (i.e., species-specific GAMs) have been shown to be useful in reducing or abolishing nonspecific staining (Houser et al., 1984). Following incubation in the secondary antibody, the specimens are placed in a PAP complex produced in the same species as the primary antibody. In the example given above, one would use mouse monoclonal PAP complex to form the third layer of the immunological sandwich over ChAT molecules in the tissue sections. If necessary, the incubations forming the second and third layers of the complex can be repeated to intensify the labeling of ChAT molecules. A final layer of the sandwich is needed to make the sites of the complexes formed by previous layers visible. This is accomplished by incubation in the peroxidase substrate and electron donor reagents, hydrogen peroxide (H202) and 3,3‘-diaminobenzidine . 4HC1 (DAB), followed by treatment with osmium tetroxide to form a chelated osmium-DAB product that indelibly marks the sites of ChAT accumulation within individual neurons and their processes. At this stage in the procedure, the stained sections are either mounted on glass slides for light microscopic examination or can be prepared further for electron microscopy. A standard for control experiments in immunocytochemistry is the use of the primary antibody solution following its absorption against pure antigen. From a practical point of view, however, this is often not possible, since pure antigen is frequently unavailable, especially in studies employing monoclonal antibodies. T h e reason for this is that such antibodies are the immunoreagents of choice for studies of antigens that are extremely difficult to purify to homogeneity (see above). Thus, the nature of the antigen precludes the use of the

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antigen-absorbed antibody control. In these circumstances, an acceptable control antibody solution is one that contains a monoclonal antibody against an epitope that is not present on the antigen of interest; the antibody is prepared in an identical manner to the experimental antibody and its immunoglobulin concentration is the same as that of the experimental antibody solution. Detailed descriptions of ChAT immunocytochemical protocols have been published by a number of investigators (Houser et al., 1983, 1984, 1985; Ingham et aL, 1985; Levey et al., 1983; Satoh et al., 1983; Wainer et ul,, 1984).

111. ImmunocytochemicalLocation of ChAT in the CNS

A. GENERAL DESCRIPTION Immunocytochemical findings from studies of vertebrate CNS indicate that ChAT has a widespread distribution within cholinergic neurons and, indeed, is present in all of the main parts of these cells, including their cell bodies, dendrites, axons, and synaptic terminals (Figs. 3 and 4). Within cell bodies, ChAT appears to be located exclusively in the cytoplasm; no immunoreaction product for this antigen is detected within nuclei (Houser et al., 1983; Sugimoto et al., 1984). In electron micrographs, ChAT-positive reaction product usually exhibits a patchy distribution throughout the soma1 cytoplasm and is associated with prbfiles of smooth endoplasmic reticulum, Golgi apparatus, small vesicular elements, and microtubules (Houser et al., 1983; Sugimoto et al., 1984). In dendrites, ChAT immunoreactivity is also associated with these same organelles with the exception of the Golgi apparatus, but its most common distribution is around microtubules. Immunoreactivity of ChAT has been observed in all major parts of axons, namely, axon initial segments, myelinated and unmyelinated intermediate segments, and preterminal axons (see, e.g., Phelps et al., 1985; Phelps and Vaughn, 1986). In all of these locations, reaction product seems to be most commonly associated with microtubules, although it can be located on the rims of small cisternae and vesiclelike structures, as well as being apparently “free” in the axoplasm. I n addition, ChAT-positive immunoperoxidase product has been shown to be concentrated in the presynaptic terminals of cholinergic neurons (Fig. 4) (Houser et al., 1983; Wainer et al., 1984). These structures are generally relatively small boutons measuring 0.5- 1.5 pm in diameter (Houser et al., 1985; Phelps and Vaughn, 1986; Wainer et

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'd FIG.3. A photomicrograph (A) and camera lucida drawing (B) of ChAT-positive cells in the neostriatum illustrating that immunoreaction product is distributed throughout the neurons. (A) Three immunoreactive somata of different shapes give rise to dendrites that branch several times within the field (arrowheads). (B) ChAT-positive neurons displaying extensive dendrites (arrows) that exhibit occasional varicosities (arrowhead). A probable axon (a) originates from a primary dendrite and branches within the section. Scale bars, 50 fim. (From Phelps ed al., 1985, with permission of Alan R. Liss, Jnc.)

al., 1984). T h e major organelle of such terminals with which ChAT is associated is the synaptic vesicle. T h e characteristic of synaptic vesicles associated with ChAT immunoreactivity are often obscured by the immunoperoxidase product itself, but when vesicles are apparent they

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FIG. 4. Electron micrographs illustrating a variety of synapses formed by ChA-1'positive presynaptic terminals in the ventral striatum. I n (A) an immunoreactive terminal forms a synaptic contact (arrow) with an unlabeled dendritic shaft. Two ChAT-negative terminals also contact this dendrite (arrowheads). The immunoreactive terminal shown in (B) forms an asymmetrical synapse (arrow) with an unlabeled dendrite, whereas the one in (C) makes a symmetrical synaptic contact (arrow) with a ChAT-negative dendritic spine that also forms an asymmetric synapse with a larger, unlabeled terminal. Scale bar for (A-C), 0.5 Fm. (From Phelps and Vaughn, 1986, with permission of Chapman and Hall, Ltd.)

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commonly are found to be clear, pleomorphic or rounded vesicles (Carlsen and Heimer, 1986; Dolabela de Lima et al., 1985; Houser et al., 1983; Phelps et al., 1985; Phelps and Vaughn, 1986; Wainer et al., 1984). Different ChAT-positive presynaptic terminals have been observed to establish either symmetric or asymmetric synapticjunctions with their postsynaptic targets (Houser et al., 1983, 1985; Wainer et al., 1984). However, symmetric contacts are by far the most common type formed by cholinergic terminals in a variety of CNS regions (Anderson et al., 1986; Bialowas and Frotscher, 1987; Carlsen and Heimer, 1986; Clarke, 1985; Dolabela de Lima and Singer, 1986; Dolabela de Lima et d,1985; Houser et al., 1985; Leranth and Frotscher, 1987; Phelps et al., 1985; Phelps and Vaughn, 1986; Wainer et al., 1984). For example, most ChAT-positive terminals observed in rat cerebral cortex form symmetric synaptic contacts exhibiting relatively thin postsynaptic densities, whereas rare ChAT-positive terminals form asymmetric synapses that are characterized by thickened postsynaptic densities as well as by subsynaptic dense bodies (Houser et al., 1985). However, in the hippocampus, the junctional characteristics formed by ChAT-positive terminals appear to vary with the type of postsynaptic element. Thus, synaptic contacts of ChAT-positive terminals with dendritic spines are asymmetric, those with dendritic shafts may be either symmetric o r asymmetric, and those with cell bodies are symmetric (Frotscher and LCrAnth, 1985; Frotscher and Leranth, 1986). It is interesting to note that transplanted cholinergic neurons form the same kinds of synapses in host hippocampus as those in intact hippocampus, but in addition appear to form axoaxonic contacts (Anderson et al., 1986). The most frequent type of postsynaptic target for ChAT-positive synaptic terminals appears to be the dendritic shafts of noncholinergic neurons. In the ventral striatum, for instance, approximately 60% of the observed ChAT-positive terminals make synaptic contacts with ChATnegative dendritic shafts. A relatively substantial proportion (26%) of ChAT-positive terminals in this region establish synaptic contacts with ChAT-negative somata, but only a small percentage (13%) with nonimmunoreactive dendritic spines (Phelps and Vaughn, 1986). Similar types of postsynaptic elements of Ch AT-positive synapses have been reported in other brain regions, although the frequencies seem to vary somewhat depending on the particular region (Bialowas and Frotscher, 1987; Carlsen and Heimer, 1986; Clarke, 1985; Dolabela de Lima and Singer, 1986; Dolobela de Lima et al., 1985; Frotscher and Leranth, 1985; Izzo and Bolam, 1988). In addition, synapses between ChAT-positive terminals and immunonegative axon initial segments have been observed in the neostriatum (Phelps et al., 1985).

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B. REGIONAL DISTRIBUTION OF CHAT Classifications of cholinergic systems in the vertebrate CNS have traditionally focused on groups of cholinergic cells that are projection neurons with long axons (Mesulam et al., 1983a,b; Woolf et al., 1984; Woolf and Butcher, 1986). Immunocytochemistry of ChAT has confirmed the cholinergic nature of such neurons but also has identified new cholinergic neuronal populations that appear to be short-axon, local circuit neurons in several CNS regions. Given these new observations and the number of projection system reviews available in the literature, it would seem useful to discuss cholinergic systems from another perspective, namely, whether they are located in regions of the CNS where (1) both intrinsic and projection cholinergic neurons may coexist; (2) cholinergic neurons are intrinsic local circuit neurons; (3) cholinergic neurons are projection neurons; or (4) cholinergic systems are largely or exclusively represented by ChAT-positive fibers and terminals. 1. ChAT-Positive Intrinsic und Projection Neurons in Spinal Cord

During the early phase of ChAT immunocytochemistry, a number of investigators localized immunoreaction product in the projection, o r motor, neurons of the spinal cord. Indeed, this demonstration was considered almost mandatory in order to provide a positive antibody control, since motor neurons are well-established cholinergic neurons in the vertebrate CNS (for reviews, see Kuhar, 1976; McCaman and McCaman, 1976; Storm-Mathisen, 1977). In addition to large a-motor neurons, smaller cells of the motor pools, putative y-motor neurons, contain ChAT-positive reaction product, as do preganglionic projection cells of the autonomic nervous system (Houser et al., 1983; Barber et al., 1984). Thus, there are different varieties of projection neurons in the spinal cord and, numerically at least, they are the main cholinergic cells in the spinal cord. However, there are at least three other varieties of ChAT-positive neurons in this region of the CNS, and their morphological characteristics suggest that they may be local circuit neurons (Barber et al., 1984; Phelps et al., 1984). Such cells will be described below following an account of the projection neurons of the spinal cord. a. Spinal Projection Neurons. ChAT immunoreaction product is present in the somata, dendrites, axons, and neuromuscular junctions of somatic motor neurons (Houser et al., 1983). These cells are arranged in distinct columns and subcolumns that are characterized by longitudinal and transverse bundles of ChAT-positive dendrites (Barber et al., 1984). While the functional significance of these dendritic bundles is not yet

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clear, it has been suggested that they may act to synchronize the action of specific groups of motor neurons (Schoenen, 1982). Another functionally intriguing observation regarding spinal motor neurons is that ChAT-positive axon terminals form synaptic contacts with large motor neurons (Houser et al., 1983) but do not appear to do so with small motor neurons (Barber et al., 1984). Since at least some of the ChATpositive terminals in the motor neuropil appear to be derived from collaterals of motor axons (Barber et al., 1984), it may be that small, putative y-motor neurons lack the recurrent collateral innervation exhibited by a-motor neurons. The preganglionic sympathetic motor neurons of the thoracolumbar spinal cord can be divided into four subgroups of cells, all of which are ChAT-positive (Barber et al., 1984). T h e main sympathetic preganglionic neurons are the principal intermediolateral cells located in the lateral horn. In addition there are smaller groups of preganglionic cells located just dorsal to the central canal (central autonomic neurons), in the intermediate gray (intercalated cells), and in the lateral funiculus (funicular intermediolateral cells) (Petras and Cummings, 1972; Petras and Faden, 1978; Chung et al., 1975). One of the most striking features of the preganglionic sympathetic system is the ladderlike arrangement of ChAT-positive dendrites formed by the constituent neurons (Fig. 5) (Barber et al., 1984). The ChAT-positive preganglionic neurons of the spinal parasympathetic system are located in the lumbosacral spinal cord, and they appear to form a single group of projection cells, the intermediolateral sacral neurons. These cells do not form the extremely well-organized ladder arrangement of dendritic bundles that is characteristic of their sympathetic counterparts. b. Spinal Local Circuit Neurons. In addition to ChAT-positive preganglionic autonomic neurons, the intermediate and central spinal gray matter contains two other groups of ChAT-positive cells. In contrast to preganglionic neurons, however, they are present at both autonomic and nonautonomic spinal levels. It is this distinction, plus differences in morphology and location, that allow these cells to be classified as separate ChAT-positive cell groups (for details see Barber et al., 1984; Phelps et al., 1984). T h e most prominent of these two cell groups are called partition neuroru because they are located in such a way as to divide, or partition, the ventral from the dorsal horn (Phelps et al., 1984; Barber et al., 1984). These cells form a network across the intermediate spinal region, spreading from the lateral gray matter to the central canal. Little is known about the function of partition cells, but their relatively large size, location within lamina VII, and course of iheir axons suggest that they may be propriospinal neurons that participate in ascending and

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FIG.5. Drawing made from ChAT irnrnunocytochernical sections of thoracic spinal coi-d. The dendrites of three subgroups of sympathetic preganglionic neurons (CA, IC, ILp) form a ladder-like arrangement in the intermediate gray matter. The “ladder rungs” are composed of characteristic rnetameric bundles of transverse dendrites from these cells. Illustration courtesy of Robert P. Barber.

descending intersegmental circuitry (see Phelps et al., 1984, for more discussion). T h e second group of cholinergic neurons have been called central canal cluster cells because they are aggregated around the central canal. These cells appear to contribute to small, longitudinal fascicles of varicose ChAT-positive processes that course rostrally and caudally in the cord near the ependymal layer. Although the function(s) of central canal cluster neurons is presently unknown, there is some circumstantial evidence that they may serve as local circuit target cells for modalityspecific cutaneous afferents (see Barber et al., 1984, for further discussion). Another group of ChAT-positive spinal neurons is located primarily in laminae III-V of the dorsal horn (Barber et al., 1984). Many of these small, dorsal horn cholinergic neurons send their dendrites dorsally to course longitudinally through lamina 111, where they intermingle with

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the ChAT-positive synaptic terminals that are concentrated in this lamina. T h e cholinergic terminals in this region form symmetric synaptic contacts, mainly with ChAT-negative dendrites (Barber et al., 1984; Houser et al., 1983). In addition, ChAT-positive terminal-like structures form a somewhat less dense band in lamina I, as well as being more sparsely scattered throughout the dorsal horn (Barber et al., 1984). The derivation(s) of the dense concentration of cholinergic terminals in lamina 111, or for that matter throughout the entire dorsal horn, has not yet been determined, but intrinsic spinal sources are suspected for the following reasons: 1. ChAT-positive elements have not been observed in the dorsal root ganglia or dorsal roots, thereby excluding primary and visceral afferents as a source of dorsal horn cholinergic terminals. 2. Supraspinal sources also can be excluded by the fact that spinal transections at rostra1 levels do not significantly reduce ChAT biochemical activity levels caudal to the lesions (Kanazawa et al., 1979). 3. There is a temporal coincidence of the developmental occurrence of ChAT-positive neurons and terminallike structures in the dorsal horn (Phelps et al., 1984). Thus, it is reasonable to suspect that the small ChAT-positive neurons of the dorsal horn are local circuit, o r perhaps propriospinal, cells that give rise to putative cholinergic synaptic terminals in laminae I and 111, as well as in the rest of the dorsal horn. The spinal cord, therefore, seems to represent a region of the CNS that contains both cholinergic projection cells and local circuit neurons. It certainly is a region that displays a much more diverse and widespread distribution of cholinergic neurons than was known prior to the advent of ChAT immunocytochemistry.

2 . Regzom Containing ChAT-Positive Intrinsic Neurons a. Cerebral Cortex. The neocortex of rat cerebrum is a region that contains small ChAT-positive neurons (Eckenstein and Thoenen, 1983; Houser et al., 1983; Eckenstein and Baughman, 1984; Houser et al., 1985). These cells are distributed throughout cortical layers II-VI but are most numerous in layers 11-111 (e.g., Houser et al., 1985). The great majority of these neurons exhibit a bipolar dendritic pattern, although there is an occasional cell with a multipolar form. It is clear, however, that all of these cells, bipolar and multipolar alike, display the morphological characteristics and synaptic inputs of nonpyramidal neurons (see Houser et al., 1985, for details). Putative cholinergic neurons similar to those in neocortex have also

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been described in limbic cerebral cortices including the hippocampal formation (Frotscher et al., 1986; Frotscher and LCranth, 1985; Matthews et al., 1983, 1987; Wainer et al., 1985), pyriform cortex, and various areas of periallocortex and proisocortex (Brady and Vaughn, 1988). In addition to this general distribution throughout rat cerebral cortex, ChAT-positive nonpyramidal neurons have been described in neocortex of cats (Stichel et al., 1987) and of fetal monkeys (Hendry et al., 1987). Furthermore, such neurons have been revealed using a number of different monoclonal and polyclonal antibodies, thus providing evidence that the antigen detected in these cells is indeed ChAT (see Houser et al., 1985, for discussion). The fact that the small ChAT-positive neurons of cerebral cortex exhibit the characteristics of nonpyramidal cells argues strongly that they are intrinsic, or local circuit, neurons because as far as is known, pyramidal neurons are the sole type of projection cell for this brain region (e.g., Jones and Wise, 1977). Thus, a dual cholinergic system appears to exist in cortex, since, in addition to the just-described intrinsic source of axon terminals, there are well-known extrinsic sources of cholinergic innervation to this brain area (Johnston et al., 1979, 1981; Mesulam et al., 1983a,b; see also below). Immunocytochemistry has shown that ChAT-positive axon terminals are distributed rather homogenously throughout all layers in motor areas of neocortex but that they exhibit laminar patterns in sensory neocortex, as well as in certain areas of limbic cortex (Brady and Vaughn, 1988; Dolabela de Lima and Singer, 1986; Houser et al., 1985; Lysakowski et al., 1986; Matthews et al., 1987; Parnavelas et al., 1986; Stichel and Singer, 1987). As mentioned above, electron microscopic studies show that ChAT-containing presynaptic terminals form both symmetric and asymmetric synapses in neocortex (Houser, 1989; Houser et al., 1985; Parnavelas et al., 1986) and in limbic cortex (e.g., hippocampus: Frotscher and LCrPnth, 1985, 1986), although symmetric junctions are predominant in both cases. Dendritic shafts are the main postsynaptic elements of ChAT-positive synapses in neocortex and hippocampus, but some dendritic spines also receive such synapses. I n neocortex, ChAT-positive presynaptic terminals form contacts with both pyramidal and nonpyramidal neurons (Clarke and Dunnett, 1986; Dolabela de Lima and Singer, 1986; Houser, 1989; Houser el al., 1985). Those on pyramidal cells contact apical and basilar dendrites but have not been observed upon somata. In contrast, ChAT-positive terminals commonly form axosomatic synapses with nonpyramidal cells. In the hippocampal formation, Ch AT-positive terminals are the presynaptic elements of synaptic junctions located on granule neurons

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and commissural cells in the hilus of the dentate gyrus, as well as on pyramidal neurons of the hippocampus proper (Frotscher and LCranth, 1985, 1986; Leranth and Frotscher, 1987). The dentate granule cells receive symmetric synaptic contacts from ChAT-positive axon terminals on both their cell bodies and dendritic shafts. The dendritic spines of granule cells, however, exhibit a somewhat more complex innervation pattern, with two types of ChAT-positive terminals establishing axospinous synapses. Some of these immunoreactive terminals form symmetric synapses with the stalks of large, complex spines, whereas others establish asymmetric junctions with the heads of small spines (Frotscher and Lerinth, 1986). Nonpyramidal neurons in the hilus of the dentate gyrus that are immunoreactive for glutamic acid decarboxylase or somatostatin receive both axosomatic and axodendritic symmetric synaptic contacts from ChAT-positive axon terminals. At least some of these postsynaptic neurons appear to be commissural cells projecting their axons to the hilus of the contralateral dentate gyrus (LCranth and Frotscher, 1987). A cholinergic innervation of neurons in the hippocampus proper also has been described. Synapses formed by ChAT-positive terminals are present upon the somata, dendritic shafts, and dendritic spines of the principal projection cells of this region, the hippocampal pyramidal neurons (Frotscher and Leranth, 1985). 'The axospinous synapses are asymmetric junctions, whereas the axosomatic contacts are symmetric. Dendritic shafts of pyramidal neurons form both symmetric and asymmetric synapses with ChAT-positive boutons. Judging from the distribution of ChAT-positive terminallike structures in light microscopic preparations (Brady and Vaughn, 1988; Houser et al., 1983; Matthews et al., 1983, 1987), it would seem likely that the basilar dendrites of hippocampal pyramids might be more heavily innervated by cholinergic inputs than their apical dendrites since the infrapyramidal band of ChATpositive staining through which basilar dendrites course is more prominent than the suprapyramidal band. Until recently, it was thought that the cholinergic innervation of the hippocampal formation was derived entirely from extrinsic sources in the basal forebrain (see below). As was mentioned above, however, ChAT immunocytochemical studies have shown that the hippocampal formation also contains endogenous ChAT-positive neurons whose nonpyramidal nature indicates that they may be local circuit neurons (Frotscher and Leranth, 1985; Houser et al., 1983; Matthews et al., 1983, 1987; Wainer et al., 1985). Therefore, it is likely that some of the cholinergic innervation of the hippocampal formation is derived from these endogenous cells. However, the number of such cells is small, and

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lesions to extrinsic hippocampal afferents cause a major reduction in both ChAT biochemical activity (Fibiger, 1982, Storm-Mathisen, 1977) and in ChAT immunoreactivity (Matthews et al., 1987). Indeed, the distinctive laminar pattern of ChAT-positive reaction product in the hippocampal formation is completely abolished by lesions of the septal area (Matthews et al., 1987). The few ChAT-positive terminallike structures remaining after septa1 lesions are usually located in the vicinity of ChAT-positive neuronal somata. These somata are located primarily in stratum lacunosum-moleculare in regio superior of hippocampus and to a lesser extent in all laminae of the dentate gyrus (Frotscher and Leranth, 1985; Matthews et al., 1987). ‘Ihus, innervation from intrinsic cholinergic neurons in the hippocampal formation would appear to be restricted to subregions surrounding the somata of these cells and therefore probably would not have the same widespread effects throughout this cortical area as would the more numerous and generally distributed afferents from the basal forebrain. Despite the fact that both have intrinsic and extrinsic cholinergic components, there appear to be some interesting differences between the cholinergic systems in neocortex and certain areas of limbic cortex. First, limbic cortical areas generally exhibit more ChAT immunoreactivity than those of neocortex (Brady and Vaughn, 1988). For example, pyriform and retrosplenial cortex, respectively, exhibit about 55 and 30% more ChAT-immunoreactive product than does neocortex. A second difference between these two classes of cerebral cortex is that ChAT-positive terminal fields are generally distributed rather homogenously throughout neocortex, whereas they exhibit laminar patterns in certain areas of limbic cortex. The most extreme example of a laminar distribution is found in the hippocampal formation, as discussed briefly above. In this cortical area, ChAT-positive terminals are concentrated at the edges of the layers containing projection neurons, that is, the hippocampal pyramids and the granule cells. Other limbic areas showing pronounced laminar arrangements of ChAT-positive immunoreactivity are entorhinal and retrosplenial cortices (Brady and Vaughn, 1988). Entorhinal cortex displays two intense bands of ChAT immunoreactivity, one located in layers 1-11 and the other in layer IV. Neurons in layer I1 (and 111) of this cortex project to the hippocampal formation (Steward, 1976), whereas cells in layer IV (and V) send their axons to other cortical areas and to basal forebrain (Van Hoesen and Yandya, 1975; Kohler and Eriksson, 1981). Thus, in certain limbic areas (e.g., hippocampal formation and entorhinal cortex), cholinergic terminals are concentrated in regions where they could form numerous synapses with projection neuron somata and their proximal dendritic trees. I n

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contrast the cholinergic innervation of neocortex appears to be more diffusely distributed throughout the entire cortical thickness with little or no special focusing upon regions of projection neurons. Thus, it may be that necortical cholinergic systems are involved with rather global influences, perhaps mediated both across (Lamour et al., 1984, Valverde, 1986) and within (Eckenstein and Baughman, 1984) cortical columns, whereas cholinergic effects in limbic areas, such as the hippocampal cortex, are more focused on projection neurons. b. Amygdaloid Complex. The basolateral amygdaloid nucleus contains the highest concentration of ChAT immunoreaction product in the deep amygdaloid nuclei (Hellendall et al., 1986). Consistent with the case presented by other investigators for the “quasi-cortical structure” of the basolateral amygdala (Carlsen and Heimer, 1986), this brain region, like neo- and limbic cortices, seems to exhibit a dual cholinergic innervation. In addition to an extrinsic system originating in the basal forebrain (Carlsen et al., 1985; see also below), the basolateral amygdala contains small ChAT-positive neurons that appear to be intrinsic local circuit neurons (Carlsen and Heimer, 1986; C. R. Houser, unpublished observations), displaying many of the same characteristics as those of ChATpositive intrinsic neurons in the cerebral cortex (Eckenstein and Baughman, 1984; Houser et al., 1983, 1985; Levey et al., 1984). Furthermore, the structural characteristic of the ChAT-positive axon terminals of cerebral cortex and basolateral amygdala are quite similar (cf. Carlsen and Heimer, 1986; Houser, 1988; Houser et al., 1985). Another part of the amygdaloid complex that displays both intense terminal staining (Brennan et al., 1986; Hellendall et al., 1986) and a few ChAT-positive cell bodies is the nucleus of the lateral olfactory tract. ChAT immunoreaction product in this nucleus is most concentrated in layer I1 (Hellendall et al., 1986). Neuronal somata displaying ChATpositive staining are not numerous, although they are scattered throughout this nucleus (P. E. Phelps, unpublished observations). c. Neostriatum (Dorsal Striatum). Another brain region that contains ChAT-positive local circuit neurons is the neostriatum, o r caudateputamen (Armstrong et al., 1983; Bolam et al., 1984; Mesulam et al., 1984; Phelps et al., 1985; Sofroniew et al., 1982; Satoh and Fibiger, 1985; Satoh et al., 1983). I n contrast to this class of neuron, generally, the intrinsic cells of the neostriatum are relatively large neurons, whose somata measure an average of 27 x 13 pm in their major and minor diameters, respectively (Phelps et al., 1985). Although a given section of the caudate-putamen may exhibit as many as 100 or more ChATpositive neurons, such cells make u p slightly less than 2% of the total neuronal population of the neostriatum (Phelps et al., 1985). However,

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the large size of these neurons and the strong possibility that their axons and axon terminals are confined to the neostriaturn (see below) indicate that they probably occupy a proportionally larger volume of this brain region than would be suspected on the basis of their numbers alone. This prediction is consistent with observations that the neostriatum contains very high biochemical levels of ChAT and ACh (Cheney et al., 1975; Hoover et al., 1978), as well as intense immunoreactivity for ChAT (Phelps et al., 1985). T h e evidence that these large cholinergic neurons are local circuit neurons comes in part from the fact that their morphological characteristics do not coincide with those of the known projection neuron of this region, the rnedium-sized spiny neuron (see, e.g., Grofova, 1975; Somogyi and Smith, 1979). Furthermore, morphological (see, e.g., Chang and Kitai, 1982) and intracellular injection (Wilson et al., 1983) studies have shown that cells with characteristics identical to those of ChAT-positive neurons have extensive local axon collaterals. These axon collaterals form symmetrical synaptic contacts within the neostriatum, as do ChAT-positive presynaptic elements (Phelps et al., 1985; lzzo and Bolam, 1988). Although it cannot be completely ruled out that some of the large cholinergic neurons of the neostriatum are projection cells (Jayaraman, 1980; Parent et al., 1981), the bulk of evidence now available indicates that they are, predominantly at least, intrinsic neurons. As described above, the axon terminals of ChAT-positive neostriatal neurons form symmetric synaptic junctions. The postsynaptic targets of such terminals are primarily dendritic shafts of noncholinergic neurons. Moreover, they have been shown to innervate the somata, dendrites and axon initial segments of rnedium-sized spiny neurons (Izzo and Bolam, 1988; Phelps et al., 1985). Previous studies have provided evidence that such neurons contain glutamic acid decarboxylase (GAD) (Fonnum et al., 1978; Oertel and Mugnaini, 1984; Ribak et al., 1979) and, as stated earlier, are neurons that project their axons from the neostriatum to other regions of the CNS. Thus, GABAergic neostriatal projection cells appear to receive a cholinergic innervation from intrinsic neurons, and this innervation is strategically placed upon the rnedium-sized spiny neurons to exert a potent influence on their output. Since cholinergic effects in the neostriatum are thought to be mostly excitatory in nature (Bernardi et al., 1976; Misgeld et al., 1984), it would appear that intrinsic cholinergic neurons function to increase the firing of inhibitory neostriatal inputs to the globus pallidus and substantia nigra, as well as to excite the intrinsic inhibitory synapses formed by the numerous axon collaterals of medium-sized spiny neurons.

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d. Ventral Striatum. The ventral striatum has been defined as that part of the forebrain that is ventral and medial to the caudate-putamen, and it is composed of the nucleus accumbens, the substriatal grey, the striatal cell bridges, and the olfactory tubercle (Heimer and Wilson, 1975). Biochemical measurements of ACh and ChAT have shown that these cholinergic markers are as abundant in the ventral as the dorsal striatum, and in some instances the ventral striatal values even exceed the dorsal striatal values (Fonnum et al., 1977; Hoover et al., 1978; Palkovits et al., 1974; Walaas and Fonnum, 1979). Similarly, immunocytochemical studies have demonstrated large Ch AT-positive neurons and numerous ChAT-containing axon terminals within all regions of the ventral striatum (Phelps and Vaughn, 1986; Satoh and Fibiger, 1985; Satoh et al., 1983). Although it is uncertain whether all of the ChATpositive neurons of this brain region are local circuit neurons, there is evidence favoring the intrinsic nature of these cells in certain ventral striatal areas. For example, hemitransections at the level of the globus pallidus resulted in no significant change in ChAT activity in nucleus accumbens, olfactory tubercle, o r neostriatum (Fonnum et al., 1977), indicating that the cholinergic fibers in these areas are not derived from ascending cholinergic systems (see below). Moreover, kainic acid destruction of intrinsic neurons of nucleus accumbens led to a 75% decrease in ChAT activity, a result favoring the notion of an intrinsic origin of ChAT in this part of the ventral striatum (Walaas and Fonnum, 1979). Finally, a correlation exists between ventral striatal areas containing high concentrations of ChAT-positive somata and those exhibiting densely immunoreactive neuropii (Phelps and Vaughn, 1986), and these observations lend support to the interpretation that many of the cholinergic terminals in the ventral striatum are derived from intrinsic neurons (Butcher and Woolf, 1982). Neurons that are immunoreactive for ChAT are located in the nucleus accumbens, substriatal gray, and olfactory tubercle. However, such cells are not evenly dispersed throughout these areas. For example, in the olfactory tubercle, they are most numerous in the multiform (or polymorphic) layer, whereas the molecular (or plexiform) layer contains very few ChAT-positive neurons. Similarly, parts of the remaining subdivisions of the ventral striatum that exhibit concentrated immunoreactive fibers and terminals contain the largest numbers of these cells (Phelps and Vaughn, 1986). Despite this heterogeneous distribution, the morphological appearance of ChAT-positive neurons in all ventral striatal subdivisions is virtually the same and bears a strong resemblance to that of the cholinergic cells of the dorsal striatum (Armstrong et al., 1983; Phelps et al., 1985; Phelps and Vaughn, 1986), although those in

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the ventral striatum are, on the average, somewhat smaller than those of the dorsal striatum (Mesulam et al., 1984; Phelps and Vaughn, 1986). T h e cholinergic neurons of the substriatal grey region of the ventral striatum receive only a few axosomatic synapses, and these contacts may exhibit either symmetric or asymmetric paramembranous densities. I n comparison, however, cholinergic dendrites are more heavily innervated than the cell bodies from which they arise, and secondary dendrites have more numerous synaptic contacts than primary shafts. As is the case for axosomatic junctions, both symmetric and asymmetric contacts occur on ChAT-labeled dendrites, but an additional type of asymmetrical synapse with pronounced subsynaptic dense bodies is observed on dendrites. ChAT-positive presynaptic elements also have been observed in apposition to the cholinergic neurons of this brain region, where they form possible synaptic relationships (Phelps and Vaughn, 1986). As in many parts of the CNS, the ChAT-positive axon terminals in the ventral striatum are small, with an average profile diameter being 0.8 pm. These boutons contain pleomorphic synaptic vesicles and in about 96% of the cases observed they establish symmetric synaptic junctions (Phelps and Vaughn, 1986). In the substriatal gray, ChATpositive boutons form contacts with several different kinds of postsynaptic elements. Most commonly, they terminate upon unlabeled dendritic shafts (-61%), although a small percentage (- 13%)innervate unlabeled dendritic spines. Axosomatic synapses formed by ChAT-positive terminals are relatively common (26%)in this brain region, and many of the postsynaptic somata belong to medium-sized ChAT-negative cells that may represent the GABAergic projection neurons of the ventral striatum (Chang and Kitai, 1985; Mugnaini and Oertel, 1985; Walaas and Fonnum, 1979). Thus, as is probably also the case in the dorsal striatum, GABAergic projection neurons receive a cholinergic innervation from intrinsic cholinergic elements located at strategic sites for modifying the projection cell output. e. Olfactory Bulb. Varicose, ChAT-positive fibers are distributed throughout much of the main olfactory bulb (J. E. Vaughn, unpublished observations). A striking pattern is formed by numerous ChATpositive terminallike structures surrounding the perimeters of the glomeruli, in contrast to the interior of these structures, where ChATpositive terminals are sparse. T h e sources of most of the cholinergic fibers and terminals in this brain area are derived, as described below, from extrinsic sources, principally in the nuclei of the diagonal band (Zaborszky et al., 1986). However, there are a few recently identified ChAT-positive somata in the olfactory bulb (J. E. Vaughn, unpublished observations) that could provide an intrinsic origin €or some of this cholinergic innervation.

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f . Retina. A sixth region of the central nervous system that contains intrinsic cholinergic neurons is the retina. The only known retinal projection cell type is the ganglion cell, and these elements have not been found to contain ChAT immunoreactivity. In contrast, two intrinsic cell populations have been shown to be ChAT-positive (Brandon, 1987; Famiglietti and Tumosa, 1987; Mariani and Hersh, 1988; Tumosa et al., 1984). These cells send their processes into characteristic sublamina of the internal plexiform layer, and ChAT-positive presynaptic terminals predominantly make contacts with ganglion cell dendrites (Brandon, 1987; Mariani and Hersh, 1988). The main synaptic input to ChATpositive processes comes from bipolar cell axon terminals. Thus, cholinergic interneurons appear to be major linking elements in the transfer of neuronal information from photoreceptors through bipolar neurons to the retinal projection-ganglion cells. g. Hypothalamus. Relatively large ChAT-positive neurons have been found in the hypothalamus, and these cells are located in a region immediately dorsolateral to the supraoptic nucleus (Mason et al., 1983). This result has been interpreted in the light of previous studies to support the possibility that the cholinergic innervation of the supraoptic nucleus is derived from local ChAT-positive neurons (Mason et al., 1983).

3. Regions Containing ChAT-Positive Projection Neurons Until recently, the locations of the cholinergic projection neurons of the CNS were described in terms of the distinct, classical nuclear groups of which they formed a part of the total population. However, several investigators are now categorizing these neurons on the bases of large, functionally related aggregations that are more or less independent of the classical nuclear groupings. For example, the cholinergic neurons of the forebrain and midbrain have been divided into eight groups, Ch 1-Ch8, based on their locations and projection patterns (Mesulam et al., 1983a,b, 1984; Mufson et al., 1986). This categorization does not include the projection motor neurons of the brainstem and spinal cord. Another global classification of cholinergic neurons has been proposed on the criteria of (1) locations, (2)common projection patterns, (3) the fact that the classical nuclear groups are not completely populated by cholinergic neurons, and (4)recent evidence that forebrain ACh neurons, at least, appear to constitute a continuous cell column (Satoh et al., 1983; Schwaber et al., 1987). This classification consists of subdividing central cholinergic neurons into four main groups: the rostra1 column of the basal forebrain, the caudal column of the pons, somatic and visceral motor neurons of the brainstem and spinal cord, and “local circuit neurons in dopamine-rich regions of the forebrain” (Satoh et al., 1983).

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Although both of these proposed classifications have conceptual merit, it may be prudent, as their proponents also caution, to await further information before a general adoption of a new classification and nomenclature. Therefore, the ChAT-positive projection neurons are described in this section on the basis of the classical nuclear groups in which they reside, with a brief note concerning their placernent in the two new classification systems. a. Medial Septum and Vertical Limb oJ’Diagona1 Band. T h e cholinergic neurons in these nuclei are included in the Chl and Ch2 groups of Mesulam et al. (1983a,b, 1984; Mufson et al., 1986) and in the rostra1 column of the basal forebrain (Satoh et nl., 1983; Schwaber et al., 1987). Numerous investigators have shown that these neurons are immunoreactive for ChAT (Houser et al., 1983; Ishida et al., 1983; Levey et al., 1983; Mesulam et al., 1983a,b; Rye et al., 1984; Satoh et al., 1983; Sofroniew et al., 1982, 1987; Woolf et al., 1984). These cells are among the smallest neurons of‘ the cholinergic basal forebrain complex, and they are interspersed with numerous ChAT-negative neurons. Nisslcounterstained immunocytochemical preparations have been used to estimate that 30-50% of the total neuronal population of the medial septal nucleus and 50-7576 of the vertical limb nucleus of the diagonal band is ChAT-positive (Mesulam et al., 1983b). These neurons provide the major cholinergic input to the hippocampus and the dentate gyrus (Amaral and Kurz, 1985; Fibiger, 1982; Mesulam et al., 1983a). As described above, lesions in the septal region produce a massive loss of ChAT-positive fibers and terminals in the hippocampal formation and completely obliterate the laminar staining of this brain region seen in ChAT immunocytochemical preparations of normal animals (Matthews et al., 1987). Electron microscopic studies of the ChAT-positive neurons of the septal region reveal that these cells exhibit very few axosomatic synaptic contacts in contrast to the ChAT-negative neurons in the same vicinity that display axosomatic synapses more frequently (Bidlowas and Frotscher, 1987). The dendrites of ChAT-positive septal neurons exhibit more synaptic .junctions than their somata, and these contacts mainly belong to the asymmetric type (Bialowas and Frotscher, 1987). I n addition to sending their axons to the hippocampal formation, ChATpositive septal neurons rnay also give rise to intrinsic axon collaterals, since ChAT-positive axon terminals are present in the septal region (Bialowas and Frotscher, 1987). These immunoreactive terminals form synaptic contacts with Ch AT-negative cell bodies and dendrites, with the latter being the more common postsynaptic target. These synapses are predominantly of the symmetric variety (Bialowas and Frotscher, 1987). b. Nucleus of’ the Horizontal Limb of the Diagonal Band. Another com-

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ponent of the cholinergic rostral column of the forebrain is contained within the horizontal limb of the diagonal band (Armstrong et al., 1983; Houser et al., 1983; Mesulam et al., 1983a, b; Rye et al., 1984; Satoh et al., 1983; Schwaber et al., 1987; Sofroniew et al., 1982, 1987; Woolf et al., 1984; Zaborszky et al., 1986). These cells have also been classified as belonging to group Ch3 of Mesulam et al. (1983a,b, 1984; Mufson et al., 1986). T h e ChAT-positive cell bodies of this group form a larger part of the total neuronal population in the medial part (50-75%) of the horizontal limb nucleus than in the lateral aspects (20-25%) (Mesulam et al., 1983b). The ChAT-positive neurons of Ch3 provide cholinergic inputs to the olfactory bulb (Mesulam et al., 1983a; Zaborszky et al., 1986) and to entorhinal cortex of the cerebrum (Woolf et al., 1984). In light and electron micrographs, profiles of Ch AT-positive neurons in the horizontal limb vary substantially in both shape and size (Dinopoulos et al., 1986). Noncholinergic synaptic afferents to ChATpositive cell bodies and proximal dendrites are not numerous, but those that are present usually display asymmetrical synaptic contacts (Dinopoulos et al., 1986). c. Nucleus Basalis. The nucleus basalis is a region of the rostral basal forebrain column (Satoh et al., 1983; Schwaber et al., 1987) that contains cholinergic neurons included in the Ch4 group (Mesulam et al., 1983a,b). Numerous investigators have shown that many of the neurons in this brain region are ChAT-positive (Armstrong et al., 1983; Houser et al., 1983; Levey et al., 1983; Mesulam et al., 1983a,b; Rye et al., 1984; Sofroniew et al., 1987; Satoh and Fibiger, 1985; Satoh et al., 1983; Wahle et al., 1984; Woolf et al., 1986). These cholinergic cells form the vast majority of the neuronal population of the nucleus basalis (-go%), and they are the largest ChAT-positive neurons in the forebrain (Mesulam et al., 1983b). In addition to morphological similarities, these cells exhibit a common projection to cerebral neocortex and amygdala (Carlsen et al., 1985; Ingham et al., 1985; Mesulam et al., 1983a; Rye et al., 1984), as well as to the nucleus reticularis thalami (Levey et al., 1987a). As mentioned above, ChAT-positive cell bodies of the basal nucleus are relatively large, but these cells appear to possess only sparsely branching dendrites (Ingham et al., 1985). The cell bodies exhibit large volumes of perikaryal cytoplasm that contain numerous organelles. Their nuclei have prominent nucleoli and are often eccentrically located. T h e somata and proximal dendrites of these cells receive very few synaptic inputs in contrast to those of ChAT-negative cells in the same vicinity. Those noncholinergic inputs that are observed form asymmetric synaptic junctions (Ingham et al., 1985) and appear to arise from neurons of the basolateral amygdala (Zaborszky et al., 1984). d. Pontomesencephalic Reticular Formation. T h e pedunculopontine (or

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cuneiform) nucleus of the pontomesencephalic reticular formation has been designated as Ch5 group (Mesulam et al., 1983b) and is included in the caudal cholinergic column of the pons (Satoh et al., 1983). Also included in this caudal column are neurons in the periventricular gray, especially those of the laterodorsal tegmentum; these cells belong to the Ch6 group of Mesulam et al. (1983b). A number of investigators have observed medium-sized ChAT-positive neurons in this brain stem region (Armstrong et al., 1983; Mesulam et al., 1983b; Satoh et al., 1983; Satoh and Fibiger, 1985; Woolf and Butcher, 1986). T h e main projection target of these neurons is the thalamus (Hallanger et al., 198’7; Mesulam et al., 1983b; Satoh and Fibiger, 1986; Sofroniew et al., 1985; Woolf and Butcher, 1986), but they also provide cholinergic inputs to the cerebral cortex, hippocampal formation, and olfactory bulb (Mesulam et al., 1983b), as well as substantia nigra (Beninato and Spencer, 1987), and superior colliculus (Beninato and Spencer, 1986). Recently, ChAT-positive neuropil staining has been reported in the substantia nigra, especially in the caudal pars reticulata (Gould and Butcher, 1986). A few, relatively large, ChAT-positive somata are also present in this brain stem region. Although these cells could be intrinsic sources of the ChAT immunoreactivity of the nigral neuropil, it is suggested that they may represent ectopic neurons of the pontomesencephalotegmental cholinergic system (Gould and Butcher, 1986). If this is the case, the ChAT-positive nigral neurons by analogy would have to be regarded as projection neurons. Further studies are required to resolve this problem. e. Medial Habenula. Several groups of investigators have observed numerous ChA’T-positive somata in the medial habenular nucleus, and this nucleus is the only one in the region of the thalamus and epithalamus that contains cholinergic cell bodies (Houser et al., 1983, 1988; Ichikawa and Hirata, 1986; Levey et a/., 1987b; Mesulam et al., 1984). The ChAT-immunoreactive somata are concentrated in the ventral part of the medial habenula; they are designated as group Ch7 (Mufson et al., 1986). Available evidence suggests that they provide a major projection to the interpenduncular nucleus (Herkenham and Nauta, 1979; Kataoka et al., 1973; Lenn et al., 1985). f. Parabigeminal Nuclew. T h e last neurvns to be currently included in the Ch classification of Mesulam and collaborators are ChAT-positive cells of the parabigeminal nucleus; they are designated as group Ch8 (Mufson et al., 1986). These cells form a compact nucleus situated along the lateral edge of the mesopontine region just ventral to the brachium of the inferior colliculus, and they comprise about 80-90% of the

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somata in this region (Mufson et al., 1986). Retrograde labeling studies indicate that the ChAT-positive parabigeminal cells provide a major cholinergic input to the superior colliculus (Mufson et al., 1986). g. Motor Neurons of Cranial Nerve Nuclei. In addition to the cholinergic projection neurons described above, a number of studies have shown that the somatic and visceral efferent components of the cranial nerves are ChAT-positive (Armstrong et al., 1983; Houser et al., 1983; Levey et al., 1983; Mesulam et al., 1984; Satoh and Fibiger, 1985; Strassman et al., 1987). For example, intensely ChAT-positive neurons are located in the somatic motor nuclei of cranial nerves III-VII and IX-XII. Such cells are also found in the nucleus ambiguus, a cell group with both somatic and special visceral motor elements. Furthermore, Ch AT-positive neurons populate the preganglionic nuclei of cranial nerves 111, VII, IX, and X (Houser et al., 1983; Satoh and Fibiger, 1985; Strassman et al., 1987). In certain cases distinct fascicles of ChAT-positive axons emerge from these brainstem nuclei, and these nerve tracts show identical trajectories to those of the associated cranial nerves. 4. ChAT-Positive Terminal Fields a. Thalamus. As mentioned above, ChAT-positive cell bodies have not been observed in the thalamus proper; the only such cells in the general vicinity of the thalamus are located in the medial habenular nucleus of the epithalamus. In contrast, however, a number of thalamic nuclei exhibit various concentrations of ChAT-positive terminals (Dolabela de Lima et al., 1985; Houser et al., 1988; Levey et al., 1987b). The highest thalamic concentrations of ChAT-immunoreactive fibers and terminals are present in the anteroventral, intralaminar, reticular, and lateral mediodorsal nuclei (Houser et al., 1988; Levey et al., 1987b). In contrast the anterodorsal, ventroposteromedial, and paraventricular nuclei display little o r no ChAT immunoreactivity (Houser et al., 1988; Levey et al., 1987b). In the visual thalamus (or metathalamus) there is a dense innervation of the dorsal lateral geniculate nucleus and the perigeniculate nucleus by ChAT-positive axons (Dolabela de Lima et al., 1985; Stichel and Singer, 1985). As described in a previous section, the cholinergic innervation of the thalamic region is derived from neurons of the pontomesencephalic reticular formation. Electron microscopic studies indicate that Ch AT-positive terminals predominantly form symmetric synapses with small and medium-sized dendrites in the thalamus proper (Houser, 1989; Houser et al., 1988) and contact both inhibitory intrinsic cells and relay neurons in the visual thalamus (Dolabela de Lima et al., 1985). 6. Interpeduncular Nucleus. T h e interpeduncular nucleus stains in-

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tensely for ChAT-positive immunoreaction product, and this product is found exclusively in small fibers and terminals (Houser et al., 1983). As discussed above, a likely source for a major portion of this cholinergic innervation is the medial habenula. Electron microscopy reveals that ChAT-positive boutons form synaptic junctions in this brainstem structure (Lenn et al., 1985; Wainer et al., 1984) and that such junctions can be classified into several different morphological groups (Lenn et al., 1985). c. Superior Col~icul~. No accounts of ChAT-positive neuronal somata have been published in the superior colliculus, although a few small ChAT-positive cells have been observed in this brain region (P. E. Phelps, unpublished observations). There is, however, substantial immunoreactivity due to ChAT-positive fibers and terminals in the interior layers of this part of the midbrain (Mufson et al., 1986, Fig. 1; P. E. Phelps and C. R. Houser, unpublished observations). T h e most intense staining is present in the intermediate gray layer. Rostrally, this immunoreactivity forms a distinct band, whereas at progressively more caudal levels, the bandlike nature of the ChAT-positive staining is interrupted by patches of ChAT-negative neuropil. N o electron microscopy has been conducted to confirm the synaptic nature of the terminallike ChATpositive staining in the superior colliculus, but as described above there is evidence that major sources of these fibers and terminallike structures are the parabigeminal nucleus (Mufson et al., 1986) and nuclei of the pontomesencephalic reticular system (Beninato and Spencer, 1986).

C. FUNCTIONAL IMPLICATIONS VERTEBRATE CNS

OF

CHAT IMMUNOCYTOCHEMISTRY IN

Possible functions of cholinergic systems in vertebrate brains have been suggested and reviewed in the past (Bartus et al., 1982; Butcher and Woolf, 1982; Fibiger, 1982; Kuhar, 1976; Squire and Davis, 1981). A number of these ideas originated before the advent of ChAT immunocytochemistry. For example, the concept that ascending cholinergic systems of the brainstem and the basal forebrain might be critical components of the reticular activating system and thus be importantly involved in arousal of neuronal activity in the cerebral cortex was formulated in large measure on the results of acetylcholinesterase histochemistry (DeFeudis, 1974; Schute and Lewis, 1967; Woolf and Butcher, 1986). Similarly, the cholinergic hypothesis of Alzheimer's disease was generated from data obtained from both ChAT biochemical and AChE histochemical studies (Bartus et al., 1982; Coyle et

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al., 1983; Whitehouse et ul,, 1982). Since these concepts are well reviewed and largely antedate ChAT immunocytochemistry, we concentrate on possible functional relationships of cholinergic neurons at cellular and synaptic levels because the resolution of ChAT immunocytochemistry uniquely allows interpretations concerning cholinergic functioning at these levels. A new concept that is arising due to the findings of ChAT immunocytochemistry is that many brain regions, particularly cortical and quasi-cortical ones, have an intrinsic cholinergic innervation in addition to previously known extrinsic synaptic inputs. This idea has grown from new identifications of ChAT-positive intrinsic neurons in many areas of neo- and limbic cerebral cortex (Brady and Vaughn, 1988; Eckenstein and Baughman, 1984; Frotscher and Leranth, 1985; Houser et al., 1983, 1985; Levey et al., 1984; Matthews et al., 1987; Wainer et al., 1985) and in a number of quasi-cortical structures such as the basolateral amygdala (Carlsen and Heimer, 1986; C. R. Houser, unpublished observations), the olfactory bulb (J. E. Vaughn, unpublished observations), and the nucleus of the lateral olfactory tract (Phelps, unpublished observations). The functional role(s) of these intrinsic ChAT-positive neurons is unknown, as obviously is their relationship to extrinsic cholinergic synaptic afferents. However, in neocortex at least, there is a hint that the intrinsic cells may have a different function than extrinsic ones, since approximately 80% of the intrinsic ChAT-positive neurons also contain the neuroactive substance vasoactive intestinal polypeptide (VIP), whereas this does not appear to be the case for extrinsic cholinergic cells that project to neocortex (Eckenstein and Baughman, 1984). Despite the fact that the functional relationships of intrinsic and extrinsic cholinergic systems are unknown, it would seem to be important that the existence of such dual cholinergic systems be considered in relation to neurological disorders in which cholinergic neurons are thought to be involved. If the principle of dual cholinergic systems manifest in lower mammals is recapitulated in primates, it would be important, for example, to see if intrinsic ChAT-positive neurons of the cerebral cortex are lost in Alzheimer’s disease, as are the extrinsic cholinergic neurons of the nucleus basalis (see, e.g., Whitehouse et al., 1982). If they are not, it might be possible in some instances to manipulate the intrinsic cholinergic cells therapeutically to compensate partially for the reduction of the extrinsic cholinergic system that occurs in this neurological disorder. Another generalization that can be derived from recent ChAT immunocytochemical investigations is that cholinergic neurons of the CNS give rise to several different types of synapticjunctions. Although it

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appears that in most brain regions the predominant kind of synapse formed by ChAT-positive terminals belongs to the symmetric category, small fractions of the total cholinergic synaptic population in a variety of brain regions can be classified as forming asymmetric synapticjunctions. In addition, as discussed earlier in this review, ChAT-positive terminals synapse with different classes of postsynaptic target neurons in a given brain region and often contact many parts of such neurons including dendritic shafts and spines, somata, and axon initial segments. T h e morphological diversity of cholinergic synapses suggests a corresponding heterogeneity of function, and this is consistent with physiological findings that indicate ACh may effect more than one type of synaptic action in several brain regions (Bernard0 and Prince, 1982; Cole and Nicoll, 1984; Dodd et al., 1981; Horn and Dodd, 1983; Krnjevik, 1981; Lamour et al., 1982; McCormick and Prince, 1986; Muller and Misgeld, 1986; Sillito and Kemp, 1983). However, there does not appear to be a simple correspondence of synaptic morphology and function at cholinergic synapses. For example, there is evidence that ACh has a different postsynaptic action on pyramidal and nonpyramidal neurons of the hippocampal formation, despite the fact that cholinergic terminals form similar synapses on both types of cells (Leranth and Frotscher, 1987). Similarly, quantitative relationships in several brain regions also suggest that a simple assignment of junctional symmetry and synaptic action is not possible for cholinergic synapses. For instance, although both excitatory and inhibitory responses to ACh have been recorded in cerebral cortex, excitatory postsynaptic effects appear to be the most common (Krnjevii-, 1981; Krnjevik and Phillis, 1963; Lamour et al., 1982; Sillito and Kemp, 1983). I n contrast, however, the vast majority of ChAT-positive terminals in cerebral cortex form symmetric synaptic contacts (Dolabela de Lima and Singer, 1986; Frotscher and Leranth, 1985; Houser et al., 1985; Wainer et nl., 1984), thus dispiaying the morphology traditionally ascribed to inhibitory synapses (for review, see, e.g., Colonnier, 1981; Eccles, 1964). A similar lack of correspondence of synaptic morphology and physiology also occurs in the neostriatum (Bernardi et al., 1976; Phelps et al., 1985; Izzo and Bolam, 1988; Misgeld et al., 1984). However, ACh appears to inhibit the inhibitory interneurons of the perigeniculate nucleus (Dingledine and Kelly, 1977; Francesconi et al., 1984; Siilito et al., 1983), and in this case the present dogma of synaptic structure and function is obeyed, since symmetric synaptic contacts are most frequently formed by ChAT-positive terminals (Dolabela de Lima et al., 1985). Nevertheless, it is clear from the examples cited above that there is no simple, one-to-one relationship that is consistent with the structural and functional heterogeneity exhibited by cholinergic synapses in the central nervous system.

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D. IMMUNOCYTOCHEMISTRY OF CHAT IN INVERTEBRATE NERVOUSSYSTEMS Several reports have described the distribution of ChAT in insect nervous system detected by staining with monoclonal anti-Drosophila ChAT antibodies (Buchner et al., 1986; Gorczyca and Hall, 1987; Ikeda et al., 1984) or polyclonal antiserum to locust enzyme (Lutz and Tyrer, 1988; Ikeda and Salvaterra, 1989). The enzyme appears to be widely distributed in the neuropil regions of sensory ganglia and brain. T h e most intense staining appears over the synaptic layers in the medulla and lobula of the optic ganglia, whereas distinct but less intense staining is also present in the lamina. T h e ChAT-positive staining appears to be qualitatively similar, but quantitatively less intense, in the nervous systems of animals carrying mutant temperature-sensitive alleles for ChAT. In general, the distribution of ChAT immunoreaction product in Drosophila is consistent with an important role for cholinergic neurotransmission in the accumulation o r processing of sensory information. An interesting aspect of ChAT immunocytochemistry in Drosophila in comparison to the more extensive studies done with vertebrates is the intrasomal distribution of reaction product. In Drosophila, little if any cell body staining has been detected. In contrast, vertebrate preparations in general show staining throughout the neuron with the most intense staining in cell soma. The reason for this apparent differential intracellular distribution of ChA’T between vertebrates and Drosophila is unknown. It may be due to technical limitations of immunocytochemical procedures and/or reagents, or to real species differences.

IV. Development of Cholinergic Neurons

A. DESCRIPTIVE STUDIES In addition to the spatial regulation of ChAT expression in the adult nervous system, the enzyme changes both qualitatively and quantitatively during the temporal course of development. Descriptive studies of the development of cholinergic neurons presently have been conducted in two general categories, those dealing with the generation or “birthdays” of such cells and those concerned with the ontogenic time of expression of the cholinergic phenotype as defined by the presence of ChAT.

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1. Biochemktry

The developmental appearance of ChAT in nervous system has been traced by measuring enzyme activity in either whole brain or microdissected brain regions from several different species (Burt and Narayanan, 1976; Dewhurst et al., 1970; Giacobini, 1972; Loh, 1976; Nadler et al., 1974; Sorimachi and Kataoka, 1974). Sexual dimorphisms in the developmental appearance of ChAT have also been noted in rats (Brown and Brooksbank, 1979; Loy and Sheldon, 1987). Using our ChAI' cDNA clone as a probe, we have also measured the developmental appearance of ChAT mRNA in Drosophila and compared the steady state mRNA levels with enzyme activity (L. Carbini and Y. M. Salvaterra, unpublished observations). Drosophilu is a holometabolous insect with a complicated life cycle. The organism passes through three larval stages and a pupal stage and emerges as an adult. During these major developmental stages, the body plan of the organism changes radically and, consequently, new demands are placed on the nervous system. Many of the neurons present in the original larval nervous system are retained throughout the life cycle, whereas others, such as the sensory neurons of the optic ganglia, are added late in the life cycle by precursor cells of the imaginal discs (Campos-Ortega, 1982). Apparently there are at least two developmental phases of increasing steady-state levels of ChAT mRNA and active enzyme to accommodate this life cycle. We have detected ChAT mRNA as early as 6 hr after oviposition, with measurable enzyme activity appearing 1-2 hr later. Interestingly, the appearance of mRNA and enzyme activity both precede the time when most neurons have undergone their final cell divisions (Hartenstein and Campos-Ortega, 1984). The ChAT' mRNA levels reach an initial maximum at the stage of the second larval instar. The level of mRNA decreases rapidly and is barely detectable during early pupation. In late pupation, mRNA levels begin to increase rapidly again and reach a plateau level in young adults. The enzyme activity during Drosophilu development shows a similar qualitative pattern but exhibits a temporal scale that lags behind the mRNA changes by several hours. Our results are consistent with the hypothesis that developmental expression of ChAT in Drosophilu is at least in part under transcriptional control. T h e two phases of rapidly increasing ChAT-specific mRNA production seem to correlate best with the times when Drosophilu neurons are making physical contact with each other, and the decreasing phase of mRNA expression correlates well with the degeneration of neural processes during pupation. The advantages of biochemically measuring ChAT mRNA and enzyme activity during development include the high sensitivity of the

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assays used, as well as the quantitative nature of the data. Unfortunately, the spatial resolution of biochemical techniques is limited by the smallest amount of tissue that can be microdissected. It is likely that ChAT activity and mRNA expression develop with different time courses in small regions and cell groups within the nervous system. T h e techniques of immunocytochemistry and in situ hybridization can be applied to localizing ChAT protein and mRNA qualitatively in smaller regions of the nervous system, including individual cells and subcellular compartments. 2. Immunocytochemistry a. Spinal Cord. As described earlier in this chapter, the rat spinal cord contains three distinct populations of intrinsic ChAT-positive neurons in addition to the spinal projection neurons represented by somatic motor and preganglionic autonomic neurons. With the exceptions of the large a-motoneurons and perhaps the principal intermediolateral preganglionic autonomic cells, it would be impossible to study the generation of these different cholinergic cell types in conventional [’Hlthymidine autoradiographs, since one could not distinguish the cholinergic neurons from surrounding noncholinergic cells. However, by combining [‘Hlthymidine autoradiography and ChAT immunocytochemistry for the same specimens, it has been possible to study the generation of the diverse subpopulations of cholinergic neurons present in the spinal cord (Fig. 6; see also Phelps et al., 1986, 1988a). The fact that the ChAT-positive cell groups in spinal cord vary in both their soma1 sizes and their positions within the known ventrodorsal neurogenic gradient of spinal cord (Hollyday and Hamburger, 1977; Langman and Hayden, 1970; Nornes and Das, 1972) allows for the testing of whether neuronal phenotype is a more potent determinant of the time of neuronal origin than a cell’s location along a neurogenic gradient. In the cervical enlargement, the large and small motor neurons are the earliest-born cholinergic cells in the spinal cord, and most of their generation occurs on embryonic day 11 (El 1; E1,day of sperm positivity of vaginal smear). Such cells are also the cells formed earliest in the spinal cord regardless of neurotransmitter phenotype. Cholinergic neurons in the intermediate region of spinal gray matter (partition cells) exhibit peak generation on E12, whereas the more dorsally located, small ChAT-positive groups (central canal cluster cells and dorsal horn neurons) are mainly formed on E13. The salient conclusions derived from this study are 1. ChAT-positive neurons are among the earliest cells generated in their respective dorsoventral subdivisions of the spinal cord.

FIG. 6. Nomarski photomicrograph demonstrating a combination of ChAT immunocytochemistry and [3H]thyniidine autoradiography. Both large and small somatic motor neurnns are double labeled with ChAT-positive reaction product in their cytoplasm and silver grains froni incorporated [SH]thymidine overlying their nuclei. Specimen was obtained from t h e cervical spinal cord of a 28-day postnatal rat that was labeled with radioisotope on embryonic day 11. Scale bar, 20 pm. (From Phelps et al., 1988a, with permission of Alan R. Liss, Inc.)

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2. All cholinergic neurons are not generated simultaneously. 3. T h e birthdays of cholinergic cells are correlated with their positions within the neurogenetic gradient. 4. Large and small ChAT-positive neurons located at the same dorsoventral level exhibit the same temporal patterns of neurogenesis (Phelps et al., 1986, 1988a). Thus, the findings of this research strongly suggest that positional information relative to neurogenic gradients is more important in the determination of the times of neuronal origin than that involved with specifying phenotypic details such as neuronal size or type of neurotransmitter expressed. The developmental time course of the expression of ChAT immunoreactivity has also been studied in spinal cord. Most of this work has been focused on early postnatal stages of development (Phelps et al., 1984), but a preliminary report of prenatal times has been presented (Phelps et al., 1988b). T h e main finding from this study so far is that the sequence of expression of ChAT immunoreactivity in spinal neurons correlates well with their sequence of neurogenesis. Thus, the earliest cells to be generated in spinal cord, the somatic motor neurons, are also the first to exhibit ChAT-positive staining. T h e next cholinergic spinal neurons to be born are the partition neurons, and they begin to express ChAT immunoreactivity shortly after motor neurons. Both motor and partition neurons display Ch AT-positive staining relatively early in the prenatal period ( E l 3 and E14-15, respectively) (Phelps et al., 1988b), whereas the later-generated central canal cluster neurons and dorsal horn cells do not express this trait until E17. At this time, these cells are lightly stained, and they are still only moderately ChAT-immunoreactive by 11-14 days postnatal (Phelps et al., 1984). 6. Basal Forebrain. The combined autoradiographic and immunocytochemical method for determining the developmental time of origin of neurons has also been applied to the rat basal forebrain (Brady et al., 1987; Semba and Fibiger, 1987, 1988). Based on Nissl studies of all neurons, this region exhibits an overall caudorostral gradient of neurogenesis (Bayer, 1985), and basal forebrain cholinergic neurons obey this general gradient. The expression of ChAT immunoreactivity has been detected as early as E 17 in neuronal somata of the septal-diagonal band complex of the rat basal forebrain (El5 in mouse) (Schambra et al., 1987), and there is a marked increase of immunostaining of these cells at perinatal stages of development (Armstrong et al., 1987). Cholinergic neurons express adultlike characteristics after postnatal day 23 (Armstrong et al., 1987). Since the earliest cholinergic neurons to be born in this part of the basal

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forebrain are generated on E12-13 (Brady et al., 1987; Semba and Fibiger, 1987, 1988), it would appear that 4-5 days of differentiation following the cessation of mitosis seem to be required for ChAT to reach immunocytochemically detectable levels in individual neurons of the basal forebrain. A shorter latency ( i e . , 2-3 days) period for the expression of ChAT immunoreactivity appears to be necessary for cells in spinal cord (Phelps et ul., 1986, 1988a,b). Furthermore, there appears to be preliminary evidence that certain cells in the forebrain may express ChAT immunoreaction product during mitosis (Schambra et al., 1987). 3 . Generalizations from Descriptive Studies

Although the number of descriptive investigations of ChAT-positive neuronal development as well as the number of brain regions examined are both very small, the first glimmerings of general principles are becoming apparent. First, it seems likely that the time of origin of cholinergic neurons in different regions of the CNS will obey the general neurogenic gradients described for their regions on the basis of conventional Nissl-stained ['Hlthymidine autoradiograms. The general gradients defined solely by this method have been sufficiently imprecise to allow for the possibility that specific subsets of neurons in a given brain region could be generated rather synchronously despite being separated widely over the general gradient (Phelps et al., 1988a). T h e fact that an identifiable subgroup of a total regional population obeys the overall gradient reinforces the significance implicit for such gradients that positional information plays a major role in determining the premier event in the life of a neuron, the time of its birth. This, in turn, determines the nature of the environment in which a neuron begins to differentiate its unique phenotypic characteristics. The second generalization that seems to be emerging from developmental studies of ChAT-positive neurons is that the latency period between the birth dates of cholinergic neurons and the expression of immunocytochemically detectable ChAT appears to vary in association with different specific subsets of cholinergic neurons. The significance of this phenomenon lies in its relationship to a third broad concept relating to cholinergic neuronal development; namely, cholinergic neurons generally may be among the very first neurons to be born in their respective CNS regions. Consequently, it may be that certain cholinergic cells, especially those with a short latency between birth and ChAT expression, are strategically placed in relative developmental time to synthesize and secrete ACh initially not as a neurotransmitter, but as an inductive or trophic factor that could be associated with regulating phenotypic characteristics of later developing neurons. While this idea is at the moment speculative, it is consistent not only with

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the early generation of cholinergic neurons, but also with the fact that the ACh-synthesizing enzyme is widely distributed throughout the major parts of cholinergic neurons. This circumstance might facilitate the diffuse broadcasting of a developmentally important signal. In addition, such a function for the early development of cholinergic neurons agrees with evidence suggesting that ACh may serve as a trophic or regulatory factor, as well as a neurotransmitter, in certain species (see Lauder, 1983, for review).

STUDIES B. EXPERIMENTAL Neurons are by nature a rather gregarious cell type. They are designed for specialization in intercellular communication via synaptic transmission. In addition, it would seem likely that a variety of normal regulatory signals must be communicated between synaptic partners in order to modulate their respective metabolic states. It is not surprising, then, that a number of intercellular regulatory events have been described as being important for regulatory control of ChAT expression. The three main types of independent regulatory strategies adopted by cholinergic systems that have so far been identified are (1) soluble tissue factors (often produced by target tissue), (2) insoluble cell surface components, and (3) nerve growth factor. All three of these systems, as well as others yet to be described, are likely developmental control points for ChAT expression and modulation. In addition, they are also likely to be important for maintaining normal cholinergic neuron function in older animals. 1 . Cholinerpc Factor

The developmental environment of vertebrate sympathetic neurons has a profound effect on their neurotransmitter phenotype. When cells that ordinarily express the adrenergic phenotype are exposed to certain environmental stimuli, they can switch their typical neurotransmitter system to a cholinergic one. Both in viva (Cochard et al., 1979; Landis and Keefe, 1983; LeDouarin, 1980) and in vitro (Patterson, 1978) observations in a variety of experimental systems have supported the use of this phenomenon as a normal developmental strategy for selecting and maintaining neurotransmitter phenotype. Moreover, the participation of factors produced by target cells for regulating neurotransmitter phenotype suggests a likely mechanism for plastic expression of this important functional characteristic of neurons (Black, 1982; Patterson, 1978). Several excellent reviews are available describing the early work

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on neurotransmitter pheontype specification and cholinergic switching (Black, 1982, Patterson, 1978). A cholinergic factor from heart cell-conditioned medium has been completely purified and characterized by Fukada (1985). The factor responsible for cholinergic switching of rat sympathetic neurons is a glycoprotein of about 45 kDa in molecular mass, and it is synthesized by the cultured heart cells and secreted into the medium. This single purified factor can induce the appearance of ChAT in normally adrenergic sympathetic neuron cultures. T h e mechanism responsible for ChAT expression is not yet known. However, specific antibodies to the factor have been produced and once adequate nucleic acid probes are available for detection of rat ChAT mRNA it will be possible to examine the hypothesis that transcription and translation of the ChAT gene are directly responsible for this newly acquired neurotransmitter phenotype. The existence of other cholinergic neuronotrophic factors which act on parasympathetic o r CNS neurons has also been described (Wallace and Johnson, 1987; Bostwick et al., 1987).

2. Cell Surface Components Neurons are in intimate physical contact with various cell types, and the cell surface molecules of these close neighbors may be intimately involved in determining and maintaining neurorial specificity. For example, sympathetic neurons in culture can differentially express a variety of different neurotransmitter phenotype profiles that depend on culture density (Adler and Black, 1985). Isolated Schwann cell or sympathetic neuron plasma membranes can mimic the effects of increased cell density (Kessler et al., 1986). In at least one case, the responsible molecule has been solubilized and partially characterized from spinal cord plasma membranes; it appears to be a small glycoprotein (Wong and Kessler, 1987). Interestingly, one report has shown that the neural cell adhesion molecule (NCAM) has some specificity for increasing ChAT levels in chick sympathetic neuron cultures and that the effects of NCAM can be blocked by addition of anti-NCAM antibodies (Rutishauser et al., 1988). 3 . NGF, Other Factors, and Alzheimer’s Disease

NGF has long been known to influence the survival and differentiation of sensory and sympathetic neurons both in culture and in uiuo (for reviews see Levi-Montalcini, 1968, 1982; Thoenen and Barde, 1980). It has come as somewhat of a surprise, however, that NGF also appears to play an important neuronotrophic role in the CNS, with possible specificity for ChAT-containing neurons (Gnahn et a / . , 1983; Hefti et aL,

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1984; Mobley et al., 1985). NGF mRNA, protein, and receptors are all present in the brains of experimental animals (for reviews see Thoenen et al., 1987; Whittemore and Seiger, 1987). In addition these functionally important molecules are distributed within the CNS in a pattern consistent with their having an important action on the large cholinergic basal forebrain neurons (Korsching et al., 1985; Seiler and Schwab, 1984; Shelton and Reichardt, 1986). This pattern of cellular distribution is intriguing, since the homologous nucleus basalis of Meynert neurons in humans are one of the primary cell groups that undergo degeneration in the brains of patients suffering from Alzheimer’s disease (Whitehouse et d., 1981, 1982). NGF has now been shown to increase the levels of ChAT activity in discrete brain regions when administered to young animals or after cholinergic lesioning paradigms (Gnahn et al., 1983; Hefti, 1986; Mobley et al., 1985). In addition it has also been shown to reveal ChAT-positive neurons that are difficult to recognize following septa1 lesions (Hagg et al., 1988). The specificity and selectivity of the NGF effect(s) on CNS neurons and ChAT expression are difficult to establish directly in vivo, but the developmental time course of NGF and ChAT activity appear to be correlated (Auburger et al., 1987). NGF receptors have also been reported to decline in aged rats (Roh and Loy, 1988). Recent in uitro experiments also seem to confirm and extend the original in viuo observations that NGF is indeed affecting cholinergic neurons specifically (Hefti et al., 1985; Martinez et al., 1987; Shelton and Reichardt, 1986). Other purified and well-characterized protein factors, such as brainderived basic fibroblast growth factor (FGF), also appear to exert similar ChAT-sparing andlor survival effects on basal forebrain cholinergic neurons (Anderson et al., 1988; Unsicker et al., 1987). In addition other neuronotrophic factors, and even insulin, have been identified in a variety of extracts, and they have some specificity for increasing the survival of cholinergic neurons (see, e.g., Barde et al., 1983; Howard and Bronner-Fraser, 1986; Iacovitti et al., 1987; Kyriakis et al., 1987). The investigation of NGF, FGF, and other neuronotrophic factors is likely to be an active and exciting area of neurobiological research in the coming years, since these factors, or others yet to be described, may have important developmental functions. Understanding these functions may be necessary for preventing neurotransmitter-specific phenotypic cell death in neurodegenerative diseases. Senile dementia of the Alzheimer’s type is often described as a disease of central cholinergic neurons. Low levels of acetylcholine, ChAT, and cholinergic neurons are a particularly constant and striking

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feature observed in the brains of patients suffering from Alzheimer’s disease (Bowen et ul., 1976; Perry et al., 1977a,b; Whitehouse et al., 1981, 1982; for reviews see Bird el al., 1983; Davies, 1979; Marchbanks, 1982; Terry and Davies, 1980). The basal forebrain cholinergic system is also thought to be important for memory processes that are impaired in Alzheimer’s disease (Bartus et al., 1982; Collerton, 1986; Coyle et al., 1983; Perry et al., 1977a). It is not clear, however, if the decreased levels of ChAT are a cause or an effect of the disease. It is relevant to note in this regard that the reduction in cortical ChAT activity seems to precede the loss of basal forebrain cholinergic cells (Perry et ul., 1982). Acetylcholine is not the only neurotransmitter system affected in Alzheimer’s disease (see, e.g., Whitehouse et al., 1985, 1987). Other brain regions in which acetylcholine is not thought to be a major neurotransmitter have also been implicated in the neurodegenerative cell groups affected in Alzheimer’s disease (Bondareff et al., 1982; Hyman et al., 1984). Nevertheless, there seems to be a strong correlation with the pathological indices of this disorder and the reduction in ChAT activity (Etienne et al., 1986; Wilcock et al., 1982). In addition the levels of cholinergic differentiation factor seem to be lower in fibroblast cultures from Alzheimer’s patients (Kessler, 1987). An especially attractive hypothesis of Alzheimer’s disease has been advanced that emphasizes the importance of growth and differentiation factors for neuronal survival (Appel, 1984). Coupled with the recent discovery of several candidates for cholinergic neuronal survival factors, there is now a clear model to test in experimental systems in which cholinergic degeneration can be induced. The ability to recognize ChA’T-containing neurons immunocytochemically or mark them with nucleic acid probes is sure to play an important part in evaluating the neurotransmitter phenotypic specificity of these models.

V. Future Directions

Part of the difficulty in evaluating the relationship of neurotransmitter deficits to specific neurological diseases is directly related to our current ignorance about the regulatory features of neurotransmitter biosynthetic enzymes. What, if any, is the dependence of neuronal survival on continued expression of a neuron’s chosen neurotransmitter? How does correct maintenance of neurotransmitter phenotype affect the survival and function of the postsynaptic targets of neurons undergoing reduced neurotransmitter production or switching of neu-

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rotransmitter phenotype? What is the chemical nature and biological function of factors necessary for the correct expression and maintenance of neurotransmitter phenotype? The answers to all these questions are sure to be central to future research efforts aimed at understanding the important regulatory features involved in ChAT expression. Figure 7 summarizes three important decisions a developing cell must make in order to become a healthy, productive cholinergic neuron. Molecular and cellular techniques are now being applied to learn the details or rules involved in making each of these decisions. Initially, a developing cell must decide whether or not to become a neuron. By definition, a positive answer to this developmental decision involves expression of neuron-specific genes. Antibody and nucleic acid probes are now being developed to detect the early spatial and temporal expression of such genes. At the next level, a positive decision to become a cholinergic neuron must involve not only transcription and translation of the ChAT gene, but also activation of a negative mechanism for preventing the expression other neurotransmitter phenotypes. Future studies will seek to identify the specific DNA sequence elements necessary for Ch AT expression in a spatially and temporally appropriate manner as well as the other factors necessary to regulate this process. These factors are likely to include not only intrinsic cellular constituents, but also factors produced by neighboring cells or target cells of the

POSITIVE

DECISION

NEGATIVE

I* 11,

111,

FIG. 7. Logic diagram showing three levels of binary decision-making, positive and negative actions, and results of those actions. The potential pathway necessary for a developing nervous system cell to become a cholinergic neuron and maintain that status is indicated by the arrows Rowing from the left of the boxed decisions.

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cholinergic neuron. The final level of decision-making involves a lifelong commitment to the decision to express ChAT. Perhaps the details of the factors required for continued expression are intrinsic to the cholinergic cell. Alternatively, they could be produced by targets and or neighboring cells. Such maintenance factors may be identical to the components regulating initial expression of ChAT, or a whole new cast of characters may be identified as we learn more about ChAT regulation in the future. All these future studies aimed at understanding the cellular and molecular logic of ChAT expression should enrich our understanding of basic neurobiology. In addition they will be essential €or designing new and rational approaches for treating a variety of neurodegenerative disorders. It will be exciting to watch and participate as the gap between neurobiology and medicine is closed.

Acknowledgments

The authors wish to acknowledge the contributions of the following colleagues, upon which a substantial part of the present chapter is based: Veronica Andressani, Robert P. Barber, Nicole Bournias-Vardiabasis, Daniel R. Brady, Luis Carbini, Garrett Crawford, Carolyn K. Houser, Nobiyuki Itoh, Victor Mutioz-Maines, Patricia F.. Phelps, J. Randall Slemnion, and Hideniatsu Sugihara. We also wish to thank the following people for technical and secretarial assistance: Marilyn Ashley, Lynn A. Brennan, Mariko Lee, Tina Nair, Noel Neighbor, Rosalba Tamayo, Christine S. Vaughn, and Sharyn Webb. Work in the authors’ laboratories was supported, in part, by grants NSI8858 and NS19482 from the NINCDS and by grant BNS8219831 from the NSF.

References

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NEUROBIOLOGY OF ZINC AND ZINC-CONTAINING NEURONS By Christopher J. Frederickson Loboratory for Neurobiology University of Texas at Dallos

Richardson, Texas 75080

I. Introduction A. Zinc in the Nervous System B. Three Pools of Zinc in the Brain C. Early History 11. Distribution of Elemental Zinc in the Brain A. Methods of Analysis B. Instrumental Assays C. Distribution of Zinc 111. Uptake and Turnover of Brain Zinc IV. Zinc and Brain Proteins A. Zinc-Containing Proteins B. Zinc-Sensitive Proteins and Enzymes C . Zinc-Protein Complexes V. Zinc and Membranes A. Stabilization of Membranes B. Membrane Pumps and Ion Channels VI. Distribution of Histochemically Reactive Zinc in the Brain A. Histochemical Methods B. T h e Histochemically Reactive Pool of Zinc C. CNS Distribution of Histochemically Reactive Zinc VII. Zinc-Containing Neurons A. Definition B. Turnover of Vesicular Zinc and Activity-Metabolism Coupling C. Anatomy of the Zinc-Containing Pathways D. Colocalization of Zinc with Transmitters and Modulators VIII. Functional Significance of Vesicular Zinc A. Zinc and Storage of Macromolecules in Vesicles B. Zinc as a Potential Modulator of Synaptic Receptors and Physiology IX. Zinc and CNS Pathology A. Brain Development B. Adult Brain Function X. Summary and Conclusions A. Zinc Metalloenzymes in the Brain B. Zinc-Containing Neurons C. T h e Zinc Signal References

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I. Introduction

A. ZINC IN

THE

NERVOUSSYSTEM

Zinc has traditionally been identified in biological literature as a trace element, a designation reflecting the fact that it was barely detectable by the analytical methods of past decades (Vallee and Galdes, 1984). Unfortunately, the label “trace” often carries the connotation “barely important,” and it is therefore worth pointing out that the amount of zinc in the brain is not a “trace” amount at all. In the first place, analytical methods are now so sensitive that biological concentrations of zinc are well above their detection limits. The true trace elements of contemporary neurobiology (e.g., selenium, chromium, vanadium) (Demmel et a/., 1982; Naylor, 1985; I,akomaa, 1985) are several orders of magnitude less abundant than zinc. In fact, zinc is among the 20 most abundant elements in the brain, following carbon, nitrogen, oxygen, and so forth, and falling between calcium, which is about six-fold more abundant than zinc, and copper, which is about one-third less abundant (Goody et al., 1975; Wallwork et al., 1983; Chan et al., 1983; Ward and Mason, 1987). Among the transition elements, zinc is second only to iron in total brain concentration. Furthermore, compared to many familiar neuroactive substance, the concentration of zinc in the brain is actually quite high. For example, the typical concentration of zinc in grey matter (about 0.15-0.2 mM) is 10- 100 times higher than that of classical neurotransmitters such as acetylcholine and the monoamines (Bradford, 1986). Compared to neuropeptides (e.g., enkephalin, dynorphin, cholecystokinin, neurotensin) there is lo3 to lo4 more zinc in the brain (Bradford, 1986). Thus, to consider zinc a trace substance in the brain is of uncertain usefulness. The sheer abundance of zinc in brain tissue is noteworthy, but it is actually a somewhat misleading parameter, fur zinc is a general-purpose molecular building block and a structural component of over 50 different enzymes found throughout the brain and other soft tissues (Vallee and Galdes, 1984; Vallee, 1985). In this respect, zinc is rather like the amino acid glutamate: Both substances are relatively abundant throughout the brain, and both are involved in a multiplicity of biochemical processes via the proteins of which they are parts. As it happens, the parallel between zinc and glutamate is especially apt because zinc, like glutamate, appears to have a specific neurosecretory role in certain CNS neurons in addition to its general role as a structural component of proteins. Not surprisingly, many of the same methodological and theoretical problems arising in the study of neuro-

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secretory glutamate and the glutamatergic neuron have arisen as well in the study of neurosecretory zinc and the zinc-containing neuron. T h e necessity of distinguishing between enzymatic zinc and neurosecretory zinc will be a recurrent theme in the present review, with zinc-containing neurons a major focus.

B. THREE POOLSOF ZINC I N

THE

BRAIN

Throughout the following review, the reader may find it useful to keep in mind three rather separate pools of CNS zinc: vesicular zinc, free zinc, and protein-bound zinc. Vesicular zinc is that which is sequestered in the presynaptic vesicles of a special class of neurons, the zinc-containing neurons, which are found primarily in limbic, cerebrocortical, and corticofugal systems (Frederickson and Danscher, 1988).This vesicular zinc can be selectively stained by several histochemical procedures and is therefore accessible to histological and histoanalytical studies. What functions zinc serves within vesicles or after physiological release into the synaptic cleft is a subject of special interest in zinc neurobiology. However, in reviewing the data on zinc per se it must be borne in mind that vesicular zinc is probably no more than 5-15% of the total zinc in the brain, and quantitative analyses of the amounts, distribution, and turnover of brain zinc will therefore be insensitive to variations in the vesicular pool. Special histochemical techniques must be used to isolate the vesicular pool for study. T h e second pool, free zinc, is an entirely hypothetical pool of ionic Zn’‘ in the cytosol o r interstitial fluid that may or may not be present in normal, healthy brain tissue. There are many zinc-binding ligands in brain tissue, and the reasonable guess is that any ionic zinc that is released (e.g., from zinc-containing vesicles) would quickly be bound. Indeed, specific peptides and proteins that may serve to sequester free zinc (much as various calcium-binding proteins sequester that ion) (Kawaguchi et al., 1987; Permentier et al., 1987) have been tentatively identified in the brain. Nonetheless, just as transient pulses of free calcium serve as important neural signals, it is plausible that similar transient zinc currents also serve as intercellular or intracellular signals. Literally dozens of neural systems that respond to the presence of ionic zinc in the milieu will be discussed later in this review. Whether the “signal” of free zinc ions is ever sent, however, remains to be determined. T h e third pool of zinc is that which is is bound firmly into the structure of the many zinc-containing enzymes in the brain. These large,

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30-400 kDa proteins generally incorporate their zinc atoms at the time of synthesis and neither donate nor accept zinc ions thereafter (Vallee, 1980, 1985; Vallee and Galdes, 1984). The enzymatic zinc is therefore a stable pool, involved only in the specific function of the zinc-containing enzymes. Quantitatively, however, the enzymatic zinc pool is quite important, because as much as 85-95% of the zinc in the brain is likely to be bound into the zinc-containing enzymes; quantitative studies of zinc levels or zinc turnover will be dominated by this enzymatic zinc pool. C. EARLY HISTORY

Evidence of zinc’s biological importance is generally traced to Raulin (1869), who showed that the common bread mold Aspergallus nzger would not survive without the element. The essentiality of dietary zinc for mammals was established in studies of rat diets in 1934 (Todd et al., 1934). The first true zinc metalloenzyme (carbonic anhydrase) was identified in 1940 (Keilin and Mann, 1940). The earliest hint of a role of zinc in the brain was Rost’s (1920) discovery that the human brain contained significant amounts of the element. Rost’s interest sprang from concern about zinc toxicity among brass foundry workers, but he correctly ascertained that there was also zinc in the tissues of subjects who had no industrial exposure to the element. Rost’s results were quickly corroborated by Bodansky (192 l ) , who found an average of about 40 parts per million (ppm) dry weight of zinc in samples from several human brains. Further instrumental assays done through the 1930s and 1940s reaffirmed and extended Rost’s and Brodansky’s initial observations that there is zinc in the mammalian brain (see, e.g., Alexander and Mayerson, 1938), but it was not until 1955 that the topic of zinc entered the general literature of the brain sciences. The latter development was the result of a seminal paper by Maske (1955), who discovered that histochemical staining for zinc yielded a vivid, selective labeling of discrete areas of the hippocampal formation. Maske’s report was followed by a spate of neurohistochemical and neuroanatomical studies in the late 1950s and early 1960s (see, e.g., Fleischhauer and Horstmann, 1957; Timm, 1958a,b; Purpura, 1959; McLardy, 1960) in which the zinc staining of certain parts of the hippocampal formation and the amygdakd was explored in some detail. These early histochemical studies laid the foundation for much of

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contemporary work on zinc in the brain. As it turns out, histochemists of that early era were (unwittingly) selectively staining the pool of zinc that is sequestered in the axonal boutons of zinc-containing neurons. What is today the most thoroughly characterized of the CNS zinc-containing fiber systems, the mossy fiber system of the hippocampus, is the same axonal system that was first stained by Maske in 1955. In addition to the early analytical and histochemical work, a third line of historically important research on zinc in the brain developed from nutritional studies done in the 1960s. Hurley and others (Hurley and Swenerton, 1966; Blamberg et al., 1960) established that zinc insufficiency can produce gross abnormalities of brain development in animals (for reviews see Prasad and Oberleas, 1970; Hurley and Schrader, 1972). At about the same time, the pioneering work of Prasad, Sandstead, Halsted, and others (Prasad et al., 1961, 1963) uncovered the sometimes devastating clinical symptoms of early dietary zinc deficiency in man. Neuropsychological deficits proved to be among the consequences of early zinc undernutrition in man, just as in laboratory animals (for reviews see Prasad, 1979; Rogers et al., 1985). T h e impact of perinatal zinc undernutrition upon developing brains continues to be an important focus of zinc research with rather direct clinical implications (Bergman et al., 1980; Sandstead, 1985; Swanson and King, 1987; Wallwork, 1987).

11. Distribution of Elemental Zinc in the Brain

A. METHODSOF ANALYSIS It is a truism that all fields of inquiry are limited by their methods, but methodological problems have been especially troublesome in research on zinc. T h e crux of the matter is that zinc is (1) an element, not a complex organic molecule, and (2) a relatively common element, present in significant amounts in virtually every corner of a laboratory. T h e first of these conditions, that zinc is an element, means that the highly selective histochemical techniques that are based on the unique structure of complex proteins, that is, immunohistochemistry or substrate-enzyme reactions, cannot be used in the study of zinc. T h e second condition, the ubiquity of zinc, means that one must be constantly vigilant lest stray zinc contaminate an assay or histochemical experiment.

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B. INSTRUMENTALASSAYS 1. Bulk Analysis a. Monitoring Contamination. Wet brain tissue contains an average of about 10 pg/g of zinc, and there are a half-dozen or more methods that allow routine detection of 1 ng of the metal (Table I). Thus, with respect to instrument sensitivity, determinating the zinc content of 0.1 mg of wet brain tissue is entirely feasible. However, instrument sensitivity is rarely the limiting factor in assaying zinc. In practice, it is uncontrolled sample contamination that limits the accuracy of zinc determinations; the problem has been discussed and reviewed repeatedly (see, e.g., Klitenick et al., 1983; Janghorbani, 1984; Vallee and Galdes, 1984; Smith et al., 1985). So-called ultrapure reagents, for example, typically contain from 0.1 to 3-4 ng/ml of zinc, and water cleansed by ion-exchange systems, 0.05-0.1 ng/ml. Moreover, glass and plasticware, regardless of cleaning techniques, will retain some zinc, which can readily move into the sample solutions, particularly into hot acidic solutions used to decompose tissue. Finally, typical urban room air contains 0.1- 1.0 pg of zinc per cubic meter, and the perspiration on finger tips has about 1 ng of zinc in each 1 p1 of fluid (Henkin, 1979).The upshot of this is that sample contamination is an unavoidable factor in any measurement of zinc. Because sample contamination cannot be avoided, it must be monitored by the use of processing blanks when assaying the zinc content of tissue. Ideally, several empty vessels are set aside during tissue collection, left empty of tissue, and then processed side-by-side with the tissuecontaining vessels through all stages of tissue preparation including addition of acids, oven drying, addition of solvents, and so forth. T h e mean zinc content of these blank vessels is then taken as the average contamination of samples, a value that can be subtracted from the observed zinc content of samples. Just as important as the mean level of contamination, however, is the variability of values recorded for the blanks. This variability represents the total error variance of the measurement procedure, and thus the variability of the blanks sets the true limit to precision in measurements of tissue zinc. As a practical guide, we have found that decomposing 50 mg pieces of brain tissue typically introduces about 40 f- 15 ng (SD) of stray zinc, and special vapor decomposition methods (Klitenick et al., 1983) can reduce this blank only to about 3 rt 2 ng of zinc per sample. This means that at least 300 ng of zinc (6 mg of dry brain tissue) are needed in the initial sample for precise determination (SDlmean = 15/300 = 5%)

151

NEUROBIOLOGY OF ZINC

TABLE I ZINCANALYTIC TECHNIQUES Method Bulk Analysis Atomic emission spectroscopy Plasma emission (PLEMS) Atomic Absorption Spectroscopy Flame (AA) Flanieless (FAA) Neutron activation analysis (NAA) Instrumental (INAA) Radiochemical (RNAA) Stable isotope mass spectrometry (SIMS) Particle-induced X-ray fluorescence (PIXE) X-ray-induced X-ray fluorescence (XRFA) Spectrochemical Analysis (SC) Absorptiometry, fluorometry Regional elemental microprobe analysis' Electron-induced X-ray fluorescence Micro PIXE Laser activated mass spectrum analysis' lon-probe mass spectrum microanalysis'

Sensitivity (g)

10-9

10- 10

Reference"

(1)

10-14

(2) (3)

10-9

(4) (5)

10-9

(6)

10-10

(7) (8)

1o-fi

(9)

10-14

(10)

10-13

(1 1) (12) (13)

10- 15 10-15

" References: (1) Wallwork rt al. (1983); Hershey el al. (1987); (2) Liska et al. (1985);Piclle el al. (1986); (3) Alcock (1984); Smeyers-Verbeke (1985); (4) Ward el al. (1987); Thompson et al. (1988); (5) Grimanis and Pertessis-Keiss (1987); (6) Klitenick el al. ( 1 983); P e k e et al. ( I 987); (7) Thorlacius-Ussing PI al. ( 1 988); Yagi pt al. (1987); (8) Merriani et ul.(1979); (9) Sethi ~t al. (1985); Mahanand and Houck (1968); (10) Falkmer et al. (1985); Dinsdale (1984); Elmes a n d Jones (3981); ( 1 1 ) Wensink el al. (1987a); L.englet el al. (1984); (12) Denoycr (1984); (13) Trottier et al. (1982); Duckett and Galle (1985). ' Absolute sensitivities of microprobes depend on concentration in the probed microregion rather than total mass of zinc; concentration sensitivities are often as poor as 50-100 ppm dry weight. ' Neither method has been proven for zinc in biological tissue; sensitivities a r e estimates, based on ionization potential of zinc.

using conventional decomposition methods, and at least 40 ng of zinc ( 1 mg of tissue) are needed when vapor decomposition is used. Needless to say, if the mean and variability of the blank are not measured directly,

both will be erroneously included in the estimated tissue content of zinc, and a biologically implausible result will be reported (e.g., 300 ? 250 p g l g of zinc in hippocampal tissue) (Valdes et al., 1982).

152

CHRISTOPHER J. FREDERICKSON

There are two approaches to minimizing the contamination of tissue during processing: (1) Minimize the amount of tissue processing, and (2) minimize contamination during processing. For minimal tissue processing, the best procedure is neutron activation (NAA) (Table I), in which dissected samples are packaged, weighed, lyophilized, reweighed, transferred to vials, irradiated with neutrons, then assayed for elements by counting y emissions from the "Zn (Ehmann et al., 1986). Except for the relatively low sensitivity of the procedure (lo-' g of zinc), the NAA procedure is attractive for those having the equipment and resources needed. The second method that requires no chemical dissolution of samples prior to assaying them is proton-induced X-ray emissions (PIXE). PIXE analysis samples are dried in a thin uniform layer onto some type of supporting film (Lenglet et al., 1984; Wensink et al., 1987a; ThorlaciusUssing et al., 1988), which is then inserted into a proton beam for assaying the zinc by analysis of the induced X-ray fluorescence. T h e tissue on the film can be either a microtome section or a dried layer of tissue homogenate. Like NAA, PIXE requires access to a particle accelerator and appropriate emission counting equipment. In addition to NAA and PIXE, the remaining methods of bulk analysis require chemical dissolution of samples and (for some) extraction of the analyte from the decomposed elemental matrix. b. Additional Sources of Analytic Error. Each instrumental method has pitfalls peculiar to it, but some of the common problems are worth mentioning briefly. Loss of analyte, for example, can easily occur during processing. Volatile organo-zinc compounds can form and evaporate from solutions, and the glass and plasticware surfaces contacted by sample solutions can adsorb significant amounts of zinc, particularly when they have been stripped of metals by soaking in hot acids. Chemical interference, or matrix effects, are also a major source of error with techniques that depend on the chemical speciation of the analyte. With atoniic absorption spectroscopy, for example, the absorption of photons depends upon the orbital states of the zinc electrons during the atomization of the sample in the flame or graphite furnace. Thus, two samples with identical zinc content but different matrices (e.g., different potassium content) can give two quite different apparent values for zinc by atomic absorption assay (Alcock, 1984; SmeyersVerbeke, 1985). Similar chemical matrix interference problems arise in procedures that require electron transfer (e.g., voltametry) or chemical binding (e.g., absorptiometry, fluorometry) between the zinc and other systems. A set of problems related to the interference problem comprises the

NEUROBIOLOGY OF ZINC

153

choice of reference o r calibration standards used in an assay procedure. In atomic absorption, for example, a calibration standard of zinc chloride in water is subject to different matrix effects than is one of sample zinc in the matrix of decomposed tissue. Thus, equal absorbances from the two solutions do not imply equal amounts of zinc. Similar problems obtain in fluorometry (different quantum yields in different matrices) and in other methods. The traditional test to guard against these effects is the method of standard additions, in which different (known) amounts of analyte are added directly to aliquots of a sample solution and the results are extrapolated to indicate the zinc in the unadulterated sample. Methods based on nuclear interactions (NAA, PIXE) are of course immune to chemical interactions, so the comparison between calibration standards and samples is less problematic. In stable-isotope dilution mass spectrometry (SIDMS), the reference material (added stable zinc isotopes) is measured simultaneously with the analyte (endogenous zinc isotopes), so the problem of matching standard to samples is not encountered. Regardless of the specific methods used, the final (and obligatory) test of absolute accuracy of any procedure is the measurement of zinc in a certified reference organic material, such as the reference materials available from the United States National Bureau of Standards.

2. Regional Analysis and Microprobe For determination of the total elemental zinc in specific cytoarchitectonic regions of the brain, one can either use a miscrodissection approach (Frederickson et al., 1983) or one of the elemental microprobe methods listed in Table I. So far as we know, the only regional microprobe method that has ever been successfully applied to zinc in CNS tissue is the micro-PIXE method, in which a proton beam has been used to probe successive 30 ,um spots in tissue sections for zinc (Lenglet et al., 1984; Wensink et al., 1987a). Other microprobing methods have been used for zinc in other tissues, however (Dinsdale, 1984; Nabarra et al., 1984), and could presumably be used for CNS zinc as well. OF ZINC C. DISTRIBUTION

1. Regzonal Distribution

Table I1 summarizes selected surveys of zinc distribution in the central nervous system. The first pattern apparent is that there is a higher concentration of zinc in grey matter than in white matter. This is a consistent finding, and values for different samples of white matter are

TABLE I1 MEASUREMENTS OF CNS ZINC

Method AA FAA PLEMS SIMS

NAA PIXE

Species Human Human Rat Human Rat Human Rat Dentate Mossy fiber Human Rat Rat Dentate Mossv fiber

Hippocampus 108

Cortex white

Spinal cord

Ponsmedulla

Cerebellar cortex

69-82 86-89 80 65

33 30 40 -

-

38 39 -

76 -

60

60-70 -

28 -

28 26

-

61 -

74

60 62

27 -

-

32

65

-

-

-

-

Amygdala

74

84 102

133

-

72-84 79 145 81 71

55 136

-

-

-

Cortex gray

-

-

-

Reference' (1) (2) (3) (4) (5)

(6) (78)

(%lo) ( 1 1)

(12)

' Cortex, cerebral cortex, gray or white matter. References: (1) Smeyers-Verbeke el al. (1974); (2) Volk el al. (1974);(3) Scheuhammer and Cherian (1981); (4) Hershey et a!. (1985); (5) Wallwork el al. (1983); (6) Kasarskis et al. (1985);(7) Frederickson el al. ( 1 982); (8) Frederickson ct al. (1983);(9) Ehmann et al. (1986);(10) Thompson et al. (1988); ( 1 1) Chan el al. (1983); (12) Wensink el al. (19874.

NEUROBIOLOGY OF ZINC

155

quite uniform regardless of the source, with corpus callosum (33 ppm) (Smeyers-Verbeke et al., 1974), fimbria (35 ppm) (Frederickson et al., 1983), and subcortical white matter (27 ppm) (Ehmann et al., 1986) all having essentially the same content of zinc. Brain regions with mixed grey and white matter have zinc contents reflecting the mixture. Thus, the spinal cord contains correspondingly more zinc in segments with a higher proportion of grey matter (sacral segments) and less zinc in the segments with a lower proportion of grey matter (Kasarskis et al., 1985a). Similarly, assays of whole cerebellum (including white matter) tend to yield lower zinc concentrations (45-55 ppm) (Chan et al., 1983; Wallwork et al., 1983) than do assays of the cerebellar cortex (6070 ppm) (Smeyers-Verbeke et al., 1974; Kasarskis et al., 1985a). T h e difference between grey and white matter is robust and reliable, but it is worth noting that the size of the difference depends in part upon one’s point of view. For example, when values for zinc are expressed as a fraction of tissue dry weight (as in this text and Table 11), the difference is about 2.5-fold: 25-30 ppm of zinc for white matter, and 60-70 ppm for grey. In contrast, if one measures micrograms of zinc per gram of tissue water, the difference is smaller (about 13 pg/g for white matter, and 17 pgig for grey) because of the lower water content of white matter (about 70%) compared to grey matter (80%).This implies that the zinc concentration of cytosol is fairly constant. The second point of the survey data in Table I1 is that there is rather little variation in the zinc content of different regions of grey matter. Cortical tissue from limbic, cerebral, and cerebellar structures alike has 50-80 pprn of zinc, with the limbic (hippocampus) values being slightly higher than other cerebral and cerebellocortical values. In fact, the largest regional difference in zinc content of CNS grey matter is the difference between the hippocampal CA4 subfield (about 150 pprn of zinc) and adjacent hippocampal grey matter (60-70 ppm), a ratio of about 2.5-fold that several groups have confirmed (McLardy, 1975; Danscher et al., 1976; Frederickson et al., 1983; Wensink et al., 1987a). Among the different vertebrate species that have been studied, the concentration of zinc in the CNS is relatively constant, with reported porcine levels being somewhat lower than those of the other species (Table 111). In early postnatal development there is a substantial increase in both the total amount of zinc in the brain (as the total mass of the brain increases) and in the concentration of zinc per gram of wet brain tissue (Table IV). These developmental changes have been confirmed in rat and human brains by several groups of investigators, but there are difficulties of interpretation that are sometimes overlooked. Specifically,

156

CHKISTOPHER J. FREDERICKSON

TABLE 111 CNS ZINC IN VARIOUSSPECIES Brain region Species

Cortex

Hippocampus

Brain stem

References"

Rat Human Rabbit cow Pig Lizard Fish Cat

60-70 60-70 66 66 46

70-85 70-80 85 70 48 80

32 38 38 39 22 35 34 44

(1) (2) (3) (4) (495) (6) (7)

-

68

(8)

References: (1) see Table 11; (2) see Table 11; (3) Kehrnan et al. (1982): (4) Wong and Fritze (1969); (5) Hesketh et al. (1985): (6) Molowny et al. (1987); (7) Maler et al. (1984); (8) Palm et al. (1985).

TABLE IV LIFESPAN Species Human (wet wt) Human (wet wt) Rat (wet wt) Mouse (wet wt) Kat (wet wt) (dry wt) (zinc/protein) (hipporam pus) Rat (wet wt) Rat (wet wt) Kai (dry wt) Rat (wet wt) Human (Wet W t ) ~~

CHANCES I N

CNS Z l N C

Early development Yes (+50%) Yes (+50%) Yes (+40%) Yes (+40%) NO

Aging

Reference"

No

(1)

NO -

(2) (3) (4)

-

-

(5)

NO

Yes (+60%) Yes (+50%) Yes (varies with region) Decline No (started postnatal day 16) Yes (varies with region)

No

-

(6) (7)

(8) (9) (10)

-

~~

"Reterences: ( I ) Hershey el al. (1985); (2) Ehniann et al. (1984); (3) Kozma and Ferkc (1979); (4) Keen and Hurley (1979): (5) Crawford and Connor (1972); ( 6 ) Szerdahelyi and Khsa (1983): (7) Lai et al. (1985b); (8) Ebadi el al. (1984a): (9) Kofod (1970); (10) V d k et al. (1974).

NEUROBIOLOGY OF ZINC

157

because the relative water content of the brain falls during early development (from about 90% to about 80% for the rat) (Crawford and Connor, 1972; Lai et al., 1985a), the proportion of total dry material in the brain doubles (rising from 10 to 20%) during that same period. Thus, when zinc concentration is expressed as a fraction of brain dry weight (or as the ratio of zinc to protein) one may find no change, or even a decrease, in the concentration of zinc during early development (Ebadi et al., 1984a). Inasmuch as the magnitudes of developmental changes in water and protein content vary among different brain regions, it follows that the developmental changes in zinc will also vary among brain regions (Crawford and Connor, 1973). Which regions show the largest developmental changes depends largely upon what parameter (wet weight, dry weight, total protein) is used for the denominator in the calculations. In contrast to early development, aging does not appear to be accompanied by consistent changes in CNS zinc concentration (Table IV). T h e data on human subjects suggest little variation in zinc from about the second through the eighth decade of life in normal subjects. Rats, likewise, show stable brain zinc levels through the age usually studied (up to 100-200 days) and apparently u p through the age of 750 days as well (Lai et al., 1985a).

2. Subcellular Compartmentalization T h e technique of separating subcellular organelles and tissue fragments by homogenization and differential centrifugation, a standard, workhorse method in neurochemistry, has not been used often for localizing zinc. This may be because of the technical difficulties of avoiding contamination (or mobilization and redistribution) of zinc during the many steps of homogenization and fractionation. In fact, in their early study of zinc in brain tissue fractions, Rajan et al., (1976) noted that “fractions which were collected from rat brains at different times showed significant variations in their metal contents,” and showed coefficients of variance of up to 72% (SDImean) for the mean zinc contents of individual tissue fractions. T h e technical challenges notwithstanding, tissue fractionation has yielded some consistent results on the subcellular distribution of zinc in the brain (Table V). For example, several studies indicate that zinc generally follows protein through differential fractionation, with the zinc-protein ratio remaining fairly constant (at about 100-200 ng of zinc/mg of protein, depending on the particular study) in all fractions. Similar findings have been reported by Crawford and Harris (1984) who

TABLE V ZINCI N SUBCELLULAR FRACTIONS Fraction"

PI Brain region

nuclear

Whole brain Hippocampus

134 58%'

Hippocampus Hippocampus Various regions Whole brain Various regions Hippocampus Hippocampus

85 34%

P2

s1

mitochondria

s2

P3

94 -

15% 107

-

-

22%'

-

-

15% 97 -

-

87 -

13O-17Od

-

30od -

150

213

-

230

100

-

50 220 196

73

-

67 4% 129

-

9%

120

-

-

-

-

-

-

-

s3 cytosol

133 16% 1 lo' 42%

-

-

-

B2 synaptosomal

Referencesb

15% -

(1)

-

(4) (5) (6)

-

120 230 177

(2) (3)

(7) (8)

(9,10)

Numbers indicate nanograms of zinc per milligram of protein; percentages are percentage of total brain zinc in fraction.

* References: (1) Colburn and Maas (1965); (2) Crawford and Connor (1973); (3)Sato el d(1984a) (4) Szerdahelyi et al. (1982), (5) Itoh etal. (1983); (6) Ebadi and Wallwork (1985); (7) Rajan et al. (1976); (8) Kalinowski et al. (1983); (9) Crawford and Harris (1984); (10) Taylor et al. (1982). Special techniques used to maximize cytosol zinc. Supernatant from single centrifugation.

NEUROBIOLOGY OF ZINC

159

analyzed nine separate fractions corresponding to synaptosomes, myelin, mitochondria, nuclei, and other organelles. In that work, the zinc concentration of various fractions was reported to be about 200 ng/mg of protein (ranging from 140 to 253 ng/mg) for eight of the nine fractions analyzed (Harris and Crawford, 198 1). Presumably, this relatively constant zinc-protein ratio reflects the abundance of zinc that is bound into the structure of zinc metalloenzymes in the brain (see below). Consistent with this interpretation, Harris and Crawford (198 1) found that 84% of zinc in brain homogenates was retained during dialysis of the homogenate across a membrane with a 12 kDa cutoff; apparently about 84% of zinc is tightly bound to proteins larger than 12 KDa, and 16%is either weakly bound, bound to smaller ligands, or (possibly) free as ionic zinc. Because histochemical data indicate that zinc is selectively concentrated in certain axonal boutons (in the vesicles of boutons), zinc enrichment of synaptosomal fractions has been sought. Rajan et al., (1976) reported variable and inconsistent concentration of zinc in some synaptosomal fractions, and Kalinowski et al., (1983) found synaptosomes to have about the same amount of zinc as the mitochondria1 fraction, but more than the crude nuclear sediment. Crawford and Harris (1984) found that the standard synaptosomal fractions from cerebellar and hippocampal tissue showed essentially no excess of zinc beyond that found in other fractions. However, a fraction prepared by those authors that was presumed to be selectively rich in large synaptosomes did show three-fold more zinc than other fractions. Interestingly, both the cerebellum (containing large synaptic glomeruli, most of which do not stain for zinc) and the hippocampus [which has large mossy fiber boutons that do stain for zinc (see below)] showed the same apparently high levels of zinc (667 and 597 ng/mg, respectively) in the large synaptosomal tissue fraction.

111. Uptake and Turnover of Brain Zinc

Whole blood contains about 7000 pg of zinc per liter (Henkin, 1979), but the amount that is freely available for transfer to the brain is only about 0.2%of that amount. Approximately 85% of the zinc in blood is in the erythrocytes, leukocytes, and platelets, and 15% (about 1000 pg/ liter) is in the serum (Henkin, 1979; Smith et al., 1985). Serum zinc, in turn, is partitioned among three compartments: protein-bound, microligand-bound, and free ionic Zn+2.The zinc bound to serum proteins

160

CHRISTOPHER J. FREDERICKSON

(chiefly a*-macroglobulin and albumin) is about 98% of total serum zinc, and microligands (e.g., cysteine, histidine, and possibly porphyrin), are generally estimated to bind 1-2% of the zinc (10-20 pghiter) (Prasad and Oberleas, 1968; Parisi and Vallee, 1969; Henkin, 1979; DiSilvestro and Cousins, 1983). The concentration of ionic Zn+2 in serum is negligible, probably no more than 0.065 pgiliter (Berthon et al., 1980) to .013 pg/liter (Magneson et al., 1987), that is, 1.0-0.2 nM. Radiotracer studies indicate that exogenous zinc distributes rapidly and proportionately among the various exchangeable compartments (albumin-bound, microligand-bound, and ionic) in the serum (Chesters and Will, 1981a,b; Magneson et al., 1987). During physiologic stress, such as endotoxin challenge, redistribution of zinc to serum ligands such as transferrin and lactoferrin is also rapid and is quantitatively substantial (Goldblum et al., 1987). The small, ultrafilterable microligands are presumed to diffuse freely into extracellular compartments (Henkin, 1979; Berthon et al., 1980), and it is therefore this fraction of serum zinc (10-20 pg/liter) that would have most direct access to the CSF and interstitial compartments of the brain. The concentration of zinc in CSF is virtually the same as the concentration of ultrafilterable (microligand-bound) zinc in serum, 10-20 pg/liter (Hershey et al., 1983, 1984; Palm et al., 1986), suggesting that the microligand zinc in serum and the zinc in CSF may be in relatively free and direct exchange, perhaps by facilitated diffusion. This is reminiscent of the situation of lead, for example, which also seems to move freely from the ultrafilterable fraction in serum into the CSF (Manton and Cook, 1984; Manton et al., 1984). In the case of zinc, the radiotracer evidence supports the notion of a rapid exchange of serum microligand zinc and CSF zinc. Kasarskis’data, for example, show that the specific activity of CSF zinc (about 30,000 counts/pg of zinc, assuming 10 pg of zindliter) is virtually the same as that of serum zinc (about 30,000 countsipg) within 15 min after an intraperitoneal injection of ‘5Zn (Kasarskis, 1984a,b). Moreover, the same data show that the serum and CSF radioactivity levels fall simultaneously by 10-fold (as the radiotracer is absorbed from serum into other tissue) (Shaikh and Lucis, 1972; Chausmer et al., 1980; Stacy and Klaassen, 1981) within 45 min after reaching peak values. Rapid equilibration of the CSF with the serum zinc is implied by these data. Curiously, the choroid plexus accumulates both lead (Manton et al., 1984; Manton and Cook, 1984) and zinc (Kasarskis, 1984a,b; Hershey et al., 198’7),even though it does not appear to present any barrier to the movement of those metals from the ultrafilterable fraction of serum into the CSF. At a concentration of 10 pgt’liter, the total amount of zinc in CSF and

NEUROBIOLOGY OF ZINC

161

interstitial fluid would be less than 0.1% of the total zinc in the brain, which averages about 10 pglg of wet weight. Changes in the concentration of the zinc in the CSF-interstitial compartment would therefore be undetectable when assaying total CNS zinc, and the possibility that extracellular zinc levels follow serum levels during induced or spontaneous fluctuations cannot be evaluated by assaying CNS zinc. Whereas equilibration between serum and extracellular zinc depots may be rapid, and “passive,” the net exchange of zinc between brain cells and serum is not. After a single systemic injection of 65Zn,the radioactivity level of the brain rises slowly (towards the higher specific activity level of serum) for about a week, then falls gradually over the next several weeks (Czerniak and Haim, 1971; Garcia del Amo et al., 1974; Sato et al., 1984b; Kasarskis, 1984a,b; Wensink et al., 1987b). T h e turnover rate of zinc in brain is therefore slow, with an estimated half-time of 22 days (Sato et al., 1984b; Wensink et al., 1987b). In general, the radiolabeled zinc that is taken into the brain after 65Zn administration is rapidly dispersed among the different organelles of the brain cells. When expressed as counts per milligram of protein, the distribution of newly incorporated “Zn is quite uniform among all subcellular fractions of brain tissue (Wensink et al., 1987b; see also Sato et al., 1984a). This is consistent with the notion that most of the zinc is incorporated into metalloproteins, which are more or less uniformly distributed throughout all organelles (see below). When turnover rates for zinc in different subcellular fractions are estimated from the clearance rate for fi5Znadministered in vivo, the results indicate that turnover is somewhat faster for zinc associated with the myelin fraction (15 days) and somewhat longer for synaptosomes (30 days) (Wensink et al., 1987b; see also Sato et al., 1984b). T h e latter result suggests that myelin-associated zinc metalloproteins may turn over more rapidly (or recycle zinc less efficiently) than synaptosomal metalloproteins. The mechanism by which zinc enters brain cells is unknown. The concentration gradient from the interstitial fluid (0.15 pM, as estimated from the cerebrospinal fluid concentration) to the interior of cells (approximately 150 p M , as estimated from the average total concentration of zinc in the brain) is about a lo3 difference, implying that energy-dependent transport of zinc is involved. Such an energydependent, high-affinity uptake process has been observed in hippocampal slices and in synaptosomes in vitro. The apparent K , values observed in those studies of 1.2 FM (Crawford and Harris, 1984), 12 p M (Howell et al., 1984), and 12-23 p M (Record et al., 1982; Dreosti and Record, 1984) are one or two orders of magnitude higher than the

162

CHRISTOPHER

1 . FREDERICKSON

0.15 p M of zinc in cerebrospinal fluid (CSF), indicating that uptake generally proceeds at 0.1-0.01 of the K , value. T h e high-affinity uptake into brain slices is slow, showing no saturation over 3 hr (Howell and Frederickson, 1984). This is comparable to zinc uptake into organs such as the pancreas ( K , 1.5 p M ) , which takes over 24 hr to saturate (Ludvigsen et al., 1979; Figlewicz et al., 1980). Wensink et al., ( 1988) have recently reported particularly elegant analyses of uptake into synaptosomes in which the K , value was determined for free, ionic zinc, as opposed to total zinc added to the medium. The resulting apparent K , was 0.25 pM (calculated free zinc). The authors point out that 0.25 pM is about 25-fold higher than their estimate of the level of free zinc in the extracellular fluid (lo&), and it is worth recalling that the estimated free zinc in serum is lower still, about 0.2-1.0 nA4 (see above). In their synaptosomal preparation, Wensink et al. (1988) observed that exogenous albumin, glutathione, and histidine all reduced uptake, implying that none of those ligands participates in a carrier-mediated transport. The possible role of other microligands (e.g., cysteine) in carrier-mediated transport into brain cells (as suggested for pancreatic cells) (Ludvigsen et al., 1979) remains unexplored. Given that extracellular zinc concentration is low in the brain and that turnover of zinc in brain cells is slow, it is to be expected that raising or lowering serum zinc will cause no major, immediate change in the total burden of zinc in the brain. This is the case. For example, dietary zinc deficiency, which causes an immediate drop in serum level, causes no general decrease in total brain zinc even when continued for several weeks (Dreosti et al., 1981; Szerdahe1yi.d al., 1982; Wallwork el al., 1983; Hesketh et al., 1985; Wensink et al., 1987a,b; Law et al., 1987; Cossack and Prasad, 1987). One possible exception to this general finding is a reported 12% decrease in cerebellar zinc in pigs (as compared to zinc-supplemented pigs) (Hesketh et al., 1985), which may be contrasted with a trend towards increased cerebellar zinc observed in zinc-deficient rat pups (Dreosti et al., 1981). Another possible exception is the reported decline in zinc in the hippocampal mossy fiber neuropil, a region in which a high proportion of the element is sequestered in the mossy axonal boutons (Frederickson et al., 1981, 1982, 1983). In their microPIXE study, Wensink et al., (1987a) found a 30% decline in mossy fiber zinc but no change in the remainder of the hippocampus in rats undergoing 90 days of zinc undernutrition. Dreosti et al. (1981) observed a reduced Timm’s metal staining in the same cytoarchitectonic zone in rats after 30 days of zinc undernutrition, whereas neither Buell et al. (1977) nor Petit and LeBoutillier (1986) saw any histochemical

NEUROBIOLOGY OF ZINC

163

change in the mossy fiber regions after comparable periods of zinc under nutrition In accord with the general stability of the total burden of CNS zinc during dietary deficiency, it is generally reported that the distribution of zinc among subcellular compartments of the brain is not altered during dietary zinc insufficiency (Szerdahelyi et al., 1982; Ebadi and Wallwork, 1985; Wensink et al., 1987b). Synthesis of 65Zn-bindingmetallothioneinlike protein in the brain (see below), is, however, apparently reduced by prior zinc undernutrition (Ebadi and Wallwork, 1985). Kasarskis ( 1984a,b) has suggested that the apparent brain sparing that keeps brain zinc high when serum level falls may involve mechanisms that retard the loss of brain zinc when serum (and possibly interstitial fluid) levels are low. T h e data of Wensink et al. (1987b) have provided direct support for that hypothesis. In the latter work, the loss of radiolabeled zinc from the brain was substantially slower in rats that had been zinc-deprived than in normal rats. A possible “pump” extruding zinc from the CSF via the choroid plexus has been proposed (Kasarskis, 1984a,b), though regulated extrusion from within cells is another possibility. One experimental approach to the mechanism(s) regulating zinc extrusion is suggested by the work of Zeneroli et al. (1984) and Kasarskis et al. (I985a), whose evidence indicates that liver dysfunction causes a rapid, profound loss of cerebral zinc. Conceivably, endotoxins liberated from the degenerating liver tissue interfere directly with the cellular mechanisms responsible for the extrusion of zinc from the brain. Raising serum zinc also fails to cause any immediate, pronounced change in total cerebral zinc concentration. Several investigators have assessed brain zinc levels by instrumental or histochemical analysis after systemic zinc loading and found that brain levels rise only slightly or not at all (Berger and O’Leary, 1975; Itoh et al., 1983a; Baraldi et al., 1986; but see Hasan and Seth, 1981; Rehman et al., 1982). Similarly, the brain metallothionein-like proteins that can be induced by intracerebral zinc administration (see below) are not induced by systemic zinc loading (Ebadi, 1986a). If, however, the zinc levels in the CSF and interstitial fluid passively follow the level of microligand-bound zinc in serum as discussed above, then it is to be expected that raising or lowering serum zinc would directly affect the cerebral extracellular zinc concentrations. In fact, “Zn is found throughout the brain within minutes after systemic administration (Kasarskis, 1984 a,b), and systemic zinc loading has immediate behavioral effects (Tokuoka et al., 1967; Porsche, 1983; Baraldi et al., 1986; Howell et al., 1986), which could reflect changes in the extracellular zinc concentration in the brain.

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IV. Zinc and Brain Proteins

There are three major categories of zinc-protein associations that are relevant to understanding zinc’s roles in the CNS: (1)zinc-containing proteins, (2)zinc-modulated proteins, and (3) zinc-protein complexes. The first category, the zinc-containing proteins, may be further divided into two subcategories, namely, zinc-containing enzymes (i.e., zinc metalloenzymes), and proteins that contain zinc but have no known enzymatic function. All of these different categories represent essentially independent roles of the zinc atom or ion; thus, whether an enzyme is zinc-containing does not determine whether it is also zincmodulated or, for that matter, whether it forms zinc-protein complexes (for reviews see Vallee, 1980, 1985; Spiro, 1983; Vallee and Galdes, 1984).

A. ZINC-CONTAINING PROTEINS Zinc is a constituent of over 50 different enzymes (over 200, counting isozymes) and of additional proteins without known enzymatic function. Vallee has proposed a specific terminology for use in describing the various roles of zinc in proteins as follows. Zinc has a catalytic role when the atom(s) are directly involved in enzymatic function and located at the active enzymatic site. A structural assignment means that the zinc atoms are necessary for maintaining protein conformation, typically stabilizing the tertiary structure of the molecule. Noncatalytic, (or undefined) has been suggested as a term for cases in which neither a catalytic nor a structural function of the zinc atom(s) in a protein has been found. Potentially important functions of proteins of this last type, in which the zinc is apparently neither a catalytic nor a structural necessity, would be scavenging, transport and/or storage of free zinc ions. 1. Metalloenzymes

The zinc metalloenzymes are functionally diverse, belonging to all six Enzyme Commission (EC) classes. Table VI shows that many key enzymes involved in nucleic acid and protein synthesis, energy metabolism, and metabolism of intracellular messenger molecules are zinc metalloenzymes. Moreover, zinc-containing enzymes are among those found in nuclei (e.g., nucleic polymerases), in mitochondria (e.g., cytochrome c oxidase, pyruvate carboxylase), in lysosomes and the Golgi apparatus (e.g., a-D-mannosidase, peptidases), associated with cell mem-

TABLE VI ZINC METALLOENZYMES' ~

Group Oxidoreductases Alcohol dehydrogenase D-Lactate dehydrogenase D-Lactate cytochrome reductase Superoxide dismutase Cytochrome c oxidase Transferases Phosphoglucomutase RNA polymerase Reverse transcriptase Mercaptopyruvate sulphur transferase Transcarboxylase Aspartate transcarbamylase Nuclear poly(A) polymerase Terminal d N T transferase Hydrolases Alkaline phosphatase Phospholipase C Dipeptidase Aminopeptidase Carboxypeptidase A Carboxypeptidase B Aspartate carboxypeptidase Carboxypeptidase (other) Collegenase Neutral protease AMP deaminase 5-Nucleotidase

EC Numberb 1.1.1.1 1.1.1.28 1.1.2.4 1.15.1.1 1.9.3.1' 2.7.5.1 2.7.7.6 2.7.7.2.8.1.2 2.1.3.1 2.1.3.2 2.7.7.3 1 3.1.3.1 3.1.4.3 3.4.3.3.4.11 .3.4.17.1 3.4.17.2 3..4.17.5 3.4.17.3.4.24.3.4.4.3.5.4.6 3.1.3.5

Reproduced with modifications from Vallee (1988), with permission. From Enzyme Commission Handbook. ' From Einarsdottir and Caughey (1984).

Group

EC Numberb

Fructose- 1,6-biphosphatase Phosphodiesterase (exonuclease) Cyclic nucleotide phosphodiesterase a-Amylase a-D-Mannosidase Angiotensin-converting enzyme Elastase Collagenase Aminocylase Dihydropyrimidine aminohydrolase Dihydroortase P-Lactamase 11 Creatininase Inorganic pyrophosphatase Nucleotide pyrophosphatase Nucleases Lyases Fructose-biphosphate aldolase Carbonic anhydrase Rhamnulose- 1-phosphate aldolase Aminolevulinate dehydratase Glyoxalase I Isomerases Phosphomannose isomerase Ligases tRNA synthetase Pyruvate carboxylase

3.1.3.1 1 3.1.4.1 3.1.4.16 2.2.1.1 3.2.1.24 3.4.15.1 3.4.2 1.11 3.4.24.3 3.5.1.14 2.4.2.2 3.5.2.3 3.5.2.6 3.5.3.3 3.6.1.1 3.6.1.9

4.1.2.13 4.2.1.1 4.1.2.19 4.2.1.24 4.4.1.5 5.3.1.8

6.4.1.1

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(:HRISTOPlIER J. FKEDERICKSON

branes (e.g., 5-nucleotidase, phospholipase C , carbonic anhydrase), and in cytosol (e.g., phosphodiesterase, phosphoglucomutase). One would therefore expect that zinc would be found in virtually all organelles and cell fractions of brain tissue. So far as we know, no metalloenzyme has ever been purified directly from brain tissue and shown to contain stoichiometric amounts of zinc; nonetheless, the likelihood that the brain would have only zinc-free isozymes of all metalloenzymes seems vanishingly small, so it is reasonable to assume that these enzymes in the brain contain zinc. MetalloenLymes bind zinc firmly (stability constants of' 10" and higher) (see, e.g., Romans et al., 1978), typically in a tridentate (catalytic site) or tetradentate (structural site) proteinaceous claw (chele), and exchange the zinc sparingly. T h e zinc in metalloenzymes is therefore a comparatively stable pool that turns over apace with the turnover of the enzymes. For example, aspartate transcarbamylase exchanges virtually none of its endogenous zinc with exogenous 65Zn during weeks of dialysis (Nelbach et al., 1972), and the half-time for exchange of bound zinc with exogenous zinc has been estimated to be 3 years for carbonic anhydrase (Romans et nl., 1978). Taken together, these considerations would suggest that virtually all cell compartments of the brain should contain a stable pool of zinc that is bound into large (30-400 kDa) proteins and has a relatively slow turnover. This seems, in fact, to be the case. As has been discussed above, the zinc content of various brain fractions is fairly constant, at about 100-200 ng of zinc per mg of protein, and most of that zinc has been found to be sequestered in large (212 kDa) proteins. (For comparison, a typical zinc metalloenzyme, carboxypeptidase, contains 2000 ng of zinc/mg) (Parisi and Vallee, 1969). 'The turnover of zinc in the brain, likewise, is compatible with the turnover rate of zinc metalloenzymes (assuming considerable recycling of zinc), with a 22-day half-time for clearance of "Zn. It has been emphasized in several reviews of the literature that zinc metalloenzymes are generally insensitive to short-term fluctuations in the bioavailability of zinc (Henkin, 1979; Bettger and O'Dell, 1981). This is not surprising in view of the tight binding of zinc into metalloenzymes. There are exceptions to this general rule, however. Table VII shows a sampling of somatic enzymes that do exhibit reduced activity following sustained zinc undernutrition, and additional enzymes specifically relevant to adult brain function that are affected by altered zinc availability are shown in Table XI (see Section VIII, B, 2, a). Moreover, the utilization of zinc is especially high in growing tissue. Thus, nucleic acid synthesis, transcription, and translation, which involve zinc atoms in multiple roles (Valle, 1986; Vallee and Falchuk, 1981, 1983; Klug and

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TABLE VII ENZYMES AFFECTEDBY ZINCDEFICIENCY in V i m Enzyme Alkaline phosphatase (serum) Alcohol dehydrogenase (liver) Aminolevulinic acid dehydratase (hepatocyte) Angiotensin converting enzyme (plasma) Carbonic anhydrase (erythrocytes) Thymidine kinase (embryos) Glutamic acid dehydrogenase (perinatal brain) RNA polymerase (liver) RNA/DNA & histone metabolic enzymes (E. gracilis)

Activity

Reference

Reduced Reduced Reduced

Adeniyi and Heaton (1980) Kfoury et al. ( 1 968) Guzelian (1982)

Reduced

White et al. ( 1 986)

Reduced

Huber and Gershoff (1973)

Reduced Reduced

Dreosti and Hurley (1975) Dreosti et al. (1981)

Reduced Alteredlreduced

Terhune and Sandstead (1972) Vallee and Falchuck (1981)

Rhodes, 1987; Berg, 1988), and protein synthesis are all adversely affected when zinc deficiency is imposed during proliferation and differentiation of organs, including the brain (for reviews see Vallee and Falchuck, 1981, 1983; Dreosti, 1984; Sandstead, 1984, 1985; Wallwork, 1987). Though transiently raising or lowering zinc levels does not affect most zinc metalloenzymes, drugs that can chelate or coordinate with zinc do. Zinc atoms that are catalytic are typically bound in a trihedral coordination, with the fourth site exposed for coordination with substrate. Molecules that complex with the exposed site inactivate the enzyme; in fact, inactivation of enzymes by chelators (e.g., dithizone, EDTA, 1- 10 phenantroline) or by anions that can bind to a zinc catalytic site (e.g., S-', CN-') is a standard test for verifying the role of metal in a suspected metalloenzyme (Spiro, 1983; Vallee and Galdes, 1984). Any of these drugs applied to brain tissue therefore has the potential action of inhibiting any zinc metalloenzyme (or other metalloenzyme), a fact that bears consideration whenever such agents are used to manipulate brain zinc. One familiar example of this type of drug action is the inhibition of the copper metalloenzyme, dopamine-P-hydroxylase, by diethyldithiocarbamate (DDC), wherein chelation of the copper results in depletion of norepinephrine (Haycock et al., 1977).

2 . Nonenzymatic Zinc-Binding Proteins Zinc has important nonenzymatic roles in nucleic acid regulation, including stabilizing the structure of RNA and DNA (Vallee, 1988;

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Vallee and Falchuck, 1981, 1983). In addition, a number of gene-control proteins have been found to contain a repeating amino acid sequence that produces a tetrahedral zinc coordination site. These so-called zinc-fingers (typically two Cys and His residues) are apparently conserved among many gene-regulating and DNA-binding proteins, giving zinc a potentially broad role in control of gene expression in the brain and other tissues (Klug and Rhodes, 1987; Berg, 1988; Vallee, 1988). Table VIII shows the zinc-containing molecules that have been isolated directly from brain tissue. The larger of these (260 kDa) have not been characterized but presumably include some of the zinc metalloenzymes in Table VI and metalloproteins mentioned above. Two of the smaller molecules have been studied in detail, namely (1) a metallothionein-like protein and (2) a small molecule that coelutes with the tripeptide glutathione. Both metallothionein and glutathione have been implicated as metal-binding molecules important in systemic metal detoxification (Brady, 1982; Gregus and Varga, 1985), and both have been proposed as possible scavenger or storage molecules for CNS zinc. Metallothionein is a small (- 6.5 kDa) protein abundant in liver and kidney that participates in both storage of trace metals (copper and zinc) and sequestering of toxic metals (mercury and cadmium) (Brady, 1982). High metal intake induces synthesis of the hepatic metallothionein, which in turn binds up to 10 gram-atoms of metal per mole. Several independent groups have identified small (515 kDa), metallothionein-

'1ABL.E V l I l ZINC-CONTAINING PKOIEINSFOUNDI N BKAIN'I'ISSUE

Locatiorl

Characterization

Brain Hippocampus I lippocampus, cerebellum

25 kDa; 210 kDa 75 kDa 60 kL)a 19 kDa (MT)" 1,500 Da (glutathione-like) 10 kDa MT MT S- 1 0OA s-100B Tubulin

Brain Brain Rrain Brain Brain Brain

MT, metallothionein-like.

Reference Itoh el (11. (1983) Record et al. ( 1982) Sato vt nl. (1984a)

(:hen arid Garither (1975) Brady (1983) Ebadi ul (11. (1984a) Raudier and Gerard (1983) Baudier ut 01. ( 1 985) Hesketh (1983)

NEUROBIOLOGY OF ZINC

169

like proteins in the cytosolic fraction of brain tissue from rats (Chen and Ganther, 1975; Itoh et al., 1983; Brady, 1983; Sat0 et al., 1984a) and monkeys (Gulati et al., 1987), but the most extensive studies of these proteins have been carried out by Ebadi and co-workers. T h e current evidence indicates that the brain metallothionein-like protein exists in two isoforms, between 6.2 and 10 kDa, which are rich in cysteine (Ebadi and Swanson, 1987; Ebadi et al., 1987) and may bind up to 7 p g of zinc per mg of protein (Ebadi, 1986a). The two isoforms have HPLC retention times virtually identical to hepatic metallothionein I and I1 (Fig. 1 ) and have been been designated metallothionein-like protein I and 11. A role of brain metallothionein-like proteins in cerebral zinc homeostasis is suggested by the fact that they are induced by intracerebral administration of free zinc ions (Ebadi et al., 1984a; Ebadi, 1986a,b) (Fig. 1). Cadmium administration does not induce synthesis of the brain metallothionein-like proteins (Ebadi and Swanson, 1987),indicating that scavenging and storing zinc, rather than metal detoxification, may be the primary function of these proteins in the brain. In view of the many potent effects of free zinc ions upon brain tissue (see below), the

123

4

5

FIG. 1. HPLC profiles of metallothionein I and I i from rat liver (A) and metallothionein-like protein I and 11 (peaks 3 and 4) from rat brain (B). Courtesy of M. Ebadi. (see Ebadi, 1986a.b. for review.)

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CHRISTOPHER J . FREDERICKSON

proposed role of the metallothionein-like protein in homeostatic regulation of free zinc could be pivotal. Brady (1983) has estimated that about 6% of brain zinc is normally bound to metallothionein-like proteins, and the amount of protein in the brain is considerably higher in adult than in newborn rats (Ebadi, 1986b). A role for these same metallothionein-like proteins in pineal zinc homeostasis has also been proposed (Awad and Ebadi, 1985). The tripeptide glutathione has also been suggested to bind toxic metals in bile (Domini and Kustin, 1983; Gregus and Varga, 1985; Postal et al., 1985) and has likewise been proposed as an important zinc-binding ligand in the brain by Sat0 and colleagues (Sato et al., 1984a-c). Sat0 et al. routinely use special procedures to liberate “occluded” cytosol from large synaptosomes during their tissue fractionation protocol. With that procedure, they have found a 1.5 kDa molecule that coelutes with zinc-glutathione and is present in their modified cytosolic fractions from both the hippocampus and the cerebellum. Approximately 5- 10% of the total zinc in individual brain regions is found by Sato et al. (1984~) to be associated with the putative glutathione, the proportion of total zinc bound to the glutathione-like molecule being higher in the hippocampus (Sato et al., 1984a-c) than in the cerebellum. Systemically administered “Zn has been found to bind preferentially to the hippocampal as compared to the cerebellar glutathione-like molecule (Sato et al., 1984a,b). Both of the latter findings have suggested an association of the putative zinc-glutathione complex in the metabolism of zinc in zinc-containing axonal terminals, which are prevalent in the hippocampus but not in the cerebellum (see below). B. ZINGSENSITIVE PROTEINS AND ENZYMES Metalloproteins, including metalloenzymes, can be rigorously defined on the basis of their tight binding of stoichiometric amounts of zinc, inactivation by metal chelators, reactivation of the metal-free apoenzyme by metal replacement, and so forth (Parisi and Vallee, 1969; Vallee, 1976, 1980, 1985; Vallee and Galdes, 1984). This is not the case with zinc-sensitive proteins; any protein that can be shown to change structure or activity in the presence of zinc salts can be called zincsensitive, zinc-modulated, or zinc-dependent. Moreover, because little is known about the level of free zinc in the brain, there is no way to insure that a physiologically relevant amount of zinc has been used in demonstrating the zinc sensitivity of a given protein. T o underscore the latter point, it may be noted that current estimates of the physiological

NEUROBIOLOGY OF ZINC

171

concentration of ionic zinc in the brain range over six orders of magnitude, for 200 pM to 300 pM. The higher value (300 pM) is the estimated concentration of extracellular zinc that might be attained during brief surges of zinc release from zinc-containing axonal boutons (see Section VII). T h e lower value (200 pM) is the estimated concentration of ionic zinc in intracellular and extracellular fluids that has been derived from studies of plasma and muscle cell cytosol (Magneson et al., 1987; see also above). Until levels of intracellular and extracellular ionic zinc are established for the CNS (both resting levels and transient levels), it seems prudent to consider most of the demonstrated effects of zinc upon brain proteins as candidate roles for the ion; nonetheless, the problem of “dose” must be kept in mind. The various CNS proteins that have been shown to be affected by zinc salts are grouped somewhat arbitrarily into three categories in the present review. Proteins with relatively direct involvement in synaptic transmission, such as enzymes of neurotransmitter metabolism and neurotransmitter receptor molecules are discussed in Section VII within the general context of zinc’s possible roles in synaptic transmission. Proteins associated with the plasma membrane are discussed in Section V, and proteins not directly associated with either synaptic transmission or neural membranes are discussed immediately following. The latter group includes tubulin, the S- 100 proteins, and calmodulin. T h e effects of zinc ions on tubulin assembly were first reported by Gaskin et al. (1976). It is now recognized that tubulin binds zinc avidly, with apparent dissociation constants of 0.9 pM (3 sites) and 16 p M (17 sites) (Eagle et al., 1983) or 110 pM (Hesketh, 1983). Bound zinc ions alter the colchicine but not the vinablastine binding site on tubulin polymers (Banerjee et al., 1982), and zinc may alter phosphorylation of tubulin (Prus and Wallin, 1983). Zinc has a biphaisc effect on the assembly of tubulin into microtubules. At low ratios of zinc to the tubulin molecule, assembly of microtubules is facilitated, whereas at high molar ratios (greater than three to one), the polymerization of tubulin into aberrant sheets of antiparallel elements is induced (Haskins et al., 1980; Hesketh, 1984a,b). Both of these phenomena have been observed in cell-free tubulin extracts, and both appear to occur in living cells. Thus, anterograde transport through axons in vitro can be dramatically faciliated by incubating axons in 5 pM of zinc salt, whereas higher concentrations (0.1-1.0 mM) retard axoplasmic flow (Edstrom and Mattsson, 1975) and induce morphologic damage and formation of tubulin sheets in cultured neurons (Gaskin et al., 1978; See also Kress et al., 1981). This latter effect is one possible cause of the death of cultured neurons caused by addition of 0.3-1 mM of zinc salt to the incubation media

172

CHRISTOPHER

1.

FKEDEKICKSON

(Yokoyama et al., 1986; Choi et nl., 1988) (Fig. 2). Tubulin extracts from the brains of zinc-deficient rats have been reported to exhibit reduced repolymerization in uitro (Hesketh, 1982). The brain-specific proteins S-100A and S- lOOB also have a relatively high affinity for zinc, with apparent dissociation constants in the range of 10-‘-10~*, and zinc-binding capacity of 4-5 atoms per molecule (S-100A) and 8-9 atoms per molecule (S-100B) (Baudier and Gerard, 1983). These proteins also bind calcium, with lower affinity (Baudier and Gerard, 1983; Baudier et al., 1985), and calcium binding is altered by prior zinc binding (Baudier and Gerard, 1983; Baudier et al., 1985; Mani and Kay, 1986). Moreover, S-100 (especially S-100B) (Deinum et al., 1983) has an inhibitory effect upon microtubule assembly that is evidently dependent upon the presence of proportional amounts of zinc (e.g.,1-3 zinc ions per S-100 molecule). For example, with 50 p M of zinc present, 40 pM of S-100 dramatically slows microtubule assembly, an effect that is immediately reversed by chelation of zinc with EDTA (Fujii et al., 1986). Fujii et al. (1986) have suggested that zinc ions could interact with the S-100 proteins to yield a “flip-flop” switch turning on either assembly or disassembly of microtubules, depending on the relative amounts of zinc and protein. Zinc interacts with calmodulin, perhaps by a relatively weak binding to a subset of the four calciuni-binding sites (Brewer, 1980; Fujii et al., 1986). Brewer has marshaled considerable evidence that zinc impedes calcium binding to calmodulin and thereby inhibits calcium-dependent calmodulin activation of enzymes such as Ca-ATPase and pholsphodiesterase (Brewer 1980; Brewer et nl., 1979). Cox and Harrison (1983) likewise suggest that zinc’s impact upon calcium-calmodulin activation may be chiefly inhibitory. Chao et al. (1984), however have emphasized that relatively low concentrations of zinc (10 pM) can stimulate calciumdependent calmodulin activation (of phosphodiesterase), with inhibition occuring only at higher (100 pM) zinc concentrations. If, in fact, zinc ions up- and down regulate calcium-calmodulin activation in uiuo, then zinc could regulate the entire spectrum of calmodulin-dependent processes in neurons and glia alike. This would give the zinc ion multiple paths for regulating CNS physiology. One recent study of zinc-deficient rats found a substantial drop in calmodulin levels in testicular tissue FIG. 2. Three clusters of cultured cortical neurons (double arrows) are shown before (A) and 24 hr after (B) a 15-min exposure to 1 mM of ZnClp in the medium. The phase contrast view shows that the cells are degenerating in (B). The same death of neurons is produced by as little as 200 of zinc with longer exposures. Scale, 20 pni. Courtesy of J . Koh and D. W. Choi. (See Choi et al., 1988.)

NEUROBIOLOGY OF ZINC

173

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CHRISTOPHER J. FREDERICKSON

after zinc deprivation (Law et al., 1987); though brain calmodulin levels were not altered according to that report (brain zinc was not altered either), the data do show a zinc dependence of the protein in vivo. Slevin et al. (1986) have presented preliminary evidence that epileptic kindling produces a lasting elevation of CNS zinc and a concomitant decrease in calmodulin-activated Ca-ATPase activity. In addition to tubulin, S-100, and calmodulin, there are a great number of traditional enzymes the activity of which is altered by the presence of zinc ions. Parisi and Vallee (1969) list over a dozen examples of enzymes that are reportedly “activated” by zinc ions in vitro; a few examples of enzymes that are similarly “inhibited” by the cation are kininase I (White et al., 1986), thymidine kinase (Zaslavsky, 1979), and DNA polymerase (Walton et al., 1982). Proteins and enzymes with direct involvement in neurotransmission or membrane function that are sensitive to zinc are discussed in later sections of this review and listed in Table XI. From these various compilations, it is obvious that the potential range of zinc’s effects on cell biochemistry is immensely broad.

C. ZINC-PROTEINCOMPLEXES Like the zinc-sensitive protein, the zinc-protein complex is a rather vaguely defined entity; it can include essentially any aggregation of metal and protein in which the metal is not tightly and stoichiometrically bound to the protein. In other words, the term “complex” covers the cases that do not fit the definition of a metalloprotein. By this definition, sheets of tubulin with zinc would be a zinc-tubulin complex. A number of proteins, peptides, and other small organic molecules have been found to form complexes with zinc in vitro and in vivo. Some examples are the complexes zinc forms with insulin (Gold and Grodsky, 1984), catecholamines and ATP (Maas and Colburn, 1965; Berneis et al., 1969; Rajan and Mainer, 1978) glutamate (Gramaccioli, 1966), 7S-nerve growth factor (’IS-NGF) (Pattison and Dunn, 1976a), and DNA/RNAtranscription factor complexes (Klug and Rhodes, 1987). One group of zinc-containing complexes that may be especially relevant to zinc’s role in the CNS are those such as zinc-insulin and zinc-7S-NGF, which are stored in the secretory granules of somatic secretory cells. These zinc storage complexes have provided a model for understanding zinc’s possible function in the secretory granules (vesicles) of zinc-containing neurons. The function of zinc in secretory granules, nature of zinc-secretory storage complexes, and possible zinc complexes present in vesicles are discussed in detail in Section VIII.

NEUROBIOLOGY OF ZINC

175

V. Zinc and Membranes

A. STABILIZATION OF MEMBRANES When dissociated tumor cells are incubated in 1-2 mM of zinc salt for 10-15 min prior to homogenization, the yield of large fragments of unbroken membranes is increased (Warren et al., 1966; Moore et al., 1976). It has been suggested that this “fixing”(Moore et al., 1976) of the membrane may be due to cross-linking or precipitation of membrane or cytoskeleton proteins (Moore et al., 1976), and interactions between zinc and membrane constituents have therefore been sought. There are zinc metalloenzymes (and therefore zinc atoms) associated with membrane fractions of tissue (see above). In addition, supraphysiological ( 1501000 pM) concentrations of zinc salts can alter the biophysical properties of membrane preparations in vitro, producing, for example, a facilitation of phospholipid vesicle fusion (Barfield and Bevan, 1985; Deleers et al., 1986b), potentiation of calcium-induced phase separation of membrane lipids (Deleers et al., 1986a), and a compaction of myelin probably mediated by the interaction of zinc with the POglycoprotein (Inouye and Kirschner, 1984). Zinc is also absorbed onto phosphatidylcholine liposomes (Sunamoto et al., 1980) and may modulate formation of lipid chylomicrons in the gut (Koo et al., 1987). Moreover, the labilization of isolated hepatic lysosomes in vitro is retarded by the addition of zinc salts (50-1000 pM) to the incubation medium, an effect that has been attributed to the stabilization of the lysosomal membrane (Chvapil et al., 1972a,b; Pfeiffer and Cho, 1980). Although the physiological relevance of some of the phenomena described above may be questioned, there is evidence to suggest that membrane stability is in fact affected by zinc status in vivo. Specifically, Chvapil has reported that systemic zinc administration (up to 44 mg/kg) can slow both lipid peroxidation (Chvapil et al., 1972a,b, 1974) and efflux of gastric lysosomal enzymes (Cho et al., 1980; Pfeiffer et al., 1980). However, Chvapil et al. (1974) have emphasized that these effects would not be expected to occur in tissues (such as the brain) that do not accumulate excess zinc intracellularly during systemic zinc loading, and Dreosti and Record (1978) have adduced evidence that even hepatic lysosomes are not made more labile by depletion of serum zinc in vivo. T h e outer membrane of the erythrocyte, however, apparently is rather sensitive to reduced serum zinc. Erythrocytes taken from zincdeprived animals exhibit both increased susceptibility to osmotic rupture (O’Dell et al., 1987) and increased fluidity of the lipid bilayer (Jay et al., 1987). As little as one day of zinc repletion reverses the fragility of the

176

C:HKISTOPHERJ. FREDERICKSOK

erythrocytes, although, interestingly, zinc ( I 8 p M ) added to the cell suspensions in zdro does not (O'Dell el al., 1987). The latter suggests that metabolic incorporation of the ion (into membrane proteins?) is a prerequisite for the effect upon the membrane. Extrapolating from the findings with erythrocytes and from other data in the literature, Bettger and O'Dell (1981) have proposed that membranes may generally be the most vulnerable of cell compartments to lowered serum zinc. The observations that erythrocyte membranes are depleted more rapidly of zinc than cytosol during zinc undernutrition (Bettger and O'Dell, 1981) and that the zinc associated with myelin has a faster turnover than the zinc of other compartments (Wensink et al., 1987b) are consistent with this idea. If the concentration of zinc in the extracellular fluids of the brain rises and falls in parallel with the concentration of zinc in the serum, then the notion that ligands on the outer surfaces of neurons might lose o r gain zinc proportionately becomes plausible. Possible changes in the fluidity of neural membranes, in turn, could cause immediate changes in various neural processes, including exocytosis and movement of physiologically important macromolecules (pumps, inonophores, receptors) in the membrane. However, it should be emphasized that there is no direct evidence as yet that (1) extracellular CNS zinc levels closely follow serum zinc levels, or (2) neural membranes are affected by physiological alterations in extracellular zinc concentrations in vivo.

B. MEMBRANE PUMPSAND ION CHANNELS In addition to the possible mechanical effects of zinc upon membranes, the cation can also alter the function of macromolecules embedded in the membrane. Of particular relevance to neural function are the effects of zinc upon membrane Na', K+-ATPase and on the calcium, sodium, and potassium channels. Zinc has been shown to affect the activity of a several ATPases (see Table XI), but the most sensitive is Na', K'-ATPase, which several groups have found to be inhibited by zinc. This inhibition has been observed with as little as 1 p M of zinc with synaptosomes or microsomes in uitro (Table XI) and after direct injection of 0.1 mg of zinc salt into the brains of rats (Donaldson et al., 1971). T h e inhibition is evidently noncompetitive (Hexum, 1974) and irreversible (Gettlefinger and Siegal, 1978) and is mimicked by equal concentrations of copper (Prakash et al., 1973; Hexum, 1974). With electroplax microsomal

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preparations, the reduced ATPase activity develops slowly, taking 10- 15 min of incubation with zinc to develop fully (Gettlefinger and Siegal, 1978). Several authors have proposed that zinc ions may induce conformational changes that inactivate the ATPase by binding to sulfhydryl groups of the molecule (Donaldson et al., 1971; Prakash et al., 1973; Watson and Beamish, 1981). Clearly, the inhibition of Na+, K+-ATPase in the brain would have profound impact, changing osmotic gradients and membrane polarization, eventually inactivating and killing cells. This may be one of the toxic mechanisms by which high concentrations (300- 1000 ,uM) of exogenous zinc kills neurons in vitro (Choi et al., 1988) and would presumably be one of the more critical actions of concentrated zinc salts injected intracerebrally (Donaldson et al., 197 1; Ebadi and Pfeiffer, 1984; Chung and Johnson, 1984; Pei and Koyama, 1986). However, it should be pointed out that neurons in culture can be incubated in up to 100 pM of zinc for over 24 hr without apparent damage (Yokoyama et al., 1986; Choi et al., 1988), and brain slices show a ouabain-sensitive high-affinity uptake of 65Zn in vitro in the presence of comparable amounts of zinc in the medium (Howell et al., 1984). These latter results suggest that the N’, K+-ATPase molecule in intact cells and tissue (as opposed to synaptosomes and microsomes) may be partially “protected” from the effects of zinc. In addition to its effects upon membrane ATPase, zinc also interacts with membrane ion channels. Voltage-sensitive channels for calcium (Kawa, 1979),chloride (Stanfield, 1970), sodium (Frelin et al., 1986), and potassium (Spaulding et al., 1986>have all been shown to be partially blocked by zinc in the extracellular fluid. Some effect has been reported at 50 pA4 of zinc (Frelin et al., 1986) but generally, the amounts of zinc required to impede ionic currents are rather high, ranging from 100 to 1000 pA4 or higher (Meves, 1976; Maetani et al., 1979; Kawa, 1979; Spaulding et al., 1986). Whether these phenomena are physiologically relevant remains uncertain.

VI. Distribution of Histochemically Reactive Zinc in the Brain

Early histochemical studies of CNS zinc were plagued by the combined facts that (1) the stains could not be shown to be specific for zinc and (2) not all zinc in the tissue could be stained (see Szerdahelyi and Kasa, 1984, for review). T h e first of these conditions is no longer true: by the proper combination of staining procedures one can now be quite

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certain in identifying zinc in most brain regions. The second condition (not all zinc can be stained) is still true but is now recognized as a major strategic advantage rather than a shortcoming of the histochemistry. The reason for this is that the pool of zinc that can be stained, the zinc that is histochemically reactive, turns out to be located almost exclusively in the presynaptic vesicles of specific CNS neurons, the zinc-containing neurons of the brain. In other words, in the same way that the new immunohistochemical methods for amino acids are specific for the “free” neurotransmitter pools (Ottersen et nl., 1986; Storm-Mathisen et al., 1983), the current zinc histochemical methods are specific for zinc in presynaptic vesicles. The difference, of course, is that stains for amino acid transmitters were expressly and intentionally developed for their specificity, whereas the zinc stains were used for decades before their selectivity for vesicular zinc was recognized.

A. HISTOCHEMICAL METHODS There are three methods of staining zinc in brain tissue: silver amplification, quinoline fluorescence, and dithizone. The sensitivities of the three methods are substantially different. Silver amplification is much more sensitive than fluorescence, which is in turn considerably more sensitive than dithizone. However, within the limits of their differing sensitivities, all three methods label exactly the same regions of the brain. Every cytoarchitectonic zone that is labeled by dithizone is also labeled in quinoline fluorescence, and those regions, in turn, are all stained by the silver amplification techniques (Table IX; Figs. 3 and 4). Any one of the methods could in theory stain some metal besides zinc, but the only metal that can produce all three histochemical reactions is zinc. Moreover, the three procedures interfere competitively, indicating that they all react with the same endogenous pool of zinc. 1. Dithironp

Dithizone (DZ; diphenylthiocarbazone) has been used for at least half a century for extraction and quantitation of metals from various organic and inorganic matrices (Stary, 1964; Irving, 1980). The dithizone molecule chelates zinc with a bidentate claw, and DZ-Zn-DZ complexes form in the presence of stoichiometric excesses of dithizone. In solution, dithizone is green, whereas the Zn-DZp chelate is a bordeaux red; both Dz and Zn-Dz:! are several orders of magnitude more soluble in organic than in aqueous media, making dithizone ideal for 0rganic:aqueous split-phase extractions of chelated metals.

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TABLE IX ZINC STAINING" Region Cerebral cortex Isocortex Laminae 1-111, V Lamina IV Allocortex Entorhinal Pyriform Cingulate

Technique*

Confirmed in boutons

Q,nT,S nT,S (barrel septa only) Q,nT,S D,Q,n'I,S QnTS

Limbic system Hippocampal formation Hippocampal mossy fibers CAI -4 pyramidal stratum CAI-4 radiatum CAI -4 oriens Induseum griseuin Dentate gyrus Granule cells Inner niolecular Middle molecular Outer molecular Subiculum Septum Medial Lateral Hypothalamus Ventromedial (core) Ventromedial (shell) Lateral Striohypothalarrius Nucleus terete Am ygdala Bed nucleus stria terminalis

No staining Q,nT,S No staining Q,nT,S nT,S L),Q,nT,S Q,nT,S

Thalamus Dorsomedial Dorsolateral Ventral

Q,nT,S nT,S (puncta)' nT,S (puncta)

D,Q,n?',S N o staining Q,nT,S Q,nT,S Q,nT,S nl'(Zn?) Q,nTS No staining Q,nT,S D,Q,n'I,S nT,S Q,nT,S

(continwd)

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TABLE IX (Continued) Region Striatum Caudate-putamen Globus pallidus Cerebellum Deep nuclei Cortex Granule cell stratum Purkinjc cell stratum Molecular Lone Brain stem Tectuni (c-olliculi) Tegrnen t tin1 Cranial nuclei Dorsal cochlear Other Spinal cord Dorsal horn Ventral horn

TechIiiq u e"

Confirnicd in houtons

Q,nT,S N o staining

Yes

nT,S (puncta)

-

n'1,S (certain glomeruli) N o staining S (vcry faint; paleocerebelluin)

Yes

-

-

-

nT,S (puncta) nT,S (puncta) Q,nT,S n'l',S (puncta)

nT,S nT,S (puncta)

Yes Yes

References: Dithizone, Fleischhauer and llorstmarin ( 1 957); Fjerdingstarl ~1 ul.( 1977); Hall et nl. (1969); Frederickson and Howell (1984); Frederickson ef ( I / . (1981); Berger and O'Leary (1975); McLardy (1960); Danscher (1984a); Maskc( 1955);Quinoline, Frederickson el nl. (19874; Frederickson and Danscher (1988); Kasarskis el nl. (1986); Savage ( I 987); Toroptsev and Eshchenko (1982). Neo-Timm's, Danscher (1981a. I984a,b); Schwerdtfeger el nl. ( 1989); Gaarskjaer ( 1 985); Perez-Clause11 and Darischci(1985). Selenium, Danscher ( 1982, l984a,b); Schwerdtfeger rt ul. (1985). Ultrastructural localization in houtons, Friedman a n d Price (1984); h i s c h e r (1981a, 1982, 1984a-d); (1989); Perez-(:lausell and Danscher (1985, 1986); Schrbcler (1979, et nl. (1985a,b); Martinez Guijarro el nl. (3984); lbara and Otsuka (1968; 1969); Haug (1967); Koznia et nl. (1978); Otsuka et ul. (1982). D, dithizone; Q, quinoliric; nT, neo-'l'imm's; S, selenium. Lightly srattered boutonlike puncta in neuropil.

When dithizone is administered intraperitoneally, it reaches the brain and forms chelates in situ,producing a bright red stain that can be seen in the brain after simply freezing and cutting sections. The red dithizonate complex could be a chelate of metals other than zinc (both iron and cadmium dithizonates are red; copper dithizonate is redviolet), but the chelate that actually forms in vivo has been extracted

FIG.3. Zinc in axonal boutons is shown by 6-methoxy-8-p-toluenesulfonamide quinoline (TSQ) fluorescence (left) and neo-Timm’s (right) in the dentate gyrus and hippocampus of a rat. Neural perikarya in the granule cell stratum and within the hilus (arrows) are unstained. G, Granule stratum; H, hilus; MF, mossy fiber neuropil; R, stratum radiatum.

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from brain (Maske, 1955; Frederickson et al., 1981; Danscher et al., 1985a) and other tissues (Stampel, 1959), and all analyses have indicated that the metal that is chelated is zinc, though trace amounts of copper (suggested to be possible contamination) have been found (Danscher et al., 1985a). Dithizone staining procedures that mask the formation of dithizonates other than zinc (addition of thiosulfate and cyanide, pH 4-5) (Mager et al., 1953) produce virtually the same pattern of staining in brain sections that is found with the intravital method (Kesslak et al., 1987; Frederickson et al., 1981), leaving no reasonable doubt that the dithizone reaction product in the brain is the zinc-dithizone chelate. Zinc has been demonstrated in the brains of 13 different species, including fish, amphibians, and mammals with the dithizone method (see references in Szerdahelyi and Kasa, 1984). Dithizone gives a rather pale staining, and the chelate decomposes easily upon exposure to solvents, light or heat. It therefore is of little use for detailed localization of zinc (Frederickson and Howell, 1984). However, the dithizone staining is quite adequate for gross localization in brain regions in which the concentration of reactive zinc is high. Furthermore, because the dithizone chelates that can be extracted from the brain contain virtually only zinc, the intravital dithizone procedure is a good competitive masking agent for use in testing the specificity of other stains. Suppression of subsequent staining by prior treatment with intravital dithizone is an essential test to determine whether a particular staining reaction is due to zinc. 2. Quinoline Fluorescence Like dithizone, quinoline is a metal chelator that has been used for many years for extraction and determination of metals (Stary, 1964). Several congeners of quinoline fluoresce when complexed with metal cations as quin-metal or quin-(metal)* chelates (Watanabe et al., 1963; Carter and Ohnesorge, 1964). For zinc histochemistry, the most useful are 2-methyl-8-hydroxy quinoline (2MQ) and 6-methoxy-8-p-toluene sulfonamide quinoline (TSQ), zinc-TSQ being much the brighter of the two fluorochromes. These fluorescent probes were first used for histo-

FIG. 4. Horizontal sections of mouse brain show zinc-containing boutons stained by neo-Timm’s and by TSQ fluorescence. The lower panel corresponds approximately to the region between the arrowheads in the upper panel. Note the limbic and cerebrocortical concentration of the stained boutons. Abbreviations as in Fig. 3 and A, amygdala; BNST, bed nucleus of the stria terminalis; Ch P, choroid plexus; CER, cerebellum; C-P, caudate-putamen; DCN, dorsal cochlear nucleus; IC, internal capsule; Inf C, inferior colliculus; Th, thalamus: I V , lamina IV of cerebral cortex.

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chemistry by Smith et al. (1969) and Hahn von Dorsche and Fiedler (1970) and were first used on brain tissue by Toroptsev and Eshchenko (1982; see also Szerdahelyi and Kiisa, 1984). Although some congeners of quinoline can be made to fluoresce when complexed with calcium and magnesium (Schacter, 1959; Watanabe et al., 1963), 2MQ and TSQ in a 0.1 M barbital buffer (pH 8-10) give no detectable emission (i.e., < 1% of the zinc-quinoline signal) when tested with up to 10 mM of calcium or magnesium salts on tissue (Frederickson et al., 1987a). No other biologically relevant cation has been found to fluoresce when chelated with TSQ, though strontium (normally undetectable in the brain) could theoretically give a weak fluorescence (<5% of the equimolar zinc signal) (Schacter, 1959; Montano et al., 198’7). Fluorescence from 2 M Q and TSQ may therefore be considered a specific marker for zinc in normal brain tissue. As would be expected, prior chelation of zinc with intravital dithizone or prior precipitation of zinc with sulphide or selenium blocks subsequent attempts to stain tissue with quinoline fluorescence (Frederickson el al., 1987a), and prior chelation with quinoline (oxine) blocks subsequent silver amplification staining (Danscher and Fredens, 1972).

3 . Silver Amplification There are three quite different variants of the basic silver amplification method: Timm’s method, neo-Timm’s, and selenium. T h e first (Timm’s) method is almost certainly not specific for zinc, whereas the latter two methods have been shown to stain only zinc, at least in most of the known instances of staining in the brain. All of the silver amplification methods are based on the original method of Timm (1958a,b), in which metals are precipitated in situ by suffusing the tissue with S2, and the insoluble metal sulfide crystals are then rendered visible by a silver development process. In the development process, the tissue is immersed in a solution of hydroquinone, silver salt, and a stabilizer, such as gum arabic, and the metal sulfide crystals catalyze the reduction of silver, resulting in formation of a metallic silver shell around the metal sulfide “seed.” Variants of Timm’s method have been used for over 30 years for labeling heavy metals (gold, mercury, lead, silver) and other transition metals (zinc, copper, iron) in tissue (Timm, 1958a,b, 1961; for reviews see Haug, 1973; Danscher, 198la; Szerdahelyi and Kisa, 1984; Danscher, 1984a-d; Danscher and M#ller-Madsen, 1985; Danscher et al., 1987b). The silver reaction product is both stable and dense, making the method suitable for both light and electron microscopic studies.

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However, the relevance of this method for zinc is limited to the specific variant developed by Danscher, the neo-Timm’s Danscher’s contribution to the development of a new Timm’s procedure was to modify the method progressively until the staining of material that could not be shown to be zinc was minimized o r eliminated. The general approach was to administer an intravital metal chelator (such as dithizone) to bind zinc in situ, then try a variant of the Timm’s procedure to see whether all staining was blocked by the prior in situ chelation (Danscher, 1981a, 1984~).Because of the evidence that dithizone chelates only zinc from the brain (Maske, 1955; Stampel, 1959; Frederickson et al., 1981; Danscher et al., 1985a), Danscher inferred that dithizone blockade of staining indicated the method was specific for zinc. Danscher’s second method of testing the specificity of staining was to perfuse a brain with sulfide, producing metal sulfides, then immerse tissue sections in dilute acids that could dissolve zinc sulfide but not, for example, copper o r mercury sulfides. Staining that was removed by the dilute acids was presumed to be due to zinc (Danscher, 1984c; see also Timm, 1961; Haug, 1973, 1975; Szerdahelyi and Kasa, 1984, for further discussion). Based on the kinds of studies described above, Danscher has proposed that staining for zinc demands the following precautions be used with the sulfide method. 1. Sulfide must be introduced (as Na2S) by carefully timed transcardial perfusion at a maximum concentration of 0.1%. Stronger sulfide solutions definitely produce staining that cannot be blocked by prior dithizone chelation and therefore may not be due to zinc. Immersion of tissue in sulfide solutions (as opposed to sulfide perfusion) likewise produces nonspecific staining. 2. Development should be done with silver lactate, not silver nitrate, because the latter readily produces nonspecific silver deposits in tissue; over development will also produce nonspecific labeling. 3. Tissue must be coated with gelatin before silver development, then washed free of the gelatin to remove stray silver grains that fall from suspension in the developer. Danscher’s newer silver amplification method, the selenium method, is based on the fact that metal selenids are also relatively insoluble and will also catalyze the formation of metallic silver grains in a developing solution (Danscher, 1982, 1984a-d). In this method, selenium is administered intravitally (as Na2Se03), and metal selenids are allowed to precipitate in situ. Subsequently, the animals are sacrificed and brains are removed, cut frozen, and developed as in the neo-Timm’s method.

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Like the neo-'Iimm's staining, the selenium staining is almost completely blocked by prior treatment of the animal with intravital dithizone; thus, it is inferred that the selenium staining is due to zinc (Danscher, 1982, 1984~). Definitive proof of the zinc specificity of the silver amplification methods would appear to require that the silver reaction product be extracted from tissue and assayed to determine what element formed the catalytic seed. Unfortunately, there may be more stray zinc, copper, and iron contaminating the silver in the developer than would be present in the catalytic seeds that are developed from the tissue.

B. THEHISTOCHEMICALLY REACTIVE POOLOF ZINC In cytoarchitectonic regions of the brain in which dithizone, TSQ, neo-'I'imm's, and selenium all produce the same pattern of staining, there can be no doubt that the reaction is due to zinc. N o other ion could produce a red dithizonate, a fluorescent TSQ chelate, and insoluble sulfides and selenides that will catalyze the reduction of silver. Moreover, because each of the zinc-chelating or zinc-precipitating reactions blocks subsequent staining by any of the others (Danscher and Fredens, 19'12; Danscher et al., 1973; Haug, 1973; Danscher, 1984c; Frederickson et al., 1987a, 1988a), the methods can be assumed to stain the same endogenous pool of zinc. Thus, the problem of identification of zinc in histological material is solved for those brain regions arid cellular processes that can be stained by both a chelation (TSQ o r dithizone) and a silver amplification (sulfide or selenium) histochemical method. Although it is clear that zinc can be unambiguously identified by contemporary histochemistry, it is equally clear that only a fraction of the zinc in the brain will stain. This can be demonstrated in several ways. First, gross anatomical distribution of staining does not match the distribution of total elemental zinc. The cerebellar cortex, for example, does not stain for zinc with dithizone or TSQ and stains only sparsely with the silver methods (Danscher, 1984d). Yet the cerebellar cortex has just as much zinc as the cerebral cortex, which stains vividly with all four zinc histochemical methods (Table IX; Figs. 4 and 5). This discrepancy

FIG.5. Zinc-containing axonal boutons are shown in horiLontal sections from a bat (left panel, neo-'l'imm's) and a niouse (right panel, selenium). 'I'he distribution of zinc containing boutons is remarkably similar in the two species. Ahhreviations as in Figs. 3 and 4; S, striatum. Courtesy of G. Danscher.

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between zinc content and zinc staining has been reported many times for normal brains (Haug, 1973; Frederickson and Howell, 1984; Lenglet et al., 1984), drug- or lesion-treated brains (Fjerdingstad et al., 1977; Szerdahelyi, 1982; Szerdahelyi and .Kasa, 1987), and for nonneural tissue as well (Elmes and Jones, 1981; Ferke et al., 1979). T h e second line of evidence that not all zinc is histochemically reactive is that staining does not match the quantitative distribution of zinc among subcellular organelles. As mentioned previously, zinc metalloenzymes are dispersed among virtually all cellular organelles, and zinc is rather uniformly distributed among all fractions of brain homogenates. Zinc staining, however, is almost completely restricted to presynaptic vesicles of specific CNS fiber systems (Lopez-Garcia et al., 1983a; Danscher, 1984a-c; P6rez-Clause11 and Danscher, 1985, 1986; see also Figs. 6 and 10 and references in Table IX). Significantly, the same ultrastructural pattern is found in many nonneural secretory cells, in which zinc staining selectively labels secretory granules and does not label membranes, nuclei, or mitochondria (see Fig. 10 and references below). Why zinc in synaptic vesicles (and other secretory granules) can be stained while zinc in other cellular compartments cannot is unknown. The plausible hypothesis is that the zinc in most organelles is bound too tightly into metalloenzymes to be easily removed for coordination or precipitation with the histologic reagents. [Note that anions and chelators can inactivate metalloenzymes without actually removing the zinc atom. (Vallee and Galdes, 1984).] This hypothesis is supported by the fact that organelles that do contain metalloenzymes (e.g., mitochondria, nuclei, membranes) (see above) generally do not stain for zinc. An example of this same phenomenon outside the nervous system is found in the erythrocyte, which contains about 60 ppm of zinc (dry weight) but does not stain for zinc (see, e.g., Smith et al., 1969; Haug, 1973); approximately 90% of the zinc in the erythrocyte is bound into carbonic anhydrase and superoxide dismutase (Yamashita et al., 1985), indicating that zinc in those metalloenzymes cannot be stained. The fact that zinc in carbonic anhydrase could not be stained in brain tissue was first noted by Timm (1958a,b). The above reasoning would suggest that the zinc atoms that can be stained are either weakly bound, o r actually free as Zn". There is some FIG. 6. A single zinc-containingbouton making synaptic contact (S) in the outer shell of the ventromedial hypothalamus.Note that even with heavy overdevelopment (large silver grains), there is virtually no zinc staining outside the bouton. These boutons arise from fibers of the stria terminalis. Courtesy J. PCrez-Clausell.

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CHRISTOPHER ,I. FRF.DERICKSON

indirect evidence for this idea, which comes from work on the secretory granules of the pancreatic P-cell. These granules, which stain vividly for zinc (Logothetopoulos et al., 1961; Pihl, 1968; Toroptsev et al., 1974), contain a precipitated insulin hexamer with two fairly tightly bound zinc atoms ( K Dabout lo-‘)), but they also sequester another 10-12 zinc atoms per mole that appear to be only weakly associated (Ku about with the insulin complex (Williamson and Williams, 1979; Epand et al., 1985). Perhaps the situation in the neural vesicle is similar, with zinc ions loosely associated with stored “packets” of neuroactive macromolecules (see Section VIII). OF HISTOCHEMICALLY REACI‘IVE ZINC C. CNS DISTRIBUTION

Figures 3, 4, 5 , and 7 and Table IX give an overview of the distribution of zinc staining in the CNS. Virtually all of the zinc-positive regions identified here and in the current literature were originally described by Haug in his 1973 monograph on Timm’s staining in the CNS of the rat. However, it is important to recognize that Haug used a

FIG. 7. Putative zinc-containing pathways are scheniatically illustrated. Shown are ( I ) hippocampal mossy fibers; (2) intrinsic cortical fibers; (3) cortirostriatal pathway; (4) aniygdalofugal stria terminals; ( 5 ) hippocainpo-septa1 prqjection; (6) perforant pathway; (7) projection from CAI t o subiculum; (8) ett‘erents to molecular zonc of DCN; (9) pyriform cortical projections to amygdala. See ‘l’ableX.

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Timm’s variant (1.0% NaZS perfusion and silver nitrate developer) that stained not only what can now be confirmed as zinc-containing cell processes but also other processes that cannot be confirmed to contain zinc by contemporary methods. I n other words, the early work of Haug and others on Timm’s staining in the brain (Timm, 1958a,b; Haug, 1973, 1974, 1975; Geneser et al., 1974; Kozma et al., 1978) showed all of the histochemically reactive zinc but may well have shown reactive substrates other than zinc as well. Since the 1970s, the major advances in this area have been ( 1 ) development of specific histochemical tests for zinc per se, (2) mapping of zinc-containing systems in a variety of species, and (3) confirmation that the zinc staining is localized in the presynaptic vesicles of specific axonal systems throughout the neuraxis. 1. Staining in the Neuropil: Zinc-Containing Axonal Boutons

a. Distribution and Localization. Much of the neuropil in the telencephalon stains vividly for zinc. This includes a variety of limbic regions, which stain especially deeply, laminae 1-111 and V of the cerebral cortex, and the entire striatum. All of these areas can be stained by TSQ as well as selenium and neo-Timm’s, and many can be stained by dithizone as well. Over a dozen of these brain regions have been examined in the electron microscope after neo-Timm’s or selenium staining, and in all cases the reaction product has been found to be restricted almost exclusively to the presynaptic boutons (Table IX). Occasional grains of silver are found in lysosomes and in axons, but the overwhelming preponderance of the neuropil stain is in boutons. One quantitative study found only 0.9% of Timm’s silver grains not directly associated with presynaptic boutons (SchroGder, 1979). Light microscopic observations with the zinc-TSQ fluorescence method are entirely consistent with this localization of zinc in boutons. The fluorescence is conspicuously absent in somata and proximal dendrites, but vivid in the neuropil (Figs. 3 and 4). Outside of the limbic and cerebrocortical regions, which have dense zinc-containing innervation, there are also many brain regions that are innervated by sparse, rather delicate plexuses of boutonlike puncta that stain by both the neo-Timm’s and the selenium methods (Haug, 1973; SchrGder, 1979, 1980; Danscher, 1984a-c) (Fig. 8). These boutons cannot be rendered visible by either dithizone (Fleischhauer and Horstmann, 1957; see also Szerdahelyi and Kasa, 1984) or TSQ fluorescence (Frederickson et al., 1987a), presumably because neither of the latter methods is sensitive enough to show individual boutons. However, the neo-Timm’s and selenium staining of these individual boutons is

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FIG. 8. Presumed zinc-containing boutons (selenium method) line the proximal dendrites and perikaryon of a reticular neuron in the cat brain stem. Scale, 50 pm.

blocked by prior treatment with intravital dithizone (Danscher, 1982, 1984b), and it is therefore reasonable to assume that they are zinccontaining boutons. Regions that are innervated by these delicate plexuses of boutonlike puncta include the neuropil of the spinal cord, much of the brain stem and medial thalamus, the superior colliculus, and parts of the granule cell stratum of the cerebellum of the rat (Danscher, 1981b, 1982, 1984a-d). In the rat cerebellum, the staining evidently labels a distinct subset of the mossy glomeruli (Danscher, 1984d). Ultrastructural studies have consistently shown that the bouton zinc is located in the vicinity of presynaptic vesicles (Haug, 1967; Ibata and Otsuka, 1969; Otsuka et al., 1982), and recent evidence indicates that the zinc is in the inner core of individual vesicles (Lopez-Garcia et al., 1983b; Friedman and Price, 1984; PCrez-Clause11 and Danscher, 1985, 1986). Only a small fraction of the vesicles in a bouton stain for zinc, and it is typically clear, round vesicles that are reactive (Perez-Clause11 and Danscher, 1985) (see Fig. 10). Whether this means that only some vesicles contain zinc or instead that the staining reaction occurs randomly in only a small percentage of vesicles cannot be decided with present methods. However, the possibility that arises is that zinc-

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containing vesicles are but a small, chemically unique subclass of vesicles, perhaps with different neurobiological roles than the rest of the vesicles in the bouton. Some investigators have emphasized that zinc-containing boutons have extensive zinc staining at the presynaptic specialization (SchrQder, 1979), in the synaptic cleft (Schrqider, 1979; Lopez-Garcia et al., 1983b; Friedman and Price, 1984), and on the outer presynaptic membrane (Friedman and Price, 1984). This distribution of zinc staining is entirely consistent with the evidence that zinc-containing vesicles are exocytosed from the boutons during synaptic activity (see below). Perhaps the relative amount of staining found in the cleft (versus that in the bouton) reflects the physiological status of the neurons just before or during the precipitation of the zinc in situ. In the cerebrum, zinc-containing boutons are consistently of Gray’s Type I morphology, making asymmetric contacts and having clear round vesicles (Haug, 1967; Friedman and Price, 1984; Martinez Guijarro et al., 1984; Perez-Clause11 and Danscher, 1985). In the spinal cord, the zinc-containing boutons are of type F (Schr@der,1979, 1980). 6. Amount of Zinc in Boutons. How much zinc is in the vesicular pool in axonal boutons is unknown, but this is clearly an important issue. Questions such as what might be colocalized with the vesicular zinc, what role zinc might have in the vesicles, and what neurobiological effects could be produced by exocytosis of the zinc could all be explored with considerably greater rigor if the amount of zinc in the boutons were known. One approach to estimating the amount of zinc in boutons is to measure total zinc in a region that is rich in zinc-containing boutons and subtract from that value the amount of zinc in a region with few such boutons. The available data indicate that the total amount of zinc in the hippocampal mossy fiber neuropil is between 145 ppm (Frederickson et al., 1983) and 136 pprn (Wensink et al., 1987a), and the amount of background zinc in the dentate gyrus about 55-79 pprn (Frederickson et al., 1983; Wensink et al., 1987a). Taking the difference gives an estimate of 200-300 pM of bouton zinc in the mossy fiber neuropil; volumetric considerations dictate that the concentration within individual boutons would necessarily be higher than 200-300 p M and the concentration within vesicles higher still. It has also been estimated that the total zinc sequestered in the mossy fiber boutons (about 66 ngihippocampus in the rat) is somewhat less than 10% of the total zinc in the hippocampus (Frederickson et al., 1983). Recent analyses in which dithizone was used to chelate bouton zinc in viuo have yielded complementary results, suggesting that the total

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amount of zinc that is chelated by dithizone (i.e., the vesicular pool) is only about 5-15% of total brain zinc (Dancher et al., 1987a). These values, interestingly, are comparable to the percentage of CNS zinc (10-1676) that Harris and Crawford (1981) and Dreosti and Record (1984) found would pass through a 12,000M , cutoff dialysis membrane. In principle, histoanalytic techniques employing zinc-dithizonate microabsorptiometry or zinc-TSQ microfluorometry could be used to assay bouton zinc without interference from the nonreactive (nonvesicular) zinc in the tissue. These methods have been used to compare relative amounts of bouton zinc in different brain regions (Frederickson et al., 1981; Frederickson and Howell, 1984), different rat strains (Savage, 1987), and after experimental manipulations (Stewart et al., 1984; Kesslak et al., 1987). Lack of suitable radiometric calibration standards has generally pecluded obtaining obsolute values for bouton zinc by fluorometry or absorptiometry, but one study using zinc-TSQ fluorometry has reported a value of 25 femtograms of zinc/lO0 pm‘ as the absolute difference between the mossy fiber neuropil and the adjacent stratum radiatum (Montdno et al., 1987). Compared to the dithizone and TSQ chelator stains, the silver amplification methods (neo-Timm’s and selenium) are less promising for quantitative histochemistry because the zinc precipitates of the latter methods are used to produce silver reaction product catalytically. Densitometric measures of silver-amplified staining have been made, however (Schwerdtfeger et al., 1985). 2. Staining of Neural Perikuqu and Glial Cells Both neural and glial perikarya can be stained with various Timm’s procedures, but whether any of that staining is due to zinc is uncertain. Haug (1973, 1974, 1975) and others (Koznia et ul., 1981; Szerdahelyi et al., 1984; Szerdahelyi and Kasa, 1986, 1987) have described both of these staining patterns in detail. As for the staining in neural perikarya, most evidence suggests that it is not due to zinc. For example, perikarya do not stain at all with the selenium method, even though that procedure is sensitive enough to render individual axonal boutons visible in light microscopy (Danscher, 1982, 1984a-c). Somata are also not stained by TSQ fluorescence in normal brains (Frederickson et al., 1987a; 1988a; 1989a) (Figs. 3 and 4). Furthermore, with neo-Timm’s staining, when prior treatment with dithizone is used as a masking test of zinc specificity, staining of the neuropil is masked, but staining of neural somata is not masked (Danscher, 1981b; see also Fredens and Danscher, 1973; Danscher et al., 1973). Finally, when sulfide-treated sections are exposed to prolonged fixation (Haug, 1973) or dilute acid rinsing (Kozma et al., 1981; Szerdahelyi and Kasa, 1984), staining in the

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neuropil and in the somata are differentially affected (but see Danscher, 1984~). The basis for the staining of glial cells is also unresolved. Certain glia can be stained by both neo-Timm’s and selenium methods (Danscher, 1981b, 1982), but they have not been observed with either dithizone or TSQ fluorescence. Moreover, prior intravital chelation with dithizone affects glial staining less than bouton staining (Haug, 1973), and rinsing sulfide-treated sections with dilute acids to remove zinc sulfide may (Danscher, 1984b,c) or may not (Kozma et al., 1981; Szerdahelyi and Khsa, 1984) prevent subsequent staining of glial cells. Glial and neural perikaryal staining could be due to metals other than zinc; copper has been suggested by the data of Szerdahelyi and Kasa (1984, 1986). Alternatively, it is possible that the silver deposits found in these cells are due to a nonmetallic substrate that can catalyze the reduction of silver. Another possibility is that the staining of the glial cells and neural somata is due to zinc that is in a different cellular organelle and/or molecular binding state than the zinc that is in presynaptic vesicles. The latter possibility would explain why the perikaryon substrate behaves differently in histochemical tests than the zinc that is in the vesicles. 3. Zinc Staining after Brain Damage Two kinds of changes in zinc staining can be observed after brain damage: loss of normal bouton staining and appearance of anomalous staining outside boutons. Loss of bouton staining will occur if axons are driven to sustained paroxysmal activity (e.g., by an excitotoxin) or if axons are cut and allowed to degenerate. These two phenomena are related, respectively, to the physiology of vesicular zinc and to the anatomical methods of‘mapping zinc-containing pathways, and they are discussed in later sections of this review. The appearance of anomalous staining for zinc outside of boutons after brain injury is discussed below. Transection of a major afferent projection to a given brain region can have several effects on zinc staining. In some instances, such as removal of septohippocampal efferents (Stewart et al., 1984; Frederickson et al., 1984; Kesslak et al., 1987) or interruption of limbic-cortical projections by destruction of the hippocampus (Berger and O’Leary, 1975), a rather dramatic increase in dithizone staining for zinc can be seen in the denervated tissue. Neither of these results has been explored with the higher-resolution silver amplification methods, so the cellular basis for the increased staining remains unknown. A parallel phenomenon in denervated tissue is the appearance of reactive astrocytes and macrophages that stain intensely by the Timm’s

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and neo-Timm’s method (see, e.g., Sunde and Zimmer, 1983; Haug, 1984). T o our knowledge, whether the Timm’s reaction in these glial cells is due to zinc has not been explored by any other method, but the possibility that glia liberate a histochemically reactive pool of zinc in response to injury is an intriguing one. A third change in staining after brain damage is the appearance of neo-Timm’s reactivity in degenerating axons. This relatively dark staining of degenerating axons develops gradually over the first few days after a lesion (Haug, 1984; Perez-Clause11et al., 1989).T h e staining could perhaps be considered a pseudo Fink-Heimer reaction, in which the degenerating fibers become agyrophilic and develop in the Timm’s solution for reasons having nothing to do with zinc; dithizone and TSQ testing could determine whether zinc is involved in this staining of degenerating axons. An exceptional case of injury-related staining of neural perikarya has also been reported recently. Neurons that degenerate as a result of prior seizures develop an anomalous neo-Timm’s reactivity. In this special case of seizure-induced neuron death, however, studies with the zinc-TSQ method indicate that the degenerating neurons are, in fact, saturated with excess zinc (Frederickson et al., 1989a). The reasons for these several types of injury-related changes in zinc staining are not understood, but the changes are clearly important methodologically and perhaps theoretically as well. The obvious methodological concern is that anomalous staining after brain injury could be misinterpreted as “normal” (i.e., bouton) staining and thus obscure significant changes occurring after brain injury. The possible theoretical significance is that the posttrauma changes in the histochemically reactive zinc pool could indicate a role of zinc in the brain’s biological response to damage. For example, it has been suggested that zinc in boutons may be associated with a 7s-NGF-like trophic factor (Crutcher and Davis, 1981; Frederickson el al., 1984); dynamic changes in the zinc pool could reflect liberation and/or accumulation of such a trophic agent after brain damage (Kesslak et al., 1987).

VII. Zinc-Containing Neurons

A. DEFINITION By analogy to other neuronal types, such as the glutamatergic neuron, or the serotonergic neuron, zinc-containing neurons may be defined as neurons that selectively concentrate zinc in their axonal

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boutons. Of course the situation here is more like that of the glutamatergic neuron than that of the serotonin-containing neuron because all neurons contain zinc and glutamate, whereas only some neurons are “zinc-containing” o r “glutamatergic.” In fact, the defining characteristic is that zinc-containing neurons sequester histochemically reactive zinc in their atonal boutons. It is the unique pool of stainable (presumably weakly bound) zinc that is concentrated in the vesicles of their boutons that defines the zinc-containing neuron.

B. TURNOVER OF VESICULAR ZINC AND ACTIVITY-METABOLISM COUPLING T h e mossy fiber axons of the hippocampus are the most thoroughly studied of the zinc-containing fiber systems. As has been mentioned previously, hippocampal slices take u p 65Zn with an apparent K , of 12-26 pA4 (Howell et al., 1984; Dreosti and Record, 1984). Within a hippocampal slice, uptake into regions innervated by mossy fibers is quantitatively greater than uptake into other regions, and uptake into the mossy fiber cells of origin (granule neurons) is intermediate (Howell et al., 1984; Howell and Frederickson, 1984). It has yet to be shown that this uptake is directly into the mossy fiber boutons, but the fact that electrical stimulation of the granule neurons selectively facilitates uptake into the mossy fiber regions suggests that the axon boutons are the likely site of uptake (Howell et al., 1984; Howell and Frederickson, 1984). The finding that the K , value for zinc uptake into hippocampal slices is significantly lower (50% lower) for tissue taken from rats which have developed a substantial mossy fiber system (14 days and older) than for tissue from rats lacking appreciable mossy fibers (7-day-old pups) further supports the notion that a substantial portion of the high-affinity uptake is into the mossy fiber boutons (Dreosti and Record, 1984). Another supporting observation is that systemically administered 65Znaccumulates preferentially in the synaptosomal fraction of hippocampal homogenates from adult rats (having mossy fibers), but not in homogenates prepared from 15-day-old pups (Wolf et al., 1984). Sat0 et al. (1984b,c) also found that 65Zn uptake in the adult hippocampus in vivo was preferentially into the subcellular fraction that they asserted contained the contents of synaptosomes, including the larger synaptosomes from mossy fibers. Release of zinc from mossy fibers has been studied by a variety of techniques in vitro and in vivo. Hippocampal slices release zinc readily in a calcium-dependent manner during either electrical stimulation of the granule neurons (Howell et al., 1984) or depolarization induced by

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adding K’ or kainic acid to the medium (Assaf and Chung, 1984).These kinetic studies do not show that such release is from mossy boutons per se, but parallel in vzzio studies indicate that this is the case. Specifically, by the use of chronic push-pull cannulae, Ben-Ari and co-workers have found that local depolarization (induced by Kt in the superfusion solution) yields a detectable release of zinc into the cannulae only when cannulae are positioned within the mossy fiber neuropil (Charton et al., 1985) and only if the mossy fiber axons are intact (Aniksztejn et al., 1987). T h e release of Linc from mossy boutons during electrophysiological activity is also suggested by several histochemical studies in which zinc staining has been found to be depleted or “bleached” in the brains of animals that have undergone prolonged, intensive paroxysmal activity just prior to sacrifice. McLardy (1962) was first to report that the mossy fiber boutons stained by the Timm’s method appeared to have undergone “shrinkage . . . (or) . . . collapse” (of the Timm’s-positive bouton material) after rats were subjected to repeated hypoglycemic or hypoxic seizures. More detailed observation has shown that the entire mossy fiber bouton system can be thoroughly bleached of Timm’s staining when rats are subjected to 24 hr of stimulation-induced hippocampal seizures (Sloviter, 1985). Virtually the same result is obtained with the ’l‘SQ method: Rats undergoing prior seizures and convulsions induced by intraperitoneal kainic acid show almost complete loss of fluorescence in the CA3 mossy fiber plexus within 8-12 hr of the onset of seizure behaviors (McCinty et al., 1988; Frederickson t t al., 1988a, 1989a). TSQ fluorescence from amygdalar boutons is also greatly diminished after seizure activity (Frederickson el al., 198%). The decrease in Timm’s staining that reportedly occurs after large doses of d-amphetamine in rats (Szerdahelyi and Kasa, 1985) and after intraventricular kainic acid in lizards (Lloret-Escrava and Perez-Clausell, 1986) may also reflect paroxysmal activity induced by those drug treatments. Similarly, the loss of mossy fiber zinc staining after trimethyl tin poisoning (Chang and Dyer, 1984) may be due to the seizure activity induced by the toxin (Sloviter, 1986). Studies in which zinc is tagged in situ within the vesicles of boutons indicate that the zinc is released by exocytosis of the zinc-rich vesicles (Perez-Clause11 and Danscher, 1986). In that study, sulfide ions were infused into the hippocampi of anesthetized rats in order to precipitate zinc in boutons as insoluble ZnS crystals. I n tissue taken from rats sacrificed shortly after the sulfide infusions, silver amplification showed that the zinc-filled vesicles were distributed uniformly through the interiors of giant mossy boutons. Quite a different result emerged, however, in brains taken from animals allowed to survive for 24 hr after

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the initial sulfide infusion. In the latter material, the silver-amplified ZnS crystals were found dispersed to the outer margins of the boutons, typically poised before the presynaptic specializations or embedded in the synaptic clefts. McLardy (1970) has reported a similar timedependent clearance of ZnS precipitate from hippocampal neuropil in behaving rats. Whether electrical stimulation would accelerate the clearance of intravitally deposited ZnS from within boutons would be important to ascertain. Taken together, the kinetic studies of turnover and the histochemical studies of zinc-containing boutons suggest that zinc is taken up into boutons by high-affinity uptake, sequestered in vesicles, and released from boutons during electrophysiological activity by exocytosis of the zinc-filled vesicles. As discussed earlier, the zinc content of these vesicles is probably well in excess of 200-300 p M . Thus, exocytosis could potentially generate sizable transient surges of zinc in the microenvironment of the boutons. Whether zinc is released from vesicles as free, Zn+2 or in some bound form is presently unknown. Autoradiographic studies with 65Zn have provided an indirect approach to the study of the turnover of zinc in mossy boutons. In general, the data indicate that the total pool of zinc associated with the mossy boutons is exchanged slowly with the systemic pool. After a single systemic injection of “Zn, the specific activity of zinc in blood peaks briefly, then falls slowly over several weeks as the tracer is excreted (see above). At the same time, the mossy fiber neuropil accumulates radiolabeled zinc, giving a dark labeling in the vicinity of the mossy fibers in autoradiographs (von Euler, 1962; Otsuka and Kawamoto, 1966; Hassler and Soremark, 1968; Crawford and Connor, 1972; Dencker and Tjalve, 1979). According to the microdensitometric analyses of hippocampal autoradiographs (Dencker and Tjalve, 1979), the radioactivity level rises gradually during the first week after 65Zn administration and falls back to 50% of peak levels over the subsequent 3 weeks. These results imply that the pool of zinc associated with the mossy fibers normally exchanges fairly slowly with the zinc in the circulation, with the bulk of the zinc released from the boutons presumably returning into them via reuptake. Unfortunately, the high y emissions (and resulting Compton electron scattering) of 65Zn preclude cellular localization of the isotope. In fact, radiographs made with X-ray film and photographed at low magnification seem to yield the best contrast between mossy fiber and adjacent neuropil (von Euler, 1962; Dencker and Tjalve, 1979),whereas emulsion methods may not even label the mossy fiber neuropil consistently (cf. Otsuka and Kawamoto, 1966; Hassler and Soremark, 1968). Nonetheless, by careful analysis of positron tracks from relatively

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thin sections, Crawford and Connor (19’72, 19’75) found that 65Zn emissions came predominantly from the region of mossy fiber terminals (stratum lucidum) in hippocampal field CA3. One especially interesting aspect of that work was that the granule neuron zone showed comparatively high “Zn activity in sections taken from young (10- 18-day-old) rat pups, a finding suggesting that substantial amounts of zinc may be transported from the granule somata into the mossy axons during the early proliferation and maturation of the granule cell-mossy axon system. The occurrence of such orthograde transport in axons is also supported by the observation that the granule neuron region takes up 65Znat a relatively high rate in vitro but is not found to contain especially high concentrations of endogenous zinc in material taken from intact animals (Frederickson et al., 1983; Howell et al., 1984; Howell and Frederickson, 1984). Slow-phase orthograde axonal transport of zinc in the optic tract has been suggested by radiotracer studies (Kasarskis, 19844.

C. ANATOMY OF THE ZINC-CONTAINING PATHWAYS ‘The zinc-containing pathways can be mapped by essentially the same techniques used to map, for example, an amine-containing neural system: Destruction of the cells of origin or efferent fibers causes a loss of the histochemically reactive bouton products as the boutons degenerate and are phagocytosed (Fig. 9). I n the case of zinc-containing boutons, the matter is somewhat complicated by injury-related zinc staining that may appearafter denervations. However, the neo-Timm’spositive astrocytes and degenerating axons that may occur in denervated zones are generally not difficult to distinguish from the zinc-containing boutons in the neuropil. Haug and coworkers were first to use degeneration methods to map a zinc-containing pathway (Haug et al., 1971; Haug, 1984). They showed that zinc staining of boutons in the mossy fiber regions disappeared permanently distal to lesions transecting the mossy fiber axons, a result that corroborated the morphological evidence that the zinc-containing boutons in that region were the mossy boutons of granule neurons axons. A cautionary point made by that study, however, was that the zinc staining vanished sooner (within 10-24 hr) than one would have expected significant degeneration of the cut axons to have occurred. This precipitous loss of the bouton zinc may have been due to paroxysmal firing of the severed fibers, while the lasting depletion reflected the subsequent degeneration of the axons.

NEUROBIOLOGY OF Z l N C

INTACT

20 1

DENERVATED

FIG.9. Loss of zinc-containing axonal houtons (neo-Timm’s method) in the ventromedial hypothalamus (VMH), nucleus terete (TE), and premammillary area (PM) after ipsilateral section of the stria terminalis is shown in a horizontal section of rat brain. (See Perez-Clause11 et al., 1989.)

Figure 7 (see Section VI, C) and Table X show some of the major zinc-containing pathways of the vetebrate forebrain. These are preliminary and highly schematic representations, based on evidence of varying types. Nonetheless, the pattern that emerges is stricking: Virtually all of the major zinc-containing systems are of limbic o r cerebrocortical origin. The limbic system is especially rich in zinc-containing components. For example, in the trisynaptic circuit that leads from the lateral entorhinal cortex to the CAI pyramidal neuron (via the mossy fibers and Schaffer collaterals), all three synapses are putatively zinc-containing; the continuing link from CAI to the subiculum has been suggested to be a zinc-containing pathway as well. Similarly, the pyriform input to the amygdala and the amygdalar projection to the basal forebrain (stria terminalis) constitutes another probable disynaptic, zinc-containing limbic circuit. These lists are by no means complete but illustrate nonetheless the richness and complexity of the many zinc-containing limbic circuits.

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TABLE X ZINC-CONTAINING PATHWAYS IN ‘THE B R A I N ~

Fiber system

Evidence

Hippocampal mossy fibers

Morphology, lesion, physiology Lesion Lesion Lesion Fibroarchitecture, lesions Lesion Lesion Fibroarchitecture Lesion Fibroarchitecture, lesions Fibroarchitecture, lesions

Amygdalofugai stria terminalis Septohippocampal Pyriform cortex-amygdala Lateral entorhinal cortex-dentate Commissurallassociational-dentate Schaffer collaterals CAI -subiculum Corticostriatal system Cerebrocorticdl interneurons and associational fibers Cochlear granule neurons-dorsal nucleus, molecular zone

~

References”

“References: (1) Haug vl nl. (1971); (2) llaug (1967); (3) Howell and Frederickson (1984); (4) Perez-Clausell cl u1. (1986); (5) C.J. Frederickson r l al. (1986); ( 6 )PPre~-C:lauscll rl ul. (1989); (7) Crawford (1986); (8) Zirnnler (1973); (9) Haug (1984); (10) Zimmer (1973; 1974); ( 1 I ) Frederickson el ul. (198%); (12) Hill and Frederickson (1988); (13) Frederickson d ul. ( 1988b, 1 Y89b).

Outside of the limbic system, most of the remaining zinc-containing terminals in the forebrain originate from cerebrocortical neurons. Thus, the zinc-containing innervation of laminae 1-111 and V of the cortex evidently arises predominantly from local cortical interneurons. This conclusion is based on the fact that the zinc staining of boutons in the cortex is little altered when all extrinsic corticopedal fibers are cut (in cortical “islands”), whereas destruction of the intrinsic neurons with ibotenic acid causes complete loss of zinc-containing boutons in the vicinity of the lesion (Hill and Frederickson, 1988). Cerebrocortical neurons are also the primary (probably exclusive) source of the zinccontaining boutons in the striatum, because the zinc staining of boutons in the latter region is lost after cerebrocortical lesions but is unaffected by neurotoxic lesions placed in the substantia nigra, thalamus, or directly in the striatum (Perez-Clause11et al., 1986; Frederickson et al., 1989~). T h e cerebrocortical and limbic circuits described above account for the preponderance of the zinc-containing boutons in the CNS, but there are also apparent zinc-containing boutons in the thalamus, brain stem, and spinal cord. In most of the latter regions, the boutons are sparsely scattered and lighly stained, and mapping of the efferent pathways has

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not been attempted. One exception to this is in the dorsal cochlear nucleus, the molecular zone of which is fairly heavily innervated with zinc-containing boutons. Lesion data indicate that these zinc-containing boutons originate from the cochlear granule neurons, the parallel fiber axons of which innervate the molecular zone of the nucleus (Frederickson et at., 1989b).

D. COLOCALIZATION OF ZINC WITH TRANSMITTERS AND MODULATORS N o neurotransmitter or neuromodulator has been shown to be exactly coextensive with zinc-containing boutons throughout the CNS. There are many substances that are colocalized with zinc in some terminals; the zinc-rich hippocampal mossy fibers’ boutons, for example, contian enkephalin and dynorphin (Stengaard-Pedersen and Larsson, 1981; Khachaturian et al., 1982; McGinty et al., 1988)and most probably glutamate-aspartate as well (Crawford and Connor, 1973; for review see Ottersen and Storm-Mathisen, 1986). However, there is also enkephalin and dynorphin in the globus pallidus (Stengaard-Pedersen and Larsson, 1981; Khachaturian et al., 1982) and glutamate in the cerebellar molecular zone (Ottersen and Storm-Mathisen, 1986), t w o regions that have essentially no zinc-containing axonal boutons. Although no identified neuroactive substance maps one-to-one with the zinc-containing terminals, it does appear that bouton zinc is closely associated with excitatory amino acid neurotransmission. Specifically, all but one of the zinc-containing pathways that have been tentatively identified are pathways that have also been suggested to be glutamatergic-aspartergic pathways (Ottersen and Storm-Mathisen, 1986; Cotman et al., 1987). (The exception is the amygdolofugal stria terminalis, for which the amino acid data are not available.) This zinc-glutamate link has been noted by others (Crawford and Connor, 1972, 1973, 1975; Storm-Mathisen, 1977; Wolf and Schmidt, 1982; Crawford, 1983, 1986). One aspect to emphasize, however, is that not all Glu-Asp pathways are zinc-containing: T h e cerebellar parallel fibers, primary acoustic afferents, and optic nerve fibers are three examples of presumed amino acid axonal systems (references in Ottersen and Storm-Mathisen, 1986) that have virtually no histochemically reactive zinc in the terminals (Table IX; Figs. 4 and 5). The anatomical data indicate that zinc-containing neurons are a specific subclass of Glu-Asp neurons, and the distribution of Glu-Asp receptors is consistent with this view. The zinc-containing terminals are not coextensive with any single receptor type, but if one considers the

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combined distribution of the N-methyl-u-aspartate (NMDA) and the kainic receptors, the match with zinc-containing terminals is reasonably good. ‘The localization of the N M D A and kainic receptors in the hippocampus, and in the different laminae of the cerebral cortex, in particular, is strikingly similar to the localization of the zinc-containing boutons [cf. Figs. 4 and 5 with Cotman et al. (1987)l.These localization data point to a specific role of vesicular zinc in modulation of Glu-Asp activation of the NMDA and kainic receptors. -1’he physiological effects of zinc upon amino acid receptors discussed below are generally compatible with this hypothesis. VIII. Functional Significance of Vesicular Zinc

Zinc could play either of two roles at the zinc-containing synapse. Within the vesicle, the cation could be an essential “storage cofactor,” necessary for stabilizing storage of the primary secretory substances in the vesicle. After release into the cleft, the zinc ion could also assume an intercellular messenger-modulator function. These t w o functions are not incompatible, and there is evidence to suggest that both may occur.

A. ZINC A N D STORAGE OF MACROMOLECULES I N VESICLES There is no direct empirical support for the storage cofactor hypothesis as it applies to vesicular zinc; in fact, what molecules might be stored with (stablilized by) zinc in neural vesicles remains completely unkown. What makes the storage cofactor idea attractive nonetheless is the precedent for it one finds in a variety of nonneural secretory cells. The histochemically reactive zinc that is found in neural vesicles is but one instantiation of a broader biological pattern. Many secretory cells sequester histochemically reactive zinc in their secretory storage granules (Fig. 10). Examples of this include pancreatic p-cells (Logothetopoulos el al., 1961; Pihl, 1968; Toroptesev and Eshchenko, 1971), salivary granular convoluted tubule (GCT) cells (Frederickson et al., 1987b), mast cells (Danscher et al., 1980), certain pituitary cells (Thorlacius-Ussing and Danscher, 1985; Thorlacius-Ussing, 1987) leukocytes (Pihl et al., 1967; Smith et al., 1969),paneth cells (Elmes andJones, 1981; Danscher et uL, 1985b), thymus cells (Nabarra et ul., 1984), and adrenal medullary cells (Thorlacius-Ussing and Kasmussen, 1986). T h e zinc staining in all of these secretory cells is very much like that in neurons. Except for occasional grains found in lysosomes (Brun and Brunk, 1970;

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FIG. 10. Zinc in various storage granules, including presynaptic vesicles, is shown by the neo-Timm’s method for electron microscopy. (A) Insulin granules in pancreatic P-cell; (B) secretory granules in GCT submaxillary salivary cell; (C) vesicles in giant mossy bouton of dentate hilus; (D) histamine-heparin granules of mast cell. Magnification varies from about 650 to 65,000. Note the absence of staining outside granules in all cells. N, nucleus. Micrographs (A) arid (D), courtesy of G. Danscher; ( C ) courtesy of J. PCrez-Clausell.

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see also preceding references), the vast preponderance of all zinc staining is in the secretory granules. Vesicular zinc could have an entirely different function than the zinc in other secretory storage granules, but it seems more likely that zinc would have essentially the same function in all secretory granules, including vesicles. In nonneural cells, zinc evidently stabilizes the storage of secretory macromolecules. For example, in pancreatic p-cells the zinc ions are necessary for maintaining the structure of the crystalline insulin precipitate. As mentioned earlier, there are as many a 12-14 zinc atoms associated with each insulin packet. T w o are enfolded within the insulin hexamer, coordinated with one histidine residue from each monomer, and the remaining 10-12 are loosely bound with the crystal. [Pihl (1968) has published especially striking electron micrographs of the zinc surrounding insulin crystals.] Removal of the zinc ions from the insulin granules by intravital chelation solubilizes the insulin and depletes the granules, often damaging the @cells in the process (Emdin et al, 1980; Gold and Grodsky, 1984; Epand et al., 1985; Falkmer et al., 1985). A second example of zinc’s stabilizing effects upon a stored protein comes from the GCT cells of the male mouse submaxillary gland, where zinc is colocalized with a number of proteins and preproteins, including 7-S-NGF (Frederickson et al., 1987b). The 7S-NGF molecule is coordinated with zinc, and removal of the zinc ions by chelation or dilution causes the molecule to dissociate into subunits, one of which is /3-NGF (Pattison and Dunn, 1976a,b). The possibility that the zinc in mast cell granules similarly stabilizes aggregates of histamine-heparin has also been supported by some evidence (Kerp, 1963; Keller and Sorkin, 1970), though not all (Uvnas et ul., 1975). A zinc-stabilized thymulin has also been suggested (Dardenne et al., 1985). In the same way that depolarization depletes axonal boutons of their zinc-containing vesicles, secretagogues deplete other secretory cells of storage granule zinc. The pancreatic p-cells (Logothetopoulos et al., 1961; Toroptsev e l al., 1974; Grodsky and Schmid-Formby, 1985), the salivary GCT cell (Frederickson et al., 1987b), and the mast cell (Keller and Sorkin, 1970) release their storage granule zinc (along with other secretory products) when secretion is induced. In neural vesicles, zinc could stabilize the storage of a neurotransmitter, cotransmitter, neuromodulator, or for that matter any type of neuroactive messenger molecule. Various authors have explored the possibility that the ion could stabilize storage of acetylcholine (Szerdahelyi et ul., 1984), catecholamines and ATP (Maas and Colburn, 1965; Berneis et at., 1969; Rajan and Mainer, 1978), glutamate (Gra-

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maccioli, 1966), or a 7S-NGF-like molecule (Crutcher and Davis, 1981; Frederickson et al., 1984; Kesslak et al., 1987) in the brain. Zinc-taurine (Van Gelder, 1983; Sturman et al., 1981) and zinc-histamine (Kerp, 1963; Kayali and Berthon, 1980) are two additional complexes that may be mentioned in this context. Specific mechanisms for stabilizing the storage of macromolecules could include direct effects upon the conformation of the macromolecule (as with insulin and 7S-NGF) as well as effects upon intravesicular pH (Epand et al., 1985), stability of vesicular membranes (Chvapil, 1973), formation of ternary ligand-metal complexes (Maas and Colburn, 1965; Rajan et d,1971), and maintenance of transmembrane ionic gradients (see, e.g., Donaldson et al., 1971; Brewer, 1980). Given that the zinc-containing neurons are evidently a specific subclass of glutamatergic-aspartergic neurons, one might suppose that the putative zinc-stabilized macromolecule in the vesicles would be either (1) a Glu-Asp-containing storage complex of some type, perhaps allowing exceptionally large amounts of amino acid to be stored at these synapses, o r (2) some cotransmitter, modulator, or neuroactive factor that is used only at the zinc-containing subclass of the Glu-Asp synapses.

MODULATOR OF SYNAPTIC RECEPTORS B. ZINC AS A POTENTIAL AND PHYSIOLOGY Regardless of what might be in vesicles along with zinc, it is clear that the zinc is released from the vesicles and could therefore influence preor postsynaptic targets directly. Both depletion of zinc and addition of zinc have been used to explore the ion’s possible synaptic functions. 1. Evidence from the Depletion Paradigm Attempts to remove or deplete the zinc from axonal boutons by lowering the total burden of somatic zinc have produced mixed results, not surprisingly in view of zinc’s manifold roles in the biology of virtually all cells and organs. Thus, dietary zinc deficiency or systemic administration of metal chelators has been reported to cause either depression of hippocampal synaptic responses (Hesse, 1979) or a dose-dependent facilitation followed by depression at higher doses (Crawford and Connor, 1975; Crawford et al., 1973). The use of seizure susceptibility as a measure of synaptic potency has also yielded conflicting findings, with systemic administration of chelators either reducing seizure susceptibility or inducing seizures, depending on as yet unknown paradigmatic factors (see Section IX).

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Focal, intracerebral infusion of zinc-binding agents has also been used by several groups in an effort to manipulate the zinc-containing synapses of the hippocampus selectively. This technique was introduced by von Euler (1962) in his exceedingly thorough study of zinc binding and synaptic function. Hydrogen sulfide dissolved in Ringer’s was applied topically to the hippocampus to precipitate zinc (as ZnS) in situ, and the effects of zinc precipitation upon a variety of synaptic inputs to the CA3 pyramidal cells were explored. T h e essential finding was that the mossy fiber-pyramid synapse was quickly and irreversibly blocked by the sulfide treatment, whereas other synaptic inputs to pyramidal neurons (commissural-CA3 neurons, temporammonic-<=A1 pyramids) were only transiently depressed and only after prolonged sulfide exposure. More recent work has not confirmed von Euler’s suggestion that binding vesicular zinc blocks synaptic transmission. Danscher et al. (1975) for example, used an acute preparation like von Euler’s, but substituted DDC, which chelates vesicular zinc (Danscher, 1984a-c), for the HZS. Danscher et al. (1975) found no diminuition at all in the mossy fiber-CA3 pyramid evoked potential. On the contrary, they found that the mossy fiber synaptic response was briefly facilitated after DDC, an effect that was also observed with the commissural synaptic input to the pyramids. EDTA, which does not penetrate cell membranes and does not affect zinc staining of boutons (Fredens and Danscher, 1973), likewise did not mimic the effect of the DDC. Doller and Crawford (1984), who used dithizone to chelate zinc from hippocampal slices in uitro, also found no depression of synaptic function except with drug levels (120 p M of dithizone) that suppressed responses to antidromic stimulation as well as transynaptic activation. Presumably, Doller and Crawford could confirm the chelation of zinc in their slices by observing formation of the red zinc dithizonate in the neuropil. Danscher et nl. (1975) verified zinc chelation in their studies by demonstrating loss of ‘I‘imm’s staining in sections taken from the hippocampal regions into which DDC had been infused. Failure to bind the vesicular zinc is therefore not a likely explanation for the failure to block synaptic transmission in these latter two studies. In view of the indirect evidence that the CA3 cornmissural pathway may be a zinccontaining pathway, von Euler’s finding that sulfide affected mossy fibers but not the commissural system may prove equally difficult to interpret. Moreover, it is worth recalling at this point that chelators and anions such as sulfide are used routinely to inactivate metalloenzymes arid metal-dependent enzymes in vztro (Vallee and Galdes, 1984). Therefore, any consistent effects of these agents upon synaptic function

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209

that may emerge will bear scrutiny with respect to possible indirect (metalloenzyme-mediated) physiochemical mechanisms.

2 . Evidence from the Mimicry Paradigm The biochemical effects of exogenous zinc ions and some of the difficulties arising in interpreting such effects have been discussed in prior sections of this review. In the following sections, effects of zinc upon various stages of synaptic transmission are reviewed, and the caveat raised previously is worth repeating. In the absence of data on the normal, physiological levels of free ionic zinc in the CNS, one should be cautious in accepting reported effects of zinc ions as relevant to normal brain function. This notwithstanding, it is clear that the ion has a vastly broad range of potential actions in the CNS. a, Synthesis, Uptake, and Release of Putative Transmitters. Zinc inhibits the release of secretory products from a variety of nonneural secretory cells including p-cells (Ghafghazi et al., 1981), mast cells (Marone et al., 1986, Harisch and Kretschmer, 1987), and eosinophils (Winqvist et al., 1985; but see Inoue et al., 1985), an effect that may be mediated by the suppression of calcium-dependent calmodulin activity (Brewer et al., 1979; Brewer, 1980) or could represent an inhibitory autoreceptor involved in the regulation of zinc-containing secretory granules. A similar inhibition of prolactin release from neurosecretory cells has been observed in response to relatively low (5-10 p M ) concentrations of exogenous zinc (Login et al., 1983; Judd et al., 1984; Mahajan et al., 1985). To our knowledge, inhibition of release of neurotransmitters or neuromodulators from neurons has not been demonstrated for zinc. In view of the precedent, the possibility would seem to be worth investigation. Uptake of amino acid neurotransmitters (GABA and glutamate) into brain slices has been shown to be inhibited u p to 40% by modest concentrations (30 and 50 pA4) of zinc salt added to the medium (Gabrielsson et al., 1986). Similar inhibition of choline and norepinephrine uptake into synaptosomes has been reported by some (Prakash et al., 1973)) whereas others have observed a facilitation of monoamine uptake (Komulainen, 1983; Tuomisto and Komulainen, 1983). Synthesis and catabolism of several tansmitter substances are also affected by zinc ions. Synthesis of GABA, for example, can be slowed by an inhibitory effect of zinc ions on the rate-limiting synthetic enzyme, glutamate decarboxylase (GAD) (Table XI). I n addition, zinc ions stimulate pyridoxal kinase (Table XI), thus leading to increased production of pyridoxal phosphate (PLP), a cofactor for both synthetic (GAD)

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CHRISTOPHER J. FREDERICKSON

TABLE XI BIOCHEMICAL EFFECTSOF ZINC Concentration Process

Neurotransmitter metabolism Cholinesterase 100,000 GAD 200 GAD 350 GAD 40 GABA-T 200 0.4 Pyridoxal kinase Pyridoxal phosphate phosphatase 500 0.01-0.1 GDH GDH 10 Other 5-Nucleotidase Protein kinase C Protein kinase C Protein phosphorylating enzymes Dolichol kinase ATPase HCO3Intestine Na+,K+ Macrophage Trout gill

~ g + ' Macrophage Trout gill Transmitter uptake Glu uptake GABA uptake Adrenalin (Adrenal medulla) Dopamine Serotonin Serotonin

Effect

(PM)

1Ra

125 50

1 10,000

5,000

90% Inhib 50% Inhib 50% Inhib 50% Inhib 50% Inhib 100% Stim 100% Inhib 20-40% Increase 90% Inhib

50% Inhib (K;) 50% Inhib 100% Increase (both Ca-dependent) 40% Inhibition 5000% Increase

Prevents EDTA inactivation

1

50% Suppression 50% Suppression 50% Suppression 50% Inhibition 50% Inhibition

1,000 750

50% Suppression 50% Suppression

30 30 1,700

20% Decrease 20% Decrease 50% Decrease

10 10

30% Decrease 10% Decrease 90% Increase

1,000 100 4 1

oral, in uiuo

Reference"

21 1

NEUROBIOLOGY OF ZINC

TABLE XI (Continued) Process

Concentration (PM)

Transmitter release Serotonin Platlets

1

Receptor binding GABA

1

GABA Baclofen Diazepam Diazepam Carboline GLUJASP glutamate glutamate (Na-independent) aspartate Opioid receptors Naloxone Naloxone En kephalinamide En kephalinamide ACh oxotremorine QNB Dopamine spiperone Steroids Androgen receptor Prostate Androgren receptor Prostate Progesterone Uterus

10 100 100 1,000 1,000

Ef'f'ect

40% Decrease

Reverses chelation-induced suppression 50% Decrease 60% Decrease 20% Increase 40% Increase 20% Decrease

131 1,000

50% Inhibition 90% Decrease

50

50% Inhibition

10 (in vivo)

70 50 10 1,000

3.9 0.13 (estimate) deficiency (in vivo) 10

Reference"

40% Elevation K I , 200% Elevation KD 50%Inhibition ( K I ,elevated) 25% Inhibition (K, elevated) Reverses chelation-induced inhibiton QNB displacement by agonists facilitated 50% Inhibition Conformational change in receptor 60% reduction in B,,,, 60% Increase

"References: (1) Chanh and Plancade (1971); (2) DeBoer et al. (1979); (3) Ebadi et al. (1981, 1984b); (4) K. W. Chung et al. (1986); (5) Ebadi and Govitrapong (1979) (6) Wolf and Schmidt (1982); (7) Meflah et al. (1984); (8) Murakami et al. (1987); (9) Gettlefnger and Siegal (1978); (10) Sakakihara and Volpe (1985); (11) Kaysen et al. (1979); (12) Mustafa et al. (1971); (13) Watson and Beamish (1981); (14) Donaldsonetal. (1971); (15): Hexum (1974);(16) Gabrielsson etal. (1986); (17) Kirshner (1962); (18) Tuomisto and Komulainen (1983); (19) Komulainen (1983); (20) Chvapil el al. (1975); (21) Baraldi et al. (1984); (22) Drew et al. (1984); (23)Mackerer and Kocknian (1978); (24) Mizuno el al. (1983); (25) Slevin and Kasarsikis (1985); (26) Mena et al. (1985); (27) Baraldi et al. (1986); (28) Stengaard-Pedersen (1982); (29) Ogawa etal. (1984); (30) Hulme el al. (1983); (31) Smith and Huger (1983);(32) DeVries and Beart (1985); (33) Wilson (1985); (34) K.W. Chungetal. (1986); (35) Habib et al. (1980).

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and catabolic (GABA transaminase) enzymes of GABA metabolism (for reviews see Chung and Johnson, 1984; S.-H. Chung et al., 1986; Ebadi et al., 198413; Ebadi and Hama, 1986). Intracranial zinc administration decreases GAD activity (Itoh and Ebadi, 1983) and decreases cerebral levels of GABA (Chung and Johnson, 1983b), effects that have been implicated in the epiletogenic action of intracranially administered zinc, as discussed below. T h e activity of glutamate dehydrogenase (GDH) has also been reported to be sensitive to zinc ions (Wolf and Schmidt, 1982). Increased CNS glutamate levels have been observed after zinc administration (Chung and Johnson, 1983b), whereas the effects of dietary deficiency of zinc upon CNS GDH activity have varied among different investigations (Prohaska et al., 1974; Dreosti et al., 1981; Cossack and Prasad, 1987). b. Receptor-Ligand Binding. Zinc has powerful effects on receptorligand binding and on receptor-ionophore function as well. A number of groups have examined the effects of exogenous zinc upon ligandreceptor binding in synaptosomal or crude membrane fractions of brain, and most have found that binding is depressed by the zinc ion. This is true for both excitatory and inhibitory amino acid ligands, at least one peptide, and two aminergic ligands. Binding of cholinergic agonists and antagonists shows differential effects of zinc, depending upon the specific ligand; binding to the benzodiazapine site on the GABA receptor, likewise, may be either depressed or facilitated, depending upon the specific ligand (Table XI). The effective doses for these effects of zinc on binding are generally high (0.5-1 .OmM), in fact approaching the 0.2-1.0 mM levels that have been shown to be fatal to cultured neurons. However, the modulation of glutamate, aspartate, and spiperone binding to receptors have been observed with doses in the range of 5-100 (Table XI). c. Receptor-Ionophore Physiology. Electrophysiological data tend to support the notion that zinc interferes with the receptor-mediated actions of certain neurotransmitters upon postsynaptic target cells. Because of the evidence linking zinc to glutamatergic transmission, much of the recent work has focused on zinc and amino acid receptors. The findings from studies of dissociated neurons in culture indicate that zinc ions cause rapid, selective, and reversible depression of responses to certain amino acid agonists. Depolarizing currents produced by NMDA, for example, are virtually completely blocked by microinjections of zinc salts into the immediate vicinity of cultured cortical (Peters et al., 1987) and hippocampal (Westbrook and Mayer, 1987) neurons, whereas responses to kainic acid and quisqualic acid are unaffected (Peters et al., 1987) or slightly potentiated (Peters et al., 1987; Westbrook and Mayer,

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NEUROBIOLOGY OF ZINC

1987) (Fig. 1 1 ) . A decrease in the NMDA-activated membrane conductance has been detected with as little as 5 pM of zinc (Westbrook and Mayer, 1987), and the effects of the ion on the NMDA response are rapidly and spontaneously reversed. The dose-response data suggest that this effect is not due to competitive displacement of NMDA from receptors; a specific zinc-binding site on the receptor that might allosterically modify receptor-channel coupling has been proposed (Westbrook and Mayer, 1987). Additional findings in the literature might be attributable to the antagonistic action of zinc upon the NMDA receptor. For example, zinc

A

50 pM Zn

f10.2 nA

3 sec

-

50 pM Zn

-

1 mM Zn

I

C

-

50 pM Zn

1 mM Zn

10.4 n A

10sec FIG. 1 1. Intracellular recordings from cultured hippocampal neurons showing the effects of brief pulses of zinc salt upon membrane responses to NMDA (A), kainate (B), and quisqualate (C). Note the almost complete blockade of the NMDA response and its rapid reversal in (A). Courtesy of M. L. Mayer.

2 14

CHRISTOPHER J . FREDEKICKSON

added to the medium protects cultured neurons from the excitotoxic action of NMDA but not from kainic or quisqualic acid toxicity (Peters et al., 1987). Also, modest levels of zinc chloride (50 pM) added to the medium significantly attenuate paired-pulse potentiation of the mossy fiber-CA3 pyramid evoked potential (Brown et al., 1985; Khulusi et al., 1986). Finally, an early report (Rozear et al., 19’71)that zinc consistently depressed firing rates of neurons recorded from acutely prepared cats could have been due to effects upon the NMDA receptor. In that work the effect of iontophoretically applied zinc (estimated at 25-50 p M in the tissue) was tested on neurons that had been induced to fire “ . . . more often (than not) by iontophoretic release of glutamate.” GABA-mediated responses are also reported to be modulated by the zinc ion, but there are apparent inconsistencies in the literature. Specifically, Smart and Constantini (1982) found that 10 pM of zinc chloride attenuated the GABA-induced conductance changes in lobster muscle by 40% but had no detectable effect on the GABA responses in the rat superior cervical ganglion preparation. In contrast, the GABAinduced currents in pyriform cortical neurons recorded in vitro by the same authors were augmented by addition of zinc ion (100 pM and up) to the medium (Smart and Constantini, 1983). Focal application by zinc of iontophoresis or pressure ejection has also yielded variable effects upon the GABA receptor. Thus, Wright (1984, 1986) found no obvious change in GABA-induced hyperpolarization of cortical neurons recorded from anesthetized rats when zinc was iontophoretically applied prior to GABA applications. Westbrook and Mayer (1987), on the other hand, observed substantial depression of the GABA-induced synaptic current by prior application of 5 pM zinc chloride solution (evidently applied by pressure ejection) in their studies o f rat hippocampal neurons maintained in dissociated cell culture. One possible explanation of the inconsistent findings is that zinc affects both the GABA and the benzodiazepine sites of the receptor (see Table XI), and the relative impact of the two zinc actions could vary among the different tissues that have been examined.

IX. Zinc and CNS Pathology

A. BRAINDEVELOPMENT Two quite separate issues must be disentangled in considering cerebral pathologies associated with zinc metabolism: the role of zinc in brain development and the role of zinc in the function of the adult brain.

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215

Concerning brain development, it is quite clear that zinc insufficiency can be devastating, producing varied dysmorphogenetic effects and frank teratology in the developing nervous system. Neuroamatomical and behavioral sequellae of early zinc undernutirtion have been confirmed in many species, including humans, and many of the effects are apparently permanent, resisting extended zinc repletion therapy. Some very thorough analyses of the cytological and morphological impact of deficiency have been done (Dvergsten et al., 1983, 1984; Record et al., 1985; Harding et al., 1988), and the role of zinc in the regulation of protein and nucleic acid synthesis has been discussed by several authors (Vallee and Falchuk, 1981; Coleman, 1983; Klug and Rhodes, 1987). T h e reader is referred to the many excellent reviews available for further information on this issue (Prasad, 1972; Gordon et al., 1981; Sandstead, 1984, 1985; Rogers et al., 1985; Wallwork, 1987; Swanson and King, 1987). B. ADULTBRAINFUNCTION Zinc has been considered in association with a diversity of neurologic and neuropsychiatric disorders through the years. Examples include autism (Shearer et al., 1982), schizophrenia (Kimura and Kumura, 1972; McLardy, 1975), cerebellar dysfunction (Henkin et al., 1975), senile dementia (Kenn and Gibb, 1986), learning disabilities (Grant et al, 1988), and aphasia (Lerman-Sagie et al., 1987). It has also been reported that zinc accumulates in the brains of patients with Pick’s disease (Constantinidis and Tissot, 1981), and some investigators have found regional alterations of zinc in the brains of Alzheimer’s patients (Ward and Mason, 1987), whereas others have found normal CNS zinc levels (Hershey et al., 1985; Ehmann et al., 1986) or isolated regional anomalies (Thompson et al., 1988) in that patient group. T h e brains of alcoholics often show marked reductions in zinc content compared to control subjects (McLardy, 1975; Kasarskis et al., 1985b), but the available evidence indicates that such losses of CNS zinc are the consequences of the cirrhotic liver damage accompanying the late stages of prolonged alcohol abuse, not an effect of alcohol per se (Kasarskis et al., 1985b; Zeneroli et al., 1984). Zinc can also be implicated indirectly in metal neurotoxicity, inasmuch as reduced zinc intake can result in an increased accumulation of toxic metal in the CNS (see, e.g., Yamaguchi and Uchiyama, 1987; Brewer et al., 1987a,b). Unfortunately, most of the behavioral and neurologic disorders suggested to involve zinc have proven difficult to explore in experimental models because there has been no satisfactory way to manipulate the

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CHRISTOPHER J . FKEDERlCKSON

brain’s zinc stores selectively. As has been discussed, dietary zinc deficiency in adults does not produce a detectable change in total brain zinc, at least not until well after the appearance of multiple somatic symptoms including weight loss, skin lesions, hair loss, and changes in endocrine, immune, gastric, and hepatic function (for reviews see Chvapil et al., 1972c,d; Spencer et al., 1976; Bettger and O’Dell, 1981). Thus it is not surprising that dietary deficiency in adults fails to produce discrete or specific neural or behavioral changes not attributable to the nonspecific effect of the somatic debilitation (Hesse el al., 1979; Mossaro et al., 1982; Chafetz et al., 1984; Tucker and Sandstead, 1984). Efforts to manipulate brain zinc by the systemic administration of metal chelators likewise have been hindered by the fact that those drugs can potentially inactivate any metalloenzyme in any tissue o r organ and generally cause considerable general debilitation (McLardy, 1970; Danscher and Fjerdingstad, 1975; Haycock et al., 1977; Drust and Crawford, 1984). One approach to the problem that appears promising is the use of focal intracerebral infusion of metal-binding drugs (R. E. Frederickson et al., 1986, 1989). We have used this procedure with rats bearing chronically implanted cannulae and have found that either chelation (using DDC) o r precipitation (using NaZS) of hippocampal zinc causes an immediate impairment of hippocampal function, as measured by performance on a spatial memory problem in a water maze. Significantly, the hippocam pal deficit is transitory and has a time course comparable to the histologically confirmed time course of the particular zinc-binding drug used. DDC chelates zinc from the vesicles of axonal boutons for only 1-3 hr (Danscher and Kebbe, 1984) and affects behavior for a comparable period. The sufide ion creates zinc sulfide precipitates in the vesicles that take a day or so to be cleared from terminals (Perez-Clause11 and Danscher, 1986), and the behavioral effects of sulfide infusions likewise and are not reversed until 24 hr after the initial drug treatment. Presumably, this approach could be extended to the study of othezinc-containing neural system in the brain Despite the methodological problems inherent in investigation of zinc’s roles in behavior, there are two neurobehavioral disorders in which zinc has been repeatedly implicated, namely, epilepsy and anorexia. 1. Zinc and Epilepsy

Several lines of research suggest a role of zinc in the pathophysiology of epilepsy. In brief overview, the evidence is as follows. First, CNS zinc levels are reportedly abnormal in the brains of seizure-prone laboratory animals, and in the brains and body fluids of human patients with

NEUROBIOLOGY OF ZINC

217

seizure-related disorders. Second, the heaviest concentrations of zinccontaining boutons are in the seizure-prone limbic regions of the brain. Third, manipulating brain zinc has effects upon seizure susceptibility. Fourth, seizures cause substantial changes in cerebral zinc. Fifth, zinc has multiple effects upon both excitatory and inhibitory amino acid neurotransmission. The foregoing encourages the general notion of a zinc role in seizure pathophysiology, but closer review of the evidence does not lead to any simple, unitary hypothesis about the underlying mechanisms. For example, whereas mice genetically prone to seizures have elevated brain zinc (Chung and Johnson, 1983a), genetically seizure-prone rats have 47% less zinc in the hippocampal mossy fiber region than control rats (Savage, 1987). Similarly, zinc in the serum of seizure-prone baboons (Papio papio) is apparently elevated (Alley and Killam, 1976; Alley et al., 198I), whereas the CSF of children undergoing “fifth-day fits” (Goldberg and Sheeny, 1982), and the serum of preeclamptic women (Brophy et al., 1985) are characterized by subnormal zinc concentration. In adult patients suffering from epileptic disorders, some investigators have found changes in serum zinc (Barbeau and Donaldson, 1974; Pippenger et al., 1979; Palm and Hallamans, 1982), but other evidence indicates that neither epilepsy nor anticonvulsant drug therapy is consistently associated with any abnormalities of serum zinc (Vasiliades and Sahawneh, 1975; Schott and Delves, 1978; Smith and Bone, 1982; Hurd et al., 1984; Werther et al., 1986). These findings suggest that the amount of zinc in the brain, CSF, and serum may vary depending on the eitology, clinical history, and status of seizure-prone individuals or populations. Oral zinc supplements have been used as a therapy for Wilson’s disease to lower the copper levels in those patients. Despite two-fold increases in serum zinc, no seizure disorders have been reported (Umeki et al., 1986; Brewer et al., 1987a,b). The reported effects of zinc manipulation on seizure activity in animals also present a complicated picture. Intracerebral zinc administration, in doses from 20 (Itoh and Ebadi, 1982; Ebadi and Pfeiffer, 1984) to 500 pg (Pei et al., 1983; Pei and Koyama, 1986), causes immediate seizures and motor convulsions, and lower intracerebral doses ( I 30-800 ng) can shorten the latencies for noise-induced seizures (Chung and Johnson, 1983b) and lower thresholds for electrically induced seizures in mice (Tokuoka et al., 1967). Systemic manipulation of zinc, however, produces mixed effects. For example, it has been reported that dietary zinc loading raises the threshold for kindling in cats (Sterman et al., 1986, 1988) and that single injections of zinc salt reduce the convulsive effects of kainic acid (Porsche, 1983) and the

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CHRISTOPHER J. FREDERICKSON

number of noise-induced seizures in mice (Howell et al., 1986). Others, however, have found that latencies for kindled seizures (Wallwork and Crawford, 1987) and thresholds for electroconvulsive seizures (Tokuoka et al., 1967) are somewhat increased by dietary zinc deficiency and that a single systemic injection of zinc salt can facilitate seizure induction by electical stimulation (Tokuoka et al., 1967). Systemic administration of chelators has also produced mixed results. EDTA and penicillamide reduced the occurence of seizures in mice (Chung and Johnson, 1984) and baboons (Alley et al., 1981), respectively. In contrast, DDC and dithizone induced hippocampal epileptiform activity (Haycock et al., 1977; Drust and Crawford, 1984), and phenanthroline congeners lowered seizure thresholds (Connor, 1983) in rats. Like the effect of zinc on seizures, the effect of seizures on brain zinc is complex. First, as has been discussed earlier, sustained paroxysmal activity causes a loss of histochemically reactive zinc from CNS axonal boutons arid a release of zinc into superfusing media in vitro and in vivo. This release of the bouton zinc might be expected to cause a slight decline in total brain zinc after seizures, but in fact the available evidence indicates that the total amount of zinc (in the hippocampus) is actually increased at 24 hr (Mody and Miller, 1985) and at 2 weeks (Slevin and Kasarskis, 1985) after seizures. A slight increase in the uptake of “Zn into the brain from the serum has also been reported to occur during pentretazol-induced seizures (Papavasiliou and Miller, 1983, Fig. 2), but a net loss of total zinc reportedly occurs after 2 hr of kainic acid-induced seizures (Assaf and Chung, 1984). A plausible synthesis of these findings is that zinc is realeased from zinc-containing boutons during seizures but also accumulates in other tissue compartments during and/or after the seizure. Kecent evidence indicates that some of the zinc that is released from presynaptic boutons during sustained paroxysmal activity is actually translocated into or onto the postsynaptic neurons, and a possible role o f the translocated zinc in the subsequent seizure-induced death of the postsynaptic neurons has been suggested (Frederickson el al., 1989a). The diverse effects of zinc on neurotransmission have been discussed in a prior section of this review, and it is clear from those data that zinc ions can produce both proconvulsive (excitatory-disinhibitory) and anticonvulsive (inhibitory-defacilatatory) changes at the synaptic level. Which of‘ those would predominate, that is, whether zinc would normally act to promote or to antagonize nascent seizures in an otherwise normal brain, would be essentially impossible to predict from the available data. Moreover, the findings in the various human and animal models of epilepsy reviewed above suggest that zinc might act

NEUROBIOLOGY OF ZINC

2 19

either as an anticonvulsant or a proconvulsant, depending on dose, route of administration, type of seizure, species, and perhaps other paradigmatic factors as well. Indeed, the picture that emerges suggests that zinc’s function could be as subtle and multifaceted as, for example, the functions of calcium in the brain. Both ions probably contribute in multiple and sometimes antagonistic ways to the modulation of overall neural excitability. 2. Zinc and Anorexia When animals are offered only a zinc-deficient diet, they decrease their total food intake. This general phenomenon has been observed in guinea pigs (Gordon and O’Dell, 1983), various domesticated fowl and mammals (see references in Henkin, 1979), and in laboratory rats. In rats, the so-called anorexia has the interesting property of recurring cyclically when only a zinc-deficient diet is available. Bouts of nearly normal food intake alternate with low intake in a 3-5-day cycle (Chesters and Quaterman, 1970; Wallwork et al., 1981). Serum zinc falls by about 60-70% during the first few days of the dietary deficiency. It then appears to fluctuate cyclically in synchrony with food intake, being lowest at the peak of feeding (i.e., just prior to a fasting phase) and somewhat higher at the end of fasting (Chesters and Quaterman, 1970; Wallwork et al., 1981; Giugliano and Millward, 1984; Kasarskis et al., 1986). Presumably, serum zinc levels rise during fasting as a consequence of the liberation of zinc (in very modest amounts) (see Giugliano and Millward, 1984) from body stores of protein that are catabolized during the fasting phase. Rising serum zinc, in turn, could trigger a feeding cycle. T h e observation that brain norepinephrine is elevated during dietary zinc insufficiency (Wallwork et al., 1982; Kasarskis et al., 1986; Reeves and O’Dell, 1984) has encouraged the idea that altered CNS metabolism of catecholamines might mediate the effect of zinc deficiency upon food intake. In support of that hypothesis, Reeves and O’Dell(l984)showed that lowering the tyrosine content of the diet (and thus, the hypothalamic catecholamine level) attenuated the decreased feeding of rats on a zinc-deficient diet. Kasarskis et al. (1985b), have adduced evidence that elevated hypothalamic catecholamines may be a result, rather than a cause, of the reduced food intake; the latter authors suggest that zinc deficiency may alter norepinephrine binding, storage, or release. Possible roles of reduced sensitivity of GABA, dopamine, and norepinephrine receptors (Essatara et al., 1984a) and of altered opioid metabolism and receptor binding (Essatara et al., 1984b) in the reduced feeding of zinc deficiency have also been suggested. Another interpretation of the reduced food intake seen during

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CHRISTOPHER J. FREDERICKSON

dietary zinc insufficiency is that the reduced eating is not a general anorexia at all but is merely an example of learned taste aversion. Learned taste aversion develops whenever an animal becomes ill in association with ingestion of a particular food; an aversion to that food (paleophobza) develops, and preference for any food with a different taste and smell (neophilia) emerges (Rozin and Kalat, 1971). As has been mentioned above, dietary zinc deficiency causes many somatic ailments and could therefore condition a taste aversion. The recent demonstration (Cannon et al., 1987, 1988) that rats exhibiting reduced intake of a zinc-deficient diet will immediately eat voraciously when offered food that tastes and smells different from the deficient food lends credence to this view. Further support for the taste aversion explanation is found in reports indicating that animals will preferentially consume zincsufficient diets over previously ingested deficient diets in two-choice paradigms (Vohra and Heil, 1969; Hughes and Dewar, 1971; Chafetz et aZ., 1984). Several authors have suggested a possible involvement of zinc in human anorexia (Bakan, 1979; Casper el d., 1980; Bryce-Smith and Simpson, 1984, 1985), but others have found no consistent zinc deficiency in anorexic patients (Ainley et al., 1986).

X. Summary and Conclusions

A. ZINC METALLOENZYMES IN THE BRAIN Zinc metalloenzymes are essential to the normal biology of brain cells, and any experimental treatment that is extreme enough to affect the metalloenzymes, such as prolonged zinc undernutrition or administration of chelators, can disrupt brain function via metalloenzymatic effects. Moreover, most of the zinc in the brain is probably bound into the metalloenzymes, and quantitative studies of elemental zinc in the brain will therefore tend to reflect the distribution and turnover of those metalloenzymes. However, except during early brain development, when the demand for zinc is especially high, it is not likely that short-term variations in the bioavailability of dietary zinc have appreciably impact upon the level or activity of zinc metalloenzymes. Thus, though they are of paramount importance in the basal metabolism and biology of the brain, the zinc metalloenzymes are not likely to mediate the rapid effects that zinc can have on brain function.

NEUROBIOLOGY OF ZINC

22 1

B. ZINC-CONTAINING NEURONS 1. Current Status

Zinc-containing neurons may be added to the growing list of chemospecific neuron types in the CNS. So far as current understanding goes, the zinc-containing neuron has roughly the same status as, for example, a typical peptidergic neuron. Thus there is good evidence that the zinc-containing neurons take up, store, and release zinc at their boutons, a preliminary picture of the anatomy of the zinc-containing systems is in hand, and a large number of zinc-sensitive “target” o r “receptor” sites have been tentatively identified in the brain. T h e fact that the zinc-containing neurons are situated predominantly within limbic and cerebrocortical circuitry raises the possibility of a role in cognitive and mnemonic processes. The association of vesicular zinc with excitatory amino acid transmitters, and with the NMDA receptor in particular, is consistent with this idea. Investigations of the effects of zinc manipulation upon long-term potentiation o r even on synaptic reorganization would appear to be fruitful avenues to explore. The fact that zinc is implicated in the pathophysiology of seizures is not wholly incompatible with a role of zinc in mnemonic processes; enduring changes in neural and synaptic excitability are doubtless fundamental aspects of both phenomena.

2. Major Unresolved Issues 1. What (if anything) is sequestered along with zinc in the vesicles? Does zinc merely stabilize storage of a primary vesicular release product o r is zinc per se the important intravesicular substance? Information on the “life cycle” of vesicular zinc could be informative on this point. If the ion is incorporated into vesicles at the Golgi apparatus, or during axonal transport, a peptide-protein-stabilizing function would be suggested. In the latter case, it could turn out that the real importance of zinc in vesicles is that it has provided a cytochemical footprint leading to the eventual discovery of another neuroactive molecule, which happens to be stabilized in vesicles by zinc. Many of the same approaches that have been used to elucidate the role of zinc in insulin storage granules (Gold and Grodsky, 1984; Nakamura et al., 1984; Hoftiezer et al., 1985) could be applied to the problem of the zinc-containing vesicles. 2. What is the complete anatomical organization of the zinccontaining system in the CNS? The current data suggest that the zinc-containing fiber systems are a

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subset of the glutamatergic pathways, but this association has not been extensively tested. There are many CNS regions innervated by zinccontaining fibers for which the cells of origin are unknown; mapping these zinc-containing fiber systems will allow rigorous evaluation of the zinc-glutamate hypothesis. Quantitative histochemical mapping of the density of zinc-containing boutons throughout the CNS would also facilitate correlational comparisons of zinc with glutamate and other synaptic substances. 3. Is zinc in all vesicles of a given terminal, or only in the small fraction that stain for zinc? T h e zinc-containing vesicles could be a distinct class, the release of which is governed by different physiological factors than control the release of transmitters and modulators from the same terminal. Such a possibility would expand the scope of zinc’s potential roles in synaptic regulation or modulation. For inquiries into many of these latter questions, a ganglionic or neuromuscular preparation would be a methodological advantage; the search for such a “model” zinccontaining synapse should be a high priority.

C. THEZINC SIGNAL 1 . Current Status One of the more exciting hypotheses about CNS zinc is that the zinc ions might function analogously to calcium ions, with transient ionic fluxes initiating cascades of physiological changes by allosteric modification of specific enzymes, receptors, o r structural proteins (Fig. 12). Because the zinc ions in vesicles appear to be rather weakly bound (histochemically reactive), exocytosis of those vesicles could be the primary source of the putative ionic zinc signals. The concentration of M or higher during release into the local zinc could reach 2-3 X microenvironment, a level sufficient to induce many of the neurobiological effects that have been discussed herein. Released ions that were taken u p into target cells, moreover, could modulate a wide range of physiological and biological processes within those cells.

2. Major Unresolved Issues 1. Are there zinc signals? Is zinc released from boutons as free or weakly-bound Zn2+,or is it released as a tightly bound constituent of a larger molecule? Presumably, the kinds of release studies that have been done in the past (see, e.g., Howell et al., 1984; Aniksztejn et al., 1987) could be

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FIG. 12. Summary of life cycle and possible functions of zinc (*) in axonal boutons: (1) high-affinity uptake of zinc ion and/or zinc-microligand (ML) complex into perikarya;

(2) zinc firmly bound into metalloenzymes; (3) possible transport of zinc in vesicles from Golgi apparatus; (4) zinc-containing vesicles containing either zinc, zinc-amino acid (A) complexes, or zinc-protein (P) complexes; ( 5 ) calcium- and impulse-dependent release of zinc, probably by exocytosis, and perhaps with corelease of amino acids, peptides, or proteins; (6)uptake into boutons, perhaps by carrier and/or receptor endocytosis; (7) zinc modulation of receptors, channels, and pumps; (8)possible transport of released ions into target cells; (9) zinc modulation of enzymes and calmodulin-activated systems in target cells; and (10) possible modulation of nucleic acid synthesis and/or transcription by incorporated zinc ions.

extended to determine the binding state of the zinc that is released from boutons. Dialysis of CNS superfusates collected during zinc release would be one approach to the problem. Alternatively, if a suitable congener of the quinoline fluorescent zinc probes (Frederickson et al., 1987a) were available, the real-time fluorometry techniques that have been used to track transient currents of ionic calcium (Taylor et al., 1986) could be used for zinc. 2. What are the physiological concentrations of ionic zinc in the brain? Resting levels of ionic zinc in the brain may be as low as 200 pM,

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whereas transient fluxes at the synapse may be six orders of magnitude higher. Given this uncertainty about the concentrations of the ion, it is presently impossible to say which of zinc’s many biochemical effects are physiologically relevant and which are not. Data on the resting and transient levels of ionic zinc in brain tissue are essential to obtain.

Acknowledgments

We thank D. W. Choi, G. Danscher, M. Ebadi, M. L. Mayer, and J. Perez-Clause11 for contributing their figures and micrographs to this review, and we thank I. L. Crawford, M. Ebadi, G. Danscher, L. A. Hershey, G. A. Howell, E. J. Kasarskis, and E. M. Wilson for suggesting material for inclusion. Joan Kruszynski’s excellent technical assistance is also gratefully acknowledged. Supported in part by MH4 1691, MH42798, and the Deafness Foundation.

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DOPAMINE RECEPTOR SUBTYPES AND AROUSAL By Ennio Ongini' and Vincenzo G. Longo Deportment of Pharmacology lstituto Superiore di Sanita

1-00161Rome, Italy

I. Introduction 11. Arousal: A Definition

111. IV. V. VI. VII.

Central Dopamine Receptors D-2 Receptors D-1 Receptors Interactions of D-1 and D-2 Receptors Conclusion References

I. Introduction

There is much evidence available on the role of the catecholamines dopamine (DA) and norepinephrine (NE) in the modulation of sleepwaking-arousal patterns. A variety of findings suggests that NE is involved in modulating the state of arousal and REM sleep (Jouvet, 1972; Hartmann, 1978; Gaillard, 1985; Jacobs, 1985). Other data indicate that DA is also concerned with activation of behavior and, in general, with the arousal state (Monti, 1982; Robbins and Everitt, 1982). Although the possible differential role of DA and NE is still a matter a debate and also of contradictory opinions, there is sufficient information to suggest that the two neurotransmitters subserve different physiological and behavioral functions. Several comprehensive reviews are specifically devoted to the subject and certainly provide an interesting forum for understanding the function of such neurotransmitters within the brain (Jouvet, 1972; Monti, 1982; Koella, 1984; Gaillard, 1985; Jacobs, 1985). As much as of this knowledge is very recent, in this review we focus mainly on the possible function of DA receptor subtypes, namely D-1 and D-2, in the modulation of the state of arousal.

* Current address: Research Laboratories, Schering-Plough SPA 1-20060 Comazzo (Milan), Italy IN1'ERNAIJONAL REVIEW O F NEUROBIOLOGY, VOL. 31

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Copyright 8 1989 by Academic Preaa, Inc. All rights of reproduction in any form reserved.

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Since the study of events induced by centrally acting dopaminergic (DAergic) agents is a classic means of investigating the pharmacology and function of DA within the CNS, this review is mainly devoted to the activity of selective agonists for D-1 and D-2 receptors. The series of experiments from our own laboratory that we report here demonstrates that DA receptor systems, especially the D-1 subtype, are primarily involved in the generation of arousal in laboratory animals.

II. Arousal: A Definition

Since the purpose of this chapter is to trace evidence now available that implicates DA receptors in the modulation of the arousal-state behavior, the concept of arousal has to be precisely defined. Behavioral responses are often evaluated in vague and broad terms, such as “alert,” “excited,” “depressed,” “sedated.” Therefore, in some instances drugs that are very different in their behavioral and neurological effects are described in a similar manner. There is a multiplicity of ways in which the concept of arousal is used, depending on how it is measured. A classic index is the rate and quality of behavioral output. Increases in spontaneous motor activity, enhanced exploratory activity, and increased response to stimuli are usually recognized as necessary components of arousal. These types of behavior are often correlated with electrical activity of the cortex characterized by low-amplitude, fast-frequency waves (EEG desynchronization). The bioelectrical measure of arousal appears to provide a highly sensitive and more reliable correlate of arousal state than do behavioral parameters. In fact, an animal might be immobile but highly aroused, as in the case of freezing response to signals that predict punishments. This is particularly true for studies involving drug administration, since there are compounds that produce dissociation between behavior and EEG activity (Longo, 1966; Bo et ul., 1979). Thus, drugs may induce EEG arousal without a corresponding change in behavioral response. In view of this, a thorough evaluation of the arousal state should rely on concomitant analysis of EEG and behavior. This is the case in studies purported to assess the sleep-waking cycle, in which each particular stage is defined by a characteristic set of EEG and behavioral measures (Parmeggiani et ul., 1985).This may lead to other definitions, such as attentive or active waking, which also closely reflect a behavioral state of arousal.

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This review refers to the concept of arousal as that described by the occurrence of desynchronized EEG activity associated with behavioral signs of attentive waking. Information regarding motor components of behavior will be mentioned specifically as behavioral activation.

111. Central Dopamine Receptors

DA was long considered only a precursor of NE until, in the early 1960s, important discoveries regarding its presence and function in the brain were made, all pointing to a role of DA in the control of psychomotor activity (Carlsson, 1987). This function of DA has been evidenced through research work on Parkinson’s disease, drug-induced stereotypies and striatal syndromes, and experimental catalepsy. A variety of data also indicates that DA systems in the brain have a role in mediating states of arousal (Joyce, 1983; Koella, 1984; Monti, 1982). Major findings have emerged from research effort involving two main strategies: ( 1) the detailed comparison of various syndromes produced by DA depletion and (2) the effect produced by drugs interacting selectively with DA-mediated neurotransmission. Generally, lesioning of discrete DA pathways by 6-hydroxydopamine (6-OH-DA) is followed by reduction of motor activity (Longo, 1973). There are, however, findings that are considerably less homogeneous and that contradict the notion of an essential role of DA in the modulation of spontaneous motor activity (Robbins and Everitt, 1982). With regard to EEG activity, similar neurotoxic lesions, or less specific electrolytic lesions, produce neither consistent changes of sleep-waking patterns nor reductions of EEG arousal (Longo, 1973; Monti, 1982). This would suggest that DA is more concerned with the output of different types of motor behavior rather than with the true process underlying EEG arousal (Monti, 1982; Koella, 1984). It should be noted, however, that the failure to disrupt EEG patterns by localized lesions of any structure does not conclusively rule out participation of that system in the normal generation of physiological events (Parmeggiani et al., 1985). In addition, the consequences of DA depletion are at variance with findings from pharmacological manipulations of DA neurotransmission. Studies based on the stimulation of DA-containing pathways by L-DOPA, a precursor of DA, have shown that drug administration produces increasing degrees of organized motor activity, irritability, and

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aggressiveness (see Bianchine, 1985). AH these effects have been attributed to a rise in DA levels within the brain, as shown in Fig. 1, taken from a study of Everett and Wiegand (1962) carried out in mice. It is noteworthy that DA levels measured in concomitance with behavioral changes were markedly increased (up to 12 times the normal levels), whereas increases as much as twofold were not accompanied by behavioral effects. These symptoms are comparable to those observed when DA (5-20 pg) was administered into cerebral ventricles in mice (Pellegrini Quarantotti et al., 1975). Also in other animal species (rat, dog, rabbit, monkey) a gradual development of increased motor activity and irritability has been described. In particular, the rabbit seems to be the species most sensitive to L-DOPA,which gives rise to excitation at doses of 10-20 mg/kg iv (Fig. 2; Gaillard et al., 1969; Florio and Longo, 1971; Ongini et al., 1987a). But the most dramatic effects are observed in animals pretreated with monoamine depletors. For instance, mice or monkeys that are sedated because of treatment with reserpine or M A 0 inhibitors are rapidly awakened by L-DOPA administration (Carlsson et al., 1957; Everett, 1961). In addition to behavioral effects, L-DOPA induces EEG desynchronization and increased waking in a variety of animals including humans (Bianchine, 1985). In rabbits, L-DOPA induces EEG changes whose duration increases when coadministration of a peripheral decarboxylase inhibitor allows the DA precursor to last

I f , 2 5 5 0 7 5 K30 DOSE IN mg/kg 1.P

J

FIG. 1 . Dopamine levels in whole mouse brain. 0 , Levels in mice given 100 rng/kg of monoamine oxidase inhibitor MO-911 (pargyline) and 4 hr later given various doses of L-DOPA. The mice were sacrificed 30 min later. 0, Levels of dopamine in mouse brain after varying doses of MO-911 4 hr prior to 100 rng/kg L-DOPA; the mice were sacrificed 30 min later. The areas between the dotted lines give the predicted dopamine ranges in mice with the described behavioral states. (From Everett and Wiegand, 1961.)

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C

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carbidopa FIG.2. EEG desynchronization induced by L-DOPA either alone or after pretreatment with carbidopa (1 or 2 mg/kg) in rabbits. A period of 15 min separated the two injections. Each treatment was given intravenously to at least five animals. Each bar represents the time spent in EEG desynchronization over a period of 60 min; *, p < 0.05; **, p < 0.01 compared to the control group (Dunnett’s t test).

longer within the brain (Fig. 2) (Gaillard et al., 1969). A variety of data has also been obtained by studying compounds that directly interact with DA receptors. Of these, apomorphine has been the most closely scrutinized (Joyce, 1983). Given at doses that interact with postsynaptic DA receptors, the compound has stimulatory actions, as shown by increased motor activity and a variety of stereotyped responses such as compulsive gnawing in rats and rabbits. Associated with behavioral stimulation, EEG desynchronization occurs in the cortex (Gessa et al., 1985; Ongini and Caporali, 1987). Altogether, several findings suggest that pharmacological stimulation of DA transmission leads to behavioral activation and a state of EEG arousal. This evidence is further supported by the many results available from studies with neuroleptics. I n fact, these compounds, by blocking DAergic neurotransmission, tend to produce opposite effects, that is, they promote occurrence of sleep and reduce arousal response to sensory stimuli (Hartmann, 1978; Longo, 1978). The critical involvement of DA is demonstrated by the ability of neuroleptics to antagonize EEG and behavioral arousal induced by L-DOPA or apomorphine (Florio and Longo, 1971; Ongini and Caporali, 1987). Although it is difficult to reconcile results from lesions with those obtained by pharmacological means, there appears to be a convincing argument favoring a possible role of DA in mediating cortical arousal and behavioral activation. Additional information on the possible impli-

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cation of DA in sleep-arousal processes derives from the area of sleep-disorders medicine. For example, evidence accumulated in the pathogenesis of narcolepsy, as evaluated in a model of genetically narcoleptic dogs, has shown that DA deficit is a critical feature underlying this pathology (Scott Bowersox et al., 1987).

IV. D-2 Receptors

In light of the discovery that DA receptors can be classified into two subtypes, D-1 and D-2, having different properties and often opposing roles (Stoof and Kebabian, 1984; Seeman and Grigoriadis, 1987), the involvement of DA receptors in various physiological responses had to be critically reassessed. The two receptors elicit different biochemical events. D- 1 stimulates adenylate cyclase, thus increasing the formation of the second messenger cyclic AMP; whereas D-2 inhibits adenylate cyclase in most tissues (Spano et al., 1978; Stoof and Kebabian, 1984). Thus, evidence exists to support the hypothesis that each receptor subtype subserves a different function in the CNS (Stoof and Kebabian, 1984). This is also suggested by the fact that the two receptor populations are differently distributed in the brain (Dawson et al., 1986). Historically, only D-2 receptors have received major consideration (Joyce, 1983) for a number of reasons. First, the most widely used neuroleptics either block D-2 receptors preferentially or interact equally with both receptors (Baldessarini, 1985; Seeman, 1987). Second, clinical doses of neuroleptics correlate closely with their ability to block D-2 receptors (Seeman, 1987). Third, DA agonists used therapeutically, such as bromocriptine, have a prominent affinity for D-2 (Joyce, 1983; Goldstein et al., 1984). Fourth, compounds selective for D-1 either display weak CNS activity (i.e., SKF 38393) (Setler et al., 1978) or were discovered later, i.e., SCH 23390 (Iorio et al., 1983) and SCH 39166 (Chipkin et al., 1988). Agonists for D-2 receptors all produce marked behavioral stimulation such as stereotyped movements and hyperactivity (Joyce, 1983; Meller et al., 1985). Since only D-2 receptors were considered to be meaningful, it should be noted that the effects of apomorphine, a drug that has equal affinity for both receptors (Billard et al., 1984), were often interpreted as the outcome of D-2 stimulation (Joyce, 1983). When tested for their ability to affect EEG activity or sleep-waking patterns, most agonists were able to produce EEG desynchronization, arousal, and states of wakefulness under a variety of experimental conditions

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(Vigouret et al., 1978). For example, bromocriptine, pergolide, or quinpirole (LY 171555), the last of which is the most selective D-2 agonist now available (Seeman and Grigoriadis, 1987) induced EEG arousal and behavioral activation in rabbits (Longo and Loizzo, 1978; Bo et al., 1979; Ongini and Caporali, 1987). At this stage, on the basis of existing data it is not possible to dissociate cortical arousal from the various stimulatory effects that are elicited by the activation of D-2 receptors. A variety of drugs can produce arousal, but a key question is how to separate drugs that lead to a state of arousal secondary to other main effects such as generation of pain, anxiety, o r behavioral discomfort (Dorow and Duka, 1986), from compounds that directly influence mechanisms underlying arousal processes. In this regard, evidence does not substantiate the conclusion that arousal depends directly on D-2 receptor stimulation. Indeed, it may occur as a consequence of the state of excitation that accompanies motor and stereotyped responses. Moreover, experiments based on interactions between quinpirole and selective antagonists provided evidence that behavioral activation, rather than EEG arousal, is sensitive to selective blockade of D-2 receptors (Ongini and Caporali, 1987). Therefore, these findings support a link between the D-2 receptor population and motor behavior, but a direct relationship with states of arousal remains to be explored further. Also, the body of evidence available on sedative effects of neuroleptics does not provide a definite clue that D-2 receptors are involved in modulating the sedation-arousal continuum. It is true that neuroleptics used therapeutically, which have a relatively higher affinity for D-2 or equal affinity for both receptors (Seeman, 1987), tend to produce sedation, as shown by increased sleep time and reduced responses to arousing stimuli (Hartmann, 1978; Baldessarini, 1985). For example, haloperidol and chlorpromazine have sedative actions in man and product EEG synchronization in laboratory animals (Florio and Longo, 1971; Longo, 1978). However, substituted benzamides, such as sulpiride or raclopride, which are the most selective D-2 antagonists now available (Seeman and Grigoriadis, 1987), appear to be relatively free of sedation liability as shown by the lack of meaningful changes in EEG and related behaviors in laboratory animals (Ogren et al., 1986; Ongini et al., 1985; Bo et al., 1988). These data suggest that D-2 receptor blockade may not be critical for decreasing arousal and producing sedation. In support of this interpretation, there are results of binding studies which suggest that most classical neuroleptics such as haloperidol, chlorpromazine, and fluphenazine, which possess sedative properties, also have relatively high

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affinity for other receptors (Peroutka and Snyder, 1980; Seeman and Grigoriadis, 1987). It is likely that interactions with other receptor systems, such as a-adrenergic o r serotonin (5HT) receptors, which also contribute to regulating sleep states (Jouvet, 1972; Monti, 1982), might well play a role in inducing their overall sedative effects (Peroutka and Snyder, 1980). Moreover, although hypomotility, sedation, and EEG synchronization occur when presynaptic receptors, which are of the D-2 type, are stimulated by low doses of apomorphine (Mereu et al., 1979), this does not provide evidence for a possible correlation between D-2 and the sedation-arousal process. In fact, following stimulation of receptors localized presynaptically, there is a reduced synthesis and release of DA, which in turn leads to a decreased DA tone at postsynaptic receptor sites. This means that both D-1 and D-2 receptors are equally influenced by the reduced presence of DA in the synapse. I n general, it appears the evidence in support of a role for D-2 receptors in the modulation of arousal is not persuasive, although these receptors clearly mediate various features of motor activation.

V. D-1 Receptors

The discovery of the first selective antagonist for D-1 receptors, SCH 23390, has rekindled interest in the function of this receptor population (Iorio et al., 1983; Barnett, 1986; Kebabian et al., 1986). Although a relatively selective agonist, SKF 38393, has been available for many years (Setler et al., 1978) and has been used for biochemical studies (Stoof and Kebabian, 1984), little attention has been devoted to its pharmacological profile. This is attributable to the weak CNS effects of SKF 38393 in intact animals and the lack of characteristic actions that may distinguish it from other more potent DA agonists, such as apomorphine. Over the last few years, because of the discovery of SCH 23390, closer investigation of the profile of SKF 38393 made it possible to discover responses produced by D-1 receptor stimulation. Molloy and Waddington (1984) first demonstrated that the compound specifically promotes some behavioral effects, such as episodes of grooming in the rat. A further series of experiments has demonstrated that the D-1 agonist induces a clear EEG desynchronization and behavioral arousal in rats and rabbits (Figs. 3 and 4) (Ongini et al., 1985, Ongini etal., 1987b). This effect on the EEG appears to be a major response occurring after selective stimulation of D-1 receptors. Moreover, the effect is prevented by minute doses of

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A PF-F P

~

r F-P

HIPP

FIG.3. Effects of the D-1 receptor against SKF 38393 on the EEG activity in the rabbit. Control recording was taken during a period of EEG synchronization with spindles interrupted by acoustic stimulation (arrow). The sample recording for SKF 38393 was taken 15 min after injection. At 2.5 mg/kg, cortical desynchronization and hippocampal theta rhythms characterized the EEG activity for at least 30 rnin. F-F, Frontal cortex; P-P, parietal cortex; rF-P, right frontoparietal cortex; HIPP, left dorsal hippocampus; calibration: 100 pV; 2 sec. (From Ongini et al., 1985.)

SCH 23390 (Fig. 5), thus showing that arousal induced by SKF 38393 is very sensitive to blockade of D-1 receptors. From the data, it would appear possible that D-1 receptors are implicated in modulating EEG and behavioral arousal. Based on this hypothesis it may be expected that selective blockade of D-1 would lead to opposite effects, that is, sedation and EEG synchronization. Instead, SCH 23390 has been found to produce little sedation, in rats and rabbits, as measured by evaluating EEG activity (Gessa et al., 1985; Ongini et al., 1985) and the response to phasic sensory stimuli (Bo et al., 1988). Moreover, either SCH 23390 or the more recently discovered D-1 antagonist SCH 39166 produce weak increases of slow-wave sleep in the rat (Trampus et al., 1989). However, in the cebus monkey a certain degree of sedation is observed with the long-term administration of either D-1 antagonist (Coffin et al., 1989). The discrepancy between the results may well depend upon different species sensitivity. Additionally, in the monkey study sedation was used as a broad measure of drug-

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FIG. 4. Dose-response relationship for EEG desynchronization induced by the D- 1 receptor agonist SKF 38393. The EEG was recorded for 60 min immediately after intravenous injection in rabbits (A) and 15 min after subcutaneous injection in rats (0). Each bar represents the mean percentage 2 SEM of the time spent in EEG desynchronization during the 60-min test; *, p < 0.05; **, p < 0.01 compared to control (Dunnett's t test). (After Ongini et al., 1985.)

induced behavioral depression, including inhibition of spontaneous motor behavior. This was done with the purpose of evaluating whether the drug was active at the doses used. Similar behavioral patterns, which closely reflect inhibition of motor activity, are also observed in rats when administered either D-1 antagonist (Gessa et al., 1985; Chipkin et al., 1988).Thus, effects found in the monkey d o not appear to conflict with results showing that blockade of D-1 receptors produces little or nu reduction of arousal. One possible interpretation of these findings is that blockade of D-1 weakly depresses per se arousal processes in normally behaving animals. Rather, D- 1 blockade prevents elevated arousal levels, such as those induced by the D-1 agonist SKF 38393, apomorphine, or L-DOPA. According to this notion, it would seem reasonable to expect that tonic arousal produced by physiological conditions is also blocked by SCH 23390. Experimental data confirm this assumption, thus providing a way of' reconciling apparently contradictory results. In fact, Fratta et al. (1987) have shown that the D-1 receptor blocker selectively prevents arousal induced by depriving rats of REM sleep. Thus, the D-1 receptor system appears to be fully operative under states of arousal generated by either drugs or tonic physiological stimuli.

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100,

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FIG. 5. Interaction between SCH 23390 and EEG activation induced by different dopaminergic agonists in the rabbit. The D-1 agonist SKF 38393 was given at 10 mg/kg, the D-2 agonist quinpirole (LY 171555) was injected at 0.5 mg/kg, and the nonselective agonist apomorphine (APO) was given at 1 mg/kg. Each value indicates the percentage of time desynchronized EEG activity was present over 30 rnin, starting immediately after injection of the agonist. SCH 23390 was administered 15 min before each agonist. All drugs were given intravenously. R, LY; 0, APO; 0 , SKF; *, p < 0.05; **, p < 0.01 compared to the respective agonist alone (Dunnett's t test).

VI. Interactions of D-1 and D-2 Receptors

The preceding account of the effects of selective drugs for either D-1 or D-2 receptors is complicated by findings of functional interactions between the two receptor sites. T h e first evidence comes from data showing that most effects induced by nonselective DAergic agents such as apomorphine or amphetamine are antagonized by SCH 23390, which selectively interacts with D-1 receptors only (Iorio et al., 1983; Waddington, 1986). Also, EEG effects produced by either apomorphine or L-DOPA are prevented by D-1 receptor blockade (Ongini and Caporali, 1987; Ongini et al., 1987a). Even the selective D-2 agonist quinpirole was found to modify behavioral effects induced by SCH 23390 (Meller et al., 1985). Moreover, a variety of data support the contention that D-1 receptors contribute to the expression of behavior initiated by D-2 (Braun et al., 1986; Walters et al., 1987). For example, the D-1 agonist SKF 38393, which by itself has little behavioral effect, produces a variety of marked effects that are more than additive when it is coadministered with D-2 agonists such as quinpirole o r bromocriptine. This includes DA

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responses such as increased locomotion, contralateral rotation, oral dyskinesia, chewing, increased locomotion, o r elevated firing rate of DA neurons (Barone et al., 1986; Braun et al., 1986; Robertson and Robertson, 1986; Arnt et al., 1987; Walters et al., 1987). Thus, the data indicate that concurrent stimulation of both receptors is necessary for the full expression of DA behavior that is typically observed following administration of nonselective agonists. Although a uniform interpretation of these results demands further experiments, it is clear that D-1 receptor tone within the CNS influences D-2-mediated responses and that the two systems might function in an interactive manner. In view of the results of experiments conducted under a variety of conditions, it is possible that a synergism may also exist for cortical arousal and EEG desynchronization induced by D-1 and D-2 agonists. According to Braun et al. (1986), synergism for the arousal state could exist because the marked responses elicited by concomitant stimulation of both D- 1 and D-2 may depend on a transition of the behavioral state from efficiency to the disorganization that typically characterizes high levels of arousal. Further studies now in progress might provide insights into this issue. Interestingly, a clear functionai interaction between the two receptors involving the sedation-arousal continuum has been found by using selective antagonists. Bo et al. (1988) have demonstrated that SCH 23390 and the D-2 receptor blocker raclopride, which administered alone do not induce consistent EEG changes, do produce marked synchronized activity and sedation when coadministered at low doses (Fig. 6). T h e approach used indirectly supports the hypothesis that a functional interaction also exists between the two receptors with regard to arousal processes. In this model, selective receptor blockade by SCH 23390, with the consequent decrease of D-1 receptor tone, either potentiates or permits a stronger expression of the EEG effects of D-2 antagonists. T h e ultimate result of such concurrent blockade of both receptor subtypes is a marked reduction of arousal. These findings, together with other results (Braun et al., 1986; Meller et al., 1985; Waddington, 1986), lend further support to the notion that endogenous D-1 receptor tone is critical for the expression of certain DAergic responses, including arousal. Taken together, the data support the concept that D-1 receptors have some role in modulating the sedation-arousal continuum. In addition, the fact that a functional interaction between the two receptors is necessary in order to have DA blockers inducing EEG synchronization and sedation provides a possible explanation as to why currently used

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FIG. 6. Duration of EEG synchronization, a correlate of sedation, in rabbits after administration of saline (C), haloperidol (HAL), SCH 23390 (SCH),raclopride (RACLO), or the combination of SCH 23390 and raclopride. Each treatment was given intravenously in a minimum of five animals. SCH 23390 was injected 15 min before administration of raclopride. Values indicate the duration in min of cortical EEG synchronization (highamplitude, low-frequency waves); *, p < 0.05; **, p < 0.01 compared to control (Dunnett's t test).

neuroleptics have, to different degrees, a sedation liability. This is in addition to the possible role played by interactions with other receptor systems (Peroutka and Snyder, 1980). It may well be that the relatively low selectivity or equal affinity for the two types of DA receptors of most sedating neuroleptics is the basis for a likely concomitant blockade of D-1 and D-2, which consequently leads to EEG synchronization and sedation. In view of these considerations, it also seems reasonable to interpret the low sedation potential of sulpiride, raclopride, or SCH 23390 (Bo et al., 1988) as dependent on their ability to block each receptor site selectively without interacting with both receptors concomitantly. VII. Conclusion

This review has focused on results concerning the possible function of D-1 or D-2 receptor populations in the modulation of the state of arousal. From the data, it appears that both D- 1 and D-2 receptors, when stimulated, can influence to different extents this physiological event. However, of the two receptors, D-1 seems more concerned with the

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generation of cortical and behavioral arousal. Overall evidence is provided by several findings. First, a state of arousal is a major response observed after D-1 receptor stimulation by the agonist SKF 38393 (Ongini et al., 1985), whereas D-2 agonists also induce marked motor activation and stereotyped movements (Joyce, 1983). Second, EEG and behavioral arousal induced by nonselective drugs such as apomorphine or L-DOPA is also very sensitive to D-1 blockade, whereas it is hardly influenced by D-2 blocking agents (Ongini and Caporali, 1987; Ongini et al., 1987a). Third, arousal induced by tonic physiological stimuli, such as REM sleep deprivation, is also very sensitive to blockade of D-1 but not to D-2 blockade (Fratta et al., 1987). Fourth, arousal is strongly reduced and shifted to marked sedation when D-1 receptors are blocked concomitantly with D-2 receptors (Bo et al., 1988). However, because most of these studies have been carried out in rabbits, which are very sensitive to DAergic drugs (Longo, 1978), there is a need to extend these observations with parallel studies in rats to assess whether modulation of arousal also occurs in this species at doses similar to those effective in other behavioral models. Altogether, there appears to be a convincing argument favoring a role of D-1 receptors in the generation of arousal. While there is evidence supporting the idea that D-2 receptors mediate motor activation, however, a possible involvement of D-2 in the mechanisms underlying arousal seems to be poorly substantiated by data and certainly less evident than the role of D- 1 receptors. Although evidence suggests that D-1 is involved in modulating arousal, the mechanism through which this occurs, or the function of D-1 as related to the regulation of normal processes, remains to be elucidated. There are some critical issues observed throughout the experiments. Whereas stimulation of D- 1 leads to cortical arousal, blockade of' the same receptors in drug-free animals induces only weak sedative effects. However, D- 1 receptor blockade reduces arousal markedly and produces sedation when a response is elicited by drugs such as SKF 38393, apomorphine, L-DOPA, or tonic stimuli, such as REM sleep deprivation. One possible interpretation of this critical finding is that D-1 receptors might subserve the expression of states of enhanced arousal. That is, D- 1 receptor populations may be physiologically activated under conditions that require or produce vigilance, alertness, and generally a more sensitive control of reactivity. This analysis does not take into consideration the fact that DAcontaining neurons have different distribution patterns within the brain, the so-called nigrostriatal, mesolimbic, and mesocortical pathways. There is every reason to suppose that these different areas participate in

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different, yet perhaps parallel, functions even though they receive their DA input from the same cell group. Although a mapping of‘ D-1 receptor distribution, as compared to 0-2, is now available (Dawson et al., 1986), it is not possible to establish whether a specific pathway is more critically involved in modulating arousal processes. T h e possible implication of prefrontal pathways in mechanisms of arousal would suggest devoting attention to these neurons first (Ida and Roth, 1987). In addition, there appear to be significantly more D-1 than D-2 binding sites in the cortical areas to which the mesencephalic DA system projects (Dawson et al., 1986). The density of D-1 receptors has been found to be conspicuous also in the human neocortex (Farde et al., 1987). Together, these considerations highlight a need for further experiments based mainly on combined electrophysiological and biochemical studies. At the cellular level, the differential function in arousal process between D-1 and D-2 receptors might well depend on the different cascade of events that are initiated by the stimulation of each receptor site. T h e formation of the second messenger CAMPand the activation of the polyphosphoinositide system could certainly be responsible for differences in the ultimate biological response. In addition it is well known that the DAergic system may be intimately related with the noradrenergic system in the control of behavior (Robbins and Everitt, 1982). Critical analysis of the possible nature of the link between the two neurotransmitters is beyond the scope of this review. However, as NE is deeply involved i n -the modulation of arousal (Monti, 1982; Koella, 1984), the possibility that D-1 or D-2 receptors might interact, at some stage, with NE should be taken into account. Moreover, experimental and theoretical reviews have proposed that multicenter anatomical systems with multiple neurochemical signatures are involved in the elaboration of the sleep-waking states (Koella, 1984; Hobson et al., 1986). This implies that possibly other neurotransmitters such as acetylcholine (Shiromani et al., 1987) or histamine (Nicholson et al., 1985) might be also involved with DA in the generation of cortical and behavioral arousal. References

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Koella, W. P. (1984). Experientza 40, 309-408. Longo, V. G. (1966). Pharmacol. Rev. 18, 965-996. Longo, V. G. (1973). Behav. Biol. 9, 397-420. Longo, V. G. (1978). I n “Principles of Psychopharmacology” (W. G. Clark and J. Del Giudice, eds.), pp. 247-260. Academic Press, New York. Longo, V. G., and Loizzo, A. (1978). Pharmacology 16, Suppl. 1, 189-192. Meller, E., Kuga, S., Friedhoff, J., and Goldstein, M. (1985). Life Sci. 36, 1857-1864. Mereu, G. P., Scarnati, E., Paglietti, E., Pellegrini Quarantotti, B., Chessa, P., Di Chiara, G., and Gessa, G. L. (1979). Electroencephalogr. Clin. Neurophysiol. 46, 214-219. Molloy, A. G., and Waddington, J. L. (1984). Psychopharmacology (Berlin) 82, 409-410. Monti, J. M. (1982). Life Sci. 30, 1145-1 157. Nicholson, A. N., Pascoe, P. A., and Stone, B. M. (1985). Neurofihamxology 24, 245-250. Ogren, S. O., Hall, H., Kohler, C., Magnusson, O., and Sjostrand, S. E. (1986). Psychopharmacology (Berlin) 90, 287-294. Ongini, E., and Caporali, M. G. (1987). Neuropharmacology 26, 355-360. Ongini, E., Caporali, M. G., and Massotti, M. (1985). Life Sci. 37, 2327-2333. Ongini, E.,Caporali, M. G., and Longo, V. G. (1987a). Neurosci. Lett. 82, 206-210. Ongini, E., Caporali, M. G., Massotti, M., and Sagratella, S. (1987b). Pharmacol. Biochem. Behav. 26,715-718. Parmeggiani, P. L., Morrison, A., Drucker-Colin, R., and McGinty, D. (1985). In “Brain Mechanisms of Sleep” (D. J. McGinty, R. Drucker-Colin, A. Morrison, and P. L. Parmeggiani, eds.), pp. 1-33. Raven, New York. Pellegrini Quarantotti, B., Scotti de Carolis, A., and Longo, V. G. (1975). Psychopharmacologaa 45,83-86. Peroutka, S. J., and Snyder, S. H. (1980). Am.J. Psychiatq 137, 1518-1522. Robbins, T . W., and Everitt, B. J. (1982). Int. Rev. Neurobiol. 23, 303-365. Robertson, G. S., and Robertson, H. A. (1986). Brain Res. 384, 387-390. Scott Bowersox, S., Kilduff, S., Faull, K. F., Zeller-De Amicis, L., Dement, W. C., and Ciaranello, R. D. (1987). Brain Res. 402, 44-48. Seeman, P. (1987). Synapse 1, 133-152. Seeman, P., and Gregoriadis, D. (1987). Neurochem. Int. 10, 1-25. Setler, P., Sarau, H. M., Zirkle, C. L., and Saunders, H. L. (1978). Eur. J. Pharmacol. 50, 419-430. Shiromani, P. J., Gillin, J. C., and Henriksen, S. (1987). Annu. Rev. Pharmacol. Toxicol. 27, 137- 156. Spano, P. F., Govoni, S., and Trabucchi, M. (1978). Adv. Biochem. Psychopharmacol. 19, 155- 165. Stoof, J. C., and Kebabian, J. W. (1984). Life Sci. 335, 2281-2296. Trampus, M., Monopoli, A., and Ongini, E. (1989). Soc. Neuroscience 15 (in press). Vigouret, J. M., Biirki, H. R., Jaton, A. L., Ziiger, P. E., and Loev, D. M. (1978). Pharmacology 16, Suppl. 1, 156-1’73. Waddington, J. L. (1986). Biochem. Pharmacol. 35, 3661-3667. Walters, J. R., Bergstrom, D. A., Carkon, J. H., Chase, T . N., and Braun, A. R. (1987). Science 236. 71 9-722.

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REGULATION OF BRAIN ATRIAL NATRIURETIC PEPTIDE AND ANGIOTENSIN RECEPTORS: QUANTITATIVE AUTORADIOGRAPHIC STUDIES By Juan M. Saavedra, Eero Castren, Jorge S. Gutkind, and Adit J. Nazarali Sedion on Pharmacology Labomtoly of Clinical Science National InstiMe of Mental Health Befheda, Maryland 20892

I. Introduction 11. Methods

A. Animals B. Tissue Preparation C. I n Vitro Labeling of Atrial Natriuretic Peptide Receptors D. In Vilro Labeling of Angiotensin I1 Receptors E. Preparation of '251-LabeledStandards F. Receptor Autoradiography G . Data Analysis H. Histological Controls 111. Results A. Distribution of Peptide Receptors in Brain B. Peptide Receptors in Hypertension C. Peptide Receptors in Alterations of Water Balance D. Peptide Receptors during Stress 1V. Discussion V. Conclusions References

I. Introduction

Angiotensin I1 (ANG 11) and atrial natriuretic peptide (ANP) are neurohormones which play important roles in the regulation of cardiovascular function and fluid balance (deBold, 1985; Vallotton, 1987). Circulating ANG I1 acts by stimulating specific receptors to induce vasoconstriction, aldosterone production, vasopressin release, and sodium retention (Laragh, 1987; Vallotton, 1987). Alterations in the peripheral renin-angiotensin system exist in animal models of genetic and experimental hypertension (Gavras et al., 1975; Chevillard and Saavedra, 1983; Niwa et al., 1985). The importance of the reninangiotensin system in human hypertension has long been recognized, INI'ERNAI'IONAL REVIEW OF NEUROBIOLOGY, VOL. 31

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and blockade of the last step of ANG I1 formation by inhibition of the angiotensin converting enzyme is one of the standard therapies in human essential hypertension (Unger et al., 1987). ANP produced in the cardiac atrium is released to the general circulation in response to atrial stretch as a consequence of increased venous return. Like ANG 11, it is involved in the coordinated control of fluid volume and cardiovascular function (Cantin and Genest, 1985; DeBold, 1985). ANP metabolism and function are altered in hypertension (Sonnenberg et al., 1983; Gutkowska et al., 1986; Morii et al., 1986a). The peripheral effects of ANP, that is, increased sodium and water excretion by the kidneys, decreased aldosterone production, vasodilation, and antihypertensive actions in both genetic and experimental hypertension, are antithetical to those of the water-conservatory peptides vasopressin and ANG I1 (deBold et al., 1981; Cartier et al., 1984; Kleinert et nl., 1984; Cantin and Genest, 1985; deBold, 1985; Garcia et al., 1985a,b; Needleman, 1986). For these reasons peripheral ANP and ANG I1 are considered to be parts of a regulatory system that are physiologically antagonistic to one another. Certain effects of the circulating peptides could be centrally mediated, since high concentration of their receptors are localized in the circumventricular organs, which are highly vascularized structures lacking a blood-brain barrier (Phillips et al., 1980; van Houten et al., 1980; Mendelsohn et al., 1984; von Schroeder et al., 1985; Quirion et al., 1984, 1986; Saavedra et al., 1986e, 1987a; Shigematsu et al., 1986). The centrally mediated actions proposed for ANG I1 include stimulation of drinking, increase in systemic blood pressure, stimulation of vasopressin release, and increase in salt appetite (Bickerton and Buckley, 1961; Bonjour and Malvin, 1970; Buggy and Johnson, 19’78; Ramsay et al., 1978; Mangiapane and Simpson, 1980a; Schiilkens et al., 1980; Simpson, 1981 Nicolaidis et al., 1983; Hartle and Brody, 1984; Phillips, 1987). Proposed actions of circulating ANP in the brain include antagonism of ANG II-induced drinking, decrease in systemic blood pressure, inhibition or blockade of vasopressin release, diuresis, inhibition of the increased salt appetite provoked by ANG I1 or present in genetically hypertensive rats, and direct effects on neuronal activity (AntunesRodrigues et al., 1985; Fitts et al., 1985; Masotto and Negro-Vilar, 1985; Nakamura et al., 1985; Obana et al., 1985; Samson, 1985a; AntunesRodrigues et al., 1986; Haskins et al., 1986; Iitake et al., 1986; Israel and Barbella, 1986; Itoh et al., 1986; Lappe el al., 1986; Shimizu et al., 1986; Takahashi et al., 1986). I n addition to receptors outside the blood-brain barrier, specific

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brain areas inside the blood-brain barrier contain ANG I1 and/or ANP receptors (Gehlert et al., 1984; Healy and Printz, 1984; Mendelsohn et al., 1984; Quirion et al., 1984, 1986; Lynch et al., 1986; Saavedra et al., 1986e; Shigematsu et al., 1986; Kurihara et al., 1987). Since peripherally formed peptides present in the general circulation are inaccessible to receptors in areas protected by the blood-brain barrier, it was considered that these receptors were part of distinct, central peptidergic systems. Indeed, all of the components of the renin-angiotensin system, including ANG 11, have been found in the brain, and immunostained ANG I1 fibers are discretely located in specific brain areas (Ganten et al., 1981; Chevillard and Saavedra, 1982; Chevillard et al., 1984; Lind et al., 1985a,b). In the case of ANP, recent experiments have demonstrated peptide localization by biochemical and immunohistological methods and its formation and release in brain (Glembotski et al., 1985;Jacobowitz et al., 1985; Kawata et al., 1985; Saper et al., 1985; Skofitsch et al., 1985; Standaert et al., 1986; Shibasaki et al., 1986; Tanaka and Inagami, 1986; Zamir et al., 1986). T h e nature of the effects observed after central administration of the peptides and the selectivity of the peptide and receptor localization suggests that the central peptide systems, like the peripheral systems, are involved in the regulation of fluid metabolism and cardiovascular function and that, centrally as well as peripherally, ANG I1 and ANP could be part of physiologically antagonistic systems. We have utilized quantitative autoradiographic techniques coupled to computerized microdensitometry (Goochee et al., 1980) to study the distribution and regulation of brain ANG I1 and ANP receptors in different animal models with pathological or physiological alterations. These include hypertension with central nervous system components (Saavedra et al., 1978; Brody and Johnson, 1980; Matsuguchi et al., 1982; Songu-Mize et al., 1982), stress, and models for altered fluid homeostasis. Autoradiographic techniques are the methods of choice to study neuropeptide receptors in small tissues and discrete brain areas in which sufficient amounts of material are difficult to obtain for conventional membrane binding methods (Young and Kuhar, 1979; Unnerstall et al., 1982). These techniques offer the additional advantage of preservation of tissue anatomy and allow a precise localization of receptor sites. The use of ‘251-labeledligands and the comparison of the results with standard curves generated with I2’I-labeled standards allowed for precise quantification of receptor number. Exposure time for the film during autoradiography did not exceed 1 week when ‘251-labeled ligands were used, due to the high radiation energy and high specific

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activity of the ligands (Israel et al., 1984, 1985a,b). In addition, the problems of differential quenching and the influence of section thickness encountered when using "-labeled ligands did not exist for 1251 (Kuhar and Unnerstall, 1985). These properties allowed rapid performance of physiological and pharmacological experiments as well as determination of complete binding kinetics with relative ease in individual brain nuclei from single rats (Israel et al., 1985b). Using quantitative autoradiography we were able to determine that specific alterations in brain ANG I1 and ANP receptors occur when blood pressure or fluid consumption are modified and that at least some of these central alterations are directly or indirectly related to changes in the peripheral peptide systems. Our results indicate a role for the central ANG I1 and ANP systems in fluid and cardiovascular control and suggest that peripheral and central peptidergic systems are associated, perhaps via peptidergic receptors located outside the blood-brain barrier in contact with the general circulation.

It. Methods

A. ANIMALS For ANP and ANG I1 receptor mapping studies, male SpragueDawley rats, 250-300 g in body weight, were purchased from ZivicMiller, Allison Park, Pennsylvania. The rats were housed under a constant temperature (24°C) with lights on from 0600 to 1800 hr and were given free access to food and water. For studies on genetic hypertension, groups of 4- and 14-week-old male spontaneously hypertensive rats (SHR) (Okamoto, 1972) and their age-matched normotensive controls, Wistar-Kyoto (WKY) rats, were obtained from Taconic Farms, Germantown, New York and maintained under normal laboratory conditions as described above. Blood pressures, measured 1 day before killing, were (in mm Hg) 90 L 4 and 115 5 in young rats and 122 +: 8 and 178 12 in adult rats, WKY and SHR, respectively ( p < 0.05). To prepare the desoxycorticosterome acetate (D0CA)-salt model of experimental hypertension, male, 14-week-old Wistar-Kyoto rats were anesthetized with sodium pentobarbital (40 mg/kg ip) and a unilateral left nephrectomy was performed through a retroperitoneal incision. After 1 week of recovery, the rats were weighed and divided randomly into four experimental groups. The DOCA-salt group received sub-

*

*

ATRIAL NATRIURETIC PEPTIDE AND ANCIOTENSIN RECEPTORS

26 1

cutaneous injections of DOCA (Percoten, Ciba Geigy Corp., Summit, New Jersey) twice weekly (25 mg/kg total per week) and a 1 % solution of NaCl for drinking water. The DOCA group received the same injections with DOCA and tap water to drink. The salt group received twiceweekly subcutaneous injections of the vehicle (0.5 ml/kg) and a 1% solution of NaCl for drinking water. T h e control group received the same injections of the vehicle and tap water to drink. Blood pressures were measured weekly in all animals, before and during treatment, with an electrosphygmomanometer (Narco Bio-Systems Inc., Houston, Texas) and photoelectric sensors (IITC Inc., Landing, New Jersey) and recorded on a Model 7 Grass polygraph. After 4 weeks of treatment, blood pressures were (in mm Hg): DOCA-salt, 180 5; DOCA alone, 122 ? 8; salt alone, 115 ? 4; and sham-operated control, 110 ? 10. After 4 weeks of treatment, rats were sacrificed by decapitation between 0900 and 1 100 hr. For studies on dehydration, two different rat models were studied. Water deprivation studies were performed in 12 male Sprague-Dawley rats, 250 g in body weight, obtained from Zivic-Miller. First, all animals were given tap water and rat chow ad libitium and were housed individually for 1 week under normal laboratory conditions as described above. Then, the rats were randomly distributed into two groups of six animals each, one water-satiated (control) Group with access to tap water ad libitum and one water-deprived group with no access to drinking water for 5 days before killing. Both groups were given access to chow ad libitum throughout the experiment. After 5 days of dehydration significant changes were observed in the body weights of the animals. Control hydrated animals gained 7.5%in body weight (from 241 ? 3 to 260 5 g); the dehydrated animals lost u p to 27% of their body weight (from 235 ? 2 to 172 ? 2 g) but did not show other signs of disease. Studies on chronic dehydration were performed in homozygous Brattleboro rats lacking the antidiuretic hormone vasopressin (Valtin and Schroeder, 196 1). Male 9-week-old homozygous Brattleboro (DI) rats, heterozygous Brattleboro (HZ) rats, and normally hydrated LongEvans (LE) controls were housed individually in metabolic cages under normal laboratory conditions as described above for 1 week after being purchased from Blue Spruce Farms, Altamont, New York. T h e mean daily water intake was 8 % 2 , 9 ? 5, and 75 2 5 ml per 100 g weight for LE, HZ, and DI rats, respectively. For studies on stress, seven male Sprague-Dawley rats (Zivic Miller), weighing 217 ? 6 g at the beginning of the experiment, were subjected to immobilization (Kvetnansky et al., 1970) for 2 hr per day (between 1000 and 1200 hr) for 10 consecutive days. Body weight and blood pressures

*

*

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IUAN M. SAAVEDKA et a!

were measured at the beginning of the experiment and 1 day before sacrifice. Control rats were killed by decapitation between 1000 and 1200 hr avoiding stressful handling. Stressed rats were decapitated immediately after the last stress period. Control rats gained 20% of body weight during the 10-day experiment (from 217 -t 7 to 261 2 9 g), whereas the body weight of the stressed rats did not change significantly (from 218 ? 6 to 221 -C 13 g, p < 0.0001 between control and stressed groups at the end of the experiment). There was no significant difference in blood pressure between groups at the end of the experiment (95 2 14 and 101 +- 18 mm Hg for control and stressed rats, respectively).

B. TISSUE PREPARATION All rats were sacrificed by decapitation and their brains immediately removed and frozen by immersion in isopentane at -30°C. Brain sections 16 pm thick were cut in a cryostat at - 14”C,thaw-mounted onto gelatin-coated glass slides, and placed under vacuum at 4°C overnight.

C. In Vitro LABELING OF ATRIAL NATRIURETICPEPTIDERECEPTORS Rat ANP receptors were labeled in vitro by incubation with (3(specific activity 1750-2050 Ci/mmol) (Amersham Corp., Arlington Heights, Illinois) (Quirion et al., 1986; Saavedra et al., 1986a; Saavedra, 1987). Tissue sections were preincubated for 15 min at room temperature in 50 mM Tris-HC1 buffer, pH 7.4, containing 100 mM NaCI, 5 mM MgC12, 0.5% bovine serum albumin, 40 pg/ml bacitracin, 4 pg/ml leupeptin, 2 pgiml chymostatin, 0.5 pglml phenylmethylsulfonylAuoride (PMSF). Tissue sections were subsequently incubated in fresh buffer for 60 min at room temperature with ‘251-labeled ANP as described below. Nonspecific binding was determined in consecutive sections incubated as above in the presence of unlabeled ANP (atrial peptide, rat, 28 amino acids) (Peninsula Laboratories, Inc., Belmont, California) at concentrations described below. After incubation, the slides were washed three times, 2 min each, in 50 mM Tris-HCl buffer, pH 7.4, at 4°C and dried under a stream of cold air. For analysis of ANP receptor concentrations and localization throughout the brain, consecutive tissue sections were incubated as above with a single 130 pM concentration of “’1-labeled ANP, and nonspecific binding was determined in the presence of 0.32 p M unlabeled ANP. For Scatchard analysis, consecutive sections from individ[ 1251]iodotyrosyl-28) ANP

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263

ual brains were incubated as above in the presence of lZ5I-labeledANP at concentrations ranging from 10 to 400 pM. I n these experiments, nonspecific binding was determined in consecutive sections in the presence of 1 p M unlabeled ANP.

D. In Vitro LABELING OF ANGIOTENSIN I1 RECEPTORS Within 48 hr of section preparation, ANG I1 receptors were labeled in vitro by incubation of sections with '251-labeled [Sar'l-ANG 11,

Peninsula Laboratories, iodinated by New England Nuclear, Boston, Massachusetts. For determination of saturation curves and Scatchard plots in the subfornical organ, area postrema, and nucleus of the solitary tract, consecutive tissue sections from single rat brains were preincubated for 15 min at room temperature in 10 mM sodium phosphate buffer, pH 7.4, containing 120 mM NaCl, 5mM NaZEDTA, 0.005% bacitracin, and 0.2% bovine serum albumin, and then incubated for 60 min in fresh buffer with six different concentrations of '251-labeled [Sar'l-ANG I1 ranging from 100 pM to 5 nM. Nonspecific binding was determined in the presence of 5 p M unlabeled ANG 11. For analysis of ANG I1 receptor density and localization throughout the brain, additional sections were incubated under the same conditions with 1 nM IZ5I-labeled[Sar'l-ANG I1 and nonspecific binding determined in the presence of 1 pkf ANG 11. After incubation, all slides were washed four times, 60 sec each, in ice-cold 50 mM Tris-HC1 buffer, pH 7.56, once for 30 sec in ice-cold water, and dried under a stream of cold air.

E. PREPARATION OF Iz5I-LABELEDSTANDARDS

Sets of 1251-labeledstandards were prepared as originally described for 3H-labeled standards (Young and Kuhar, 1979; Israel et al., 1985b). Known amounts of increasing concentrations of '251-labeled ANG I1 were thoroughly mixed with rat brain tissue aliquots previously ground to a paste and degassed by repeated mixing under vacuum. T h e aliquots were placed as blocks of tissue on microtome specimen holders and frozen on dry ice. Tissue sections, 16 pm thick, were cut in a cryostat at -14°C and thaw-mounted onto subbed glass slides. Parallel sets of standards obtained from consecutive sections were used for determination of protein content and radioactivity.

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F. RECEPTORAUTORADIOGRAPHY

Autoradiographic images of ANP and ANG I1 receptors were obtained by placing the previously incubated sections in X-ray cassettes (CGR Medical Corp., Baltimore, Maryland) and apposing them against 'H-labeled Ultrofilm (LKB Industries, Rockville, Maryland) at room temperature for 2-5 days. The optimum exposure time for each area, producing optical densities between 0.3 and 1.2 U, was determined in preliminary experiments. Sections presenting both areas of a very high density and areas of a very low density of binding sites were exposed twice, to obtain optimum readings for both types of areas. A complete set of '251-labeledstandards was exposed with each film. T h e films were developed at 4°C for 4 min with undiluted D19 developer (Eastman Kodak Co., Rochester, New York). G. DATAANALYSIS

Optical densities were measured by computerized microdensitometry (Goochee et al., 1980) for both '251-labeled standard images and specific areas of brain section images from each film. After determination of the standard curve for each film (In optical densities X 100 versus In disintegrations per minute in standards), the optical densities of the brain areas studied were interpolated from the straight line of the standard curve to obtain the corresponding dpm bound to the tissue (Israel et al., 1985b). Results were corrected for the decay of I2'I. Calculation of the molar quantities of ligand bound to the tissue were determined after correction for the protein content of the standards. Protein concentrations were assumed to be uniform in 16-pm sections throughout the brain. Determination of kinetic parameters was performed according to Munson (1983). Data are presented as X SEM. Student's t-test for independent means was employed for statistical analysis; p values of less than 0.05 were considered significant.

*

H. HISTOLOGICAL CONTROLS

To localize and identify precisely the areas containing ANP and ANG I1 receptors, tissue sections adjacent to those used for autoradiography were stained with Luxol fast blue or hematoxylin-eosin. Identification of discrete brain areas was performed according to Konig and Klippel (1963) and Paxinos and Watson (1982).

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111. Results

A. DISTRIBUTION OF PEPTIDE RECEPTORS IN BRAIN 1. Angiotensin II Receptors ANG I1 receptors were highly localized in 24 areas of the rat brain (Table I). A 20-fold difference in relative receptor concentration was noted between the areas with highest concentration, the circumventricular organs organon vasculosum laminae terminalis (OVLT) and subfornical organ, and the area with the lowest concentration of ANG I1 receptors, the subiculum (Table I). In the rostra1 limbic system, ANG I1 receptors were located in the nucleus of the lateral olfactory tract and at all levels of the lateral olfactory tract (Table I). High levels of ANG I1 receptors occurred in the circumventricular organs, which include the OVLT, the subfornical organ and the area postrema (Table I and Fig. 1). In the hypothalamus, ANG I1 receptors were located in a few nuclei. The highest relative density was located in the suprachiasmatic nucleus followed by the periventricular nucleus and the median preoptic nucleus. Relatively high concentrations of receptors also occurred in the parvocellular part of the paraventricular nucleus and in the median eminence. The dorsomedial nucleus contained a somewhat lower concentration of ANG I1 receptors (Table I ) . Among the limbic cortical areas, the pyriform cortex was the only area containing ANG I1 receptors (Table I). ANG I1 receptor distribution was discrete in the subthalamic areas, where the subthalamic nucleus was the only area showing ANG I1 receptors, and also in the mesencephalic areas, where ANG I1 receptors were restricted to the superior colliculi and the subiculum (Table I). In the pons, the only area showing a measurable number of ANG 11 receptors was the locus coeruleus (Table I). A number of brainstem areas contained high levels of ANG I1 receptors with the highest relative concentrations present in the nucleus of the solitary tract (NTS), the area postrema, the dorsal motor nucleus of the vagus, and the nucleus of the commissural tract (Table 1 and Fig. 1). Receptors were also present, although in lower relative concentrations, in the trigeminal tract and in the inferior olivary nuclei (Table I and Fig. 1). Sagittal sections of the brain revealed ANG I1 binding in a continuous band situated along the subependymal space, connecting the subfornical organ and the OVLT and involving the median preoptic nucleus and the lamina terminalis (Fig. 2). Caudally, the ANG I1

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JUAN M. SAAVEDRA et

a.1.

TABLE I QUANTITATIVE DISTRIBUTION OF ANGIOTENSIN 11 A N D ATRIALNATRIURETICPEPTIDE IN RECEPTORS

THE

RAT BRAIN" ~~

Apparent receptor density (fmol/mg protein)

Arcah

Arigiotensin I 1

Rostra1 limbic system Nucleus of the lateral olfactory tract Lateral olfactory tract

36 f 4 45 ? 4

Circumventricular organs Subfornicdl organ Organon vasculosum laminae terminalis Area postrema

Atrial nat riu ret ic peptide 40 23

2 2

1 2

228 ? 33 258 2 22

52 t 6 53 -+ 1

119 f 18

54 -+ 5

97 ? 13 62 f 7 161 2 9 22 2 4 5 5 ? 10 98 f 7 ND

ND 32 2 1 ND ND 14 f 3 43 1 55 f 8

Limbic cortex Pyriform cortex Fimbria of the hippocampus Medial habenular nucleus Fasciculus retroflexus

36 t 5 ND ND ND

ND 20 f 2 272 I 23 t 2

Subthalamus-metathalamus Subthalamic nucleus

40 -c 4

ND

Mesencephalic regions Superior colliculi Subiculum

16 3 13 ? 0.4

ND ND

Pons Locus coeruleus

26 f 6

ND

141 2 18 29 4 ND

13 5 2 ND 13 2

Hypothalamus Periventricular nucleus Paraventricular nucleus Suprachiasmatic nucleus Dorsornedial nucleus Median eminence Median preoptic nucleus Supraoptic nucleus

Brainstem Dorsal motor nucleus of the vagus Trigeminal tract (sp 5) Hyppoglossal nucleus

*

*

*

*

267

ATRIAL NATRIURETIC PEPTIDE AND ANGIOTENSIN RECEPTORS

TABLE I (Continued) Apparent receptor density (fmol/mg protein)

Angiotensin I1

Atrial natriuretic peptide

Nucleus of the solitary tract Anterior (bregma - 13.3 mm) Intermediate (bregma - 13.8 mm) Posterior (bregma - 14.5 mm)

140 k 18 172 k 18 73 ? 6

17? 1 -

Commissural tract of the nucleus of the solitary tract Inferior olivary nuclei Choroid plexus

135 2 12 32 ? 5 ND

Areab

* From Saavedra et aL(1986e) and Kurihara et al. (1987).

’

Nomenclature and coordinates are according to Konig and Klippel (1963) for the forebrain areas and Paxinos and Watson (1982) for the pons and brainstem areas. ‘ Autoradiographic determinations were performed in tissues from five single animals assayed individually after incubation with 1 nM ‘251-labeled[Sar’l-ANG I1 or 130 pM [“’I]-ANP as described in Methods.

receptor band was in direct continuity with ANG I1 receptors highly localized to the periventricular and paraventricular nuclei and to the median eminence (Fig. 2). In addition to localization of specific ANG I1 receptors, the sites were characterized in the subfornical organ, the nucleus of the solitary tract, and the area postrema by incubation of consecutive tissue sections obtained from single rat brains with increasing concentrations of lZ5Ilabeled [SARI]-ANG 11. All three areas showed a single class of saturable, high-affinity receptors. 2 . Atrial Natriuretic Peptide Receptors The highest concentrations of ANP receptors were present in the circumventricular organs, including the OVLT, the subfornical organ, and the area postrema (Fig. 3 and Table I). In these areas, nonspecific binding was less than 25% of the total binding. In addition to the circumventricular organs, ANP receptors were localized in selected hypothalamic and limbic system nuclei. In the

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JUAN M. SAAVEDRA el al.

FIG. 1. Angiotensin I1 receptor distribution in the rat brain. (A-E) Frontal to caudal coronal secrions, total binding. OVLT, Organon vasculosum laminae terminalis; MPN, median preoptic nucleus;SFO, subfornical organ; PVN, paraventricular nucleus; AP, area postrema; NTS, nucleus of the solitary tract; 10,inferior olive. (From Nazarali et al., 1987.)

hypothalamus, the highest concentration of ANP receptors was located in the supraoptic nucleus, followed by the median preoptic nucleus. Moderate ANP receptor concentrations were also detected in the paraventricular nucleus (Fig. 3 and Table I). Lower numbers of ANP receptors were located in the median eminence (Table I). In the rostra1 limbic system, ANP receptors were located in the olfactory areas, especially in the olfactory bulb and at all levels of the lateral olfactory tract. Other limbic system areas, such as the fimbria of the hippocampus, the medial habenular nucleus, and the fasciculus retroflexus, also have moderate numbers of ANP receptors (Table 1). In the brainstem, in addition to the area postrema, small numbers of ANP receptors were located in the nucleus of the solitary tract, the dorsal motor nucleus of the vagus, and the hypoglossal nucleus (Table I). No other brainstem areas presented significant numbers of A N P receptors.

ATRIAL NATRIURETIC PEPTIDE AND ANGIOTENSIN RECEPTORS

269

A

I ME

hpv

C

B

e

I

ME

ME

FIG. 2. Angiotensin I1 receptors in forebrain sagittal sections. (A) Midsagittal section; (B) close-up of (A); (C) adjacent section stained with Luxol fast blue. SFO, subfornical organ; pome, median preoptic nucleus; CA, anterior commissure; hpv, periventricular nucleus; PVH, paraventricular nucleus; ME, median eminence; OVLT, organon vasculosum laminae terminalis; SGS, superficial grey layer superior colliculus; AP, area postrema; NTS, nucleus of the solitary tract. (From Shigematsu et al., 1986.)

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JUAN M. SAAVEDRA et al.

A12130p

t

I

A7470p

I

FIG. 3. Atrial natriuretic peptide receptors in rat forebrain. Numbers correspond to antero-posterior coordinates according to Konig and Klippel ( 1 963). LMIO, Olfactory bulb, lamina medular interna; LPIB, Olfactory bulb, lamina plexiforme interna; VL, lateral ventricle; CP, choroid plexus; OVLT, organon vasculosum laminae terminalis; TOL, lateral olfactory tract; pome, median preoptic nucleus; VIII, third ventricle; SO, supraoptic nucleus; SFO, subfornical organ. (From Kurihara et al., 1987.)

ATRIAL NATRlURETlC PEPTIDE AND ANGIOTENSIN RECEPTORS

27 1

In addition to specific nuclei within the brain parenchyma, the choroid plexus contains high numbers of ANP receptors (Figs. 3 and 4 and Table I ) . ANP receptors could also be detected in the ependymal layer of the ventricles, vasculature, and pial membranes, but these structures were too thin to allow a reliable quantification (Fig. 3 and 4). Determination of saturation curves and Scatchard analysis of ANP receptors in the subfornical organ, the area postrema, and the choroid plexus revealed the presence of a single class of saturable, high-affinity ANP receptors (Table 11). B. PEPTIDERECEPTORSI N HYPERTENSION 1. Genetic Hypertension

a. Angzotensin Receptors. Both young (4-week-old) and adult (14week-old) SHR had higher ANG 11 receptor concentrations in selected forebrain and brainstem areas when compared to age-matched WKY rats (Table 111). ANG I1 receptors in SHR were significantly higher in the median preoptic nucleus, subfornical organ, paraventricular nucleus, and nucleus of the solitary tract than those of WKY. No significant differences were found in the olfactory bulb, suprachiasmatic nucleus, inferior olive, and area postrema. In the subfornical organ, similar results were found after determination of saturation curves and Scatchard plots. SHR exhibited significantly more binding sites for ANG I1 than normotensive rats at both ages. The receptor affinity was lower in young and adult SHR than in WKY rats (Table 11). 6. Atrial Natriuretic Peptide Receptors. Both young (4-week-old) and adult (14-week-old) SHR had much lower numbers of ANP receptors than their normotensive controls in the subfornical organ, the choroid plexus (Fig. 4), the area postrema, and the nucleus of the solitary tract (Table I1 and IV). Scatchard analyses were performed in the subfornical organ and the choroid plexus of both young and adult rats and in the area postrema of young animals and revealed that SHR had much lower B,,, values than age-matched WKY (Table 11). The affinity for ANP binding was also lower in the subfornical organ of adult SHR compared to adult WKY rats. T h e other areas studied, however, did not show differences in the affinities for ANP binding between SHR and the WKY rats (Table 11).

2 . DOCA-Salt Hypertension a. Angiotensin ZZ Receptors. Treatment with salt alone significantly increased ANG I1 binding only in median preoptic nucleus and sub-

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FIG.4. Atrial natriuretic peptide receptors in subfornical organ and choroid plexus of' adult spontaneously hypertensive rats. Top: Wistar-Kyoto rats; bottom: spontaneously hypertensive rats. Arrows point to the subfornical organ and the choroid plexus. Incubation conditions and exposure times were identical for both figures. (From Saavedra, lY86a.)

273

ATRIAL NATRIURETIC PEPTIDE AND ANGIOTENSIN RECEPTORS

TABLE I1

KINETICSOF ATRIALNATRIURETIC PEPTIDE AND ANCIOTENSIN I1 RECEPTORS IN SUBFORNICAL ORGAN OF SPONTANEOUSLY HYPER.I.ENSIVE RATS (SHR) AND NORMOTENSIVE CONTROLS (WKY)" Binding capacity, B,, (fmol per mg protein) Peptide and area

Age (weeks)

WKY

4 14 4 4 14

85 -+ 12 124 t 11 110f 10 74 f 13 1 2 0 2 14

4

444 f 85 848 f 136

Atrial natriuretic peptide Subfornical organ Area postrema Choroid plexus Angiotensin I1 Subfornical organ

14

Affinity constant, Kd (nM)

SHR

WKY

SHR

0.12 f 0.2 0.09 -t_ 0.2 0.05 2 0.01 0.1 1 a 0.02 0.14 f 0.05

0.14 2 0.06 0.23 t 0.Og6 0.05 2 0.01 0.09 f 0.02 0.16 f 0.02

1.15 f 0.20 735 5 91b 1390 2 6-1&c 0 . 5 3 0.11

3.13 f 0.62b 1.12 2 0.236

38 -t 4' 73 5 lob 54 4 66 21 -t 3b 61 lob

*

*

From Saavedra et al. (1986a). Significant difference compared with WKY rats (P < 0.05).

.

TABLE 111 APPARENT ANGIOTENSIN I1 RECE~TOR CONCENTRATIONS IN BRAIN NUCLEI OF SPONTANEOUSLY HYPERTENSIVE AND WISTAR-KYOTO RAT SO.^ ~~~~~

Apparent concentration &mg protein) ~

~~

Area

Strain

4 weeks old

14 weeks old

Olfactory bulb

WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR

55 c 5 65 2 8 116 c 5 146 ? 8< 155 c 12 274 ? 22" 102 +- 5 1 2 6 c 11 89 +. 4 119 2 8c 74 +- 4 111 t 8c 74 9 86 +- 8 1 0 3 2 12 100 +- 8

66 t 4 64 f 3 90 t 5 129 6' 1 5 8 k 18 255 f 13' 110 f 6 101 f 9 110 f 6 155 t 7c 92 f 4 126 f 8c 105 2 8 119 f 8 40 f 5 44 5

Median preoptic nucleus Subfornical organ Suprachiasmatic nucleus Paraventricular nucleus Nucleus of the solitary tract Area postrema Inferior olive

*

*

*

~~

From Gutkind et al. (1988a). Brain sections were incubated with 3 nM '251-labeled[Sar'l-ANG 11. Groups consisted of six-eight animals, assayed individually. Significantly different from age-matched WKY rats ( p < 0.05). a

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TABLE IV ATRIALNAIRIURETIC PEPTIDE RECEPTORS IN BRAINSTEM NUCLEI OF SPONTANEOUSLY RATS" HYPERTENSIVE Apparent binding site concentration (fmol/mg protein) Area Area postrema Nucleus of the solitary bct

Age (weeks)

WKY

SHR

4 14

120 f 13 206 ? 15

4 0 2 8b 104 & 14*

4

ND 51&3

ND 6 f 2'

14

"Tissues from groups of six rats were incubated, assayed individually with a single 0.3 nM concentration of '*'I-labeled ANP. ND, not dctectable. Significant difference ( p < 0.05) between SHK and age-matched WKY rats.

fornical organ (?*& V). DOCA administration alone significantly increased ANG Ii-receptors only in median preoptic nucleus, subfornical organ, and paraventricular nucleus (Table V). In the forebrain of DOCA-salt hypertensive rats, the number of ANG I1 receptors was significantly higher than that of control rats in the median preoptic nucleus, subfornical organ, and in paraventricular nucleus (Table V ) . DOCA-salt treatment increased ANG I1 binding in the median preoptic nucleus to the same extent as DOCA or salt treatment alone. In subfornical organ and paraventricular nucleus, however, the increase in ANG I1 binding in DOCA-salt hypertensive rats was significantly greater than that from either salt or DOCA treatment alone (Table V). None of the treatment regimens produced significant alterations in the concentrations of ANG I1 receptors in other forebrain areas, the olfactory bulb, or in the suprachiasmatic nucleus (Table V). In the brainstem, DOCA-salt hypertensive rats had increased ANG I1 binding in the nucleus of the solitary tract and in the area postrema. Neither salt alone nor DOCA alone produced any significant modification of ANG I1 binding in brainstem nuclei (Table V). b. Atrial Natriuretic Peptide Receptors. Alterations in ANP receptor

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TABLE V APPARENTANCIOTENSIN I1 RECEPTOR CONCENTRATIONS I N BRAIN OF DOCA-SALT HYPERTENSIVE RATS",~ Area Olfactory bulb

Median preoptic nucleus

Subfornical organ

Suprachiasmatic nucleus

Paraventricular nucleus

Nucleus of the solitary tract

Area postrema

Group control DOCA salt DOCA-salt control DOCA salt DOCA-salt control DOCA salt DOCA-salt control DOCA salt DOCA-salt control DOCA salt DOCA-salt control DOCA salt DOCA-salt control DOCA salt DOCA-salt

Apparent concentration (fmol/mg protein)

*

45 5 56 2 6 61 2 8 61 ? 6 131 9 166 f lo' 175 f 9" 182 6' 175 ? 8 229 f 12c 232 f 1lC 271 f 11"' 124 ? 14 142 f 20 113 9 128 f 18 78 2 8 118 6' 105 9 149 ? 6',d 81 2 8 77 f 6 109 9 151 2 73 7 62 ? 5 91 +.6 125 f 1 0 " ~ ~

* *

*

* *

* *

From Gutkind et al.( 198813). Brain sections from eight animals assayed individually were incubated with 3 nM '251-labeled[Sar'l-ANG. Significant difference (p < 0.05) when compared with the control group. *Significant difference ( p < 0.05) when compared with DOCA only and salt only groups.

concentrations occurred in subfornical organ and choroid plexus. Treatment with salt alone produced large increases ( + 8 5 % ) in ANP receptors in these structures (subfornical organ +85%, choroid plexus + 5776, salt-treated versus controls) (Table VI). Treatment with DOCA alone did not modify the concentration of ANP receptors (Table VI). Surprisingly, hypertensive DOCA-salt treated rats did not show differ-

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ences in ANP receptor concentrations when compared to controls, and the ANP receptor number was significantly lower than that present in the group treated with salt alone (Table VI).

C. PEPTIDE RECEPTORS IN ALTERATIONS OF WATERBALANCE 1. Water Depn'vation

a. Anpotensin II Receptors. Only the subfvrnical organ showed a significant increase in receptor density after dehydration (Table VII). Increased ANG I1 receptor concentrations were also observed in the median preoptic nucleus, the paraventricular nucleus, and the OVLT of the dehydrated animals, although these results were not significant (Table VII). There were no significant changes after dehydration in ANG I1 binding in the area postrema, both the anterior and medial portions of the nucleus of the solitary tract, as well as the inferior olive. b. Atrial Natnuretic Peptide Receptors. Large increases in the maximum binding capacity (BmaX)of ANP receptors were found in the subfornical organ and the choroid plexus of water-deprived rats compared to water-satiated controls (Table VIII). N o differences in affinity were detected in the subfornical organ of dehydrated rats. However, the b i a i n g affinity was decreased in the choroid plexus of water-deprived animals compared to water-satiated controls (Table VIII).

TABLE VI APPARENT ATRIAL NATRIURETIC PEPTIDE RECEPTORCONCENTRATIONS I N BRAIN OF DOCA-SALT HYPERTENSIVE RATS" Group Controls DOCA Salt DOCA-salt

Subfornical organ Choroid plexus

34 39 63 38

t2 23 f 6h f3

37 f 3 41 ? 3 58 k 6b 41 3

*

' Brain sections from seven animals, assayed individually, were incubated with 0.3 nM '%Ilabeled ANP. Significant difference ( p < 0.05) when compared to control, DOCA-treated, and DOCA-salttreated groups.

'

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ATRIAL NATRIURETIC PEPTIDE AND ANGIOTENSIN RECEPTORS

TABLE VII APPARENT ANCIOTENSIN I1 RECEPTOR DENSITY IN RAT BKAIN OF CONTROL AND WATER-DEPRIVED RAT^ SPKACUE-DAWLEY Apparent receptor density* (fmol/mg protein) Control Dehydrated

Brain nuclei Subfornical organ Organon vasculosum of the lamina terminalis Median preoptic nucleus Paraventricular nucleus Area postrema Nucleus of the solitary tract (anterior portion, bregma - 13.8 mm)d Nucleus of the solitary tract (medial portion, bregma - 14.3 rnm)d Inferior olive a

171 2 12 185 -I- 15 142 f 14 116 2 12 140 f 9 136 2 7

373 f 10' 210 -c 20 185 f 7 135 f 10 136 2 10 126 f 9

166 2 8

171

118

* 10

-I-

Change (%)

118 13 30 17 -3 -8 3

9

l l O + 16

-6

From Nazarali et al. (1987). as mean f SEM for groups of six animals. Significantly different from controls ( p < 0.01). Data from Paxinos and Watson (1982).

' Data are represented

2. Brattleboro Rats a. Atrial Natriuretic Peptide Receptors. DI rats had a much higher number of ANP receptors in the subfornical organ than their LE controls (Fig. 5 ) . HZ rats had receptor concentrations sf intermediate value between those of LE controls and DI rats. When water deprived for 4 days, LE controls showed upregulation of ANP receptors in the subfornical organ, and the maximum concentration attained after water deprivation in LE was similar to that present in DI rats (Table IX). In contrast, there were no differences in binding affinities between any of the groups studied (Table IX). TABLE VIII ATRIALNATRIURETIC PEPTIDERECEPTORS IN THE BRAIN OF WATER-DEPRIVED SPRAGUE-DAWLEY RATS" Subfornical organ Group

B rnax ( f m o l h g protein)

Control Dehydrated

76 rt 10 139 2 23'

a

Results are expressed as means

Choroid plexus K d

B

mdx

(d) (fmol/mg protein) 0.08 2 0.01 0.12 2 0.02 f

SEM.

'Significantly different from controls ( p < 0.05).

6 6 a 12 126 2 gb

Kd

(a) 0.08 f 0.01 0.14 2 0.02'

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JUAN M. SAAVEDKA e6 al.

Bound (pM)

FIG. 5 . Atrial natriuretic peptide receptors in the subfornical organ of Long-Evans and homozygous Brantleboro rats. Filled circles, Long-Evans rats; open circles, homozygnus Brattleboro rats. Data represent a typical experiment which was replicated five to eight times per group (see Table IX). (From Saavedra, l986b.)

TABLE IX BRAINATRIALNATRIURETIC PEPTIDERECEPTORS I N LON(;-EVANS,B R A T T L E B ~ K ~ AND WATER-DEPRIVED RATS' Kd

(N)

Strainb ~~

Binding capacity, Em,, (fmol/mg protein)

~

LE control LE water-deprived HZ DI

0.06 k 0.01 0.07 2 0.01 0.06 f 0.005 0.08 rf: 0.01

*

29 f 2 74 rf: 58'd 44 2 1' 67 rf: Fd

SEM. I.E, Long-Evans; HZ, Brattleboro heterozygotes; D1, Brattleboro homozygous diabetic rats. ' Significantly different from LE controls ( p < 0.005). Significantly different from HZ rats ( p < 0.005). * Values are means

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279

D. PEPTIDERECEPTORS DURING STRESS Anpotensin Receptors

In the paraventricular nucleus, receptors were localized predominantly in the medial part of the nucleus, corresponding to the parvocelM a r subdivision. Binding concentration in the magnocellular paraventricular nucleus was too low to be quantified by the method used. Concentration of ANG I1 receptors was significantly higher in both parvocellular paraventricular nucleus (Fig. 6) and in subfornical organ of chronically stressed rats when compared to that of the control rats. There was no significant alteration in the binding affinity (Fig. 7 and Table X). ANG I1 binding concentration in anterior pituitary slices incubated at a single 2 nM concentration of the ligand was not altered significantly by repeated stress (118 +- 8 and 117 2 4 fmol/mg protein in control and stressed rats, respectively).

FIG.6 . Angiotensin I1 receptors in the paraventricular nucleus of stressed rats. (A and B) Typical sections from stressed and control rats, respectively. (C) adjacent section stained with toluidine blue. (D) Nonspecific binding. P, parvocellular paraventricular nucleus; M, magnocellular paraventricular nucleus; OT, optic tract. (From Castren and Saavedra, 1988.)

280

JUAN M. SAAVEDRA

-.-

el nl.

K

a)

-

100

200

'%Labeled [SARI]-ANGIOTENSINI1 (nM)

FIG. 7. Angiot sin I1 receptors in subfornical organ of' stressed rats. Typical saturation curves and Scat9 ard plots (inserts) from individual rats, repeated seven times per group (see Table X). Filled circles, stressed rats. Open circles, control rats. (From Castren and Saavedra, 1988.)

"ih

TABLE X ANGIOIENSIN RECEPTOR CONCENTRATION IN BRAIN OF CHRONICALLY STRESSED RATS",~ ~

Control

Stressed

Bma,

Kd

Area

(fmolimg protein)

(nM)

B max (fmoltmg protein)

Subfornical organ Paraventricular nucleus (parvocellular subdivision)

230 -C 8 63 t 7

0.71 2 0.08 0.54 0.09

311 2 13' 105 t 11'

*

Kd

(nM)

0.78 0.61

* 0.07 5

0.06

From Castren and Saavedra (1988). Kinetic paramaters were calculated from seven different Scatchard plots per group, measured individually in different rats. Significantly different from controls ( p < 0.05).

ATRIAL NATRIURETIC PEPTIDE AND ANGIOTENSIN RECEPTORS

28 1

IV. Discussion

Brain ANG I1 and ANP receptors are selectively localized. Some of the ANG I1 and ANP receptors are located outside the blood-brain barrier, in the circumventricular organs. These observations indicate that peripherally formed, circulating peptides could influence brain function by stimulation of their circumventricular organ receptors. The circumventricular organs contain ANG II-sensitive neurons and their lesion prevents the centrally mediated drinking response and blood pressure increase of peripherally injected peptide (Knowles and Phillips, 1980; Ueda et al., 1972; Nicolaidis et al., 1983; Sayer et al., 1984; Lind et al., 1983; Mangiapane and Simpson, 1980b; Johnson, 1985; Johnson et al., 1978). These structures contain large amounts of the angiotensinconverting enzyme and ANG I1 immunopositive fibers (Chevillard et al., 1984; Saavedra and Chevillard, 1982; Correa et al., 1985; Lind et al., 198513). Thus, both binding of circulating ANG I1 and local peptide formation could occur in circumventricular structures. The issue of the existence of an endogenous brain ANG I1 system, controversial for a decade, was finally settled with the demonstration of the presence in the brain of all components of the system (Saavedra et al., 1982; Phillips, 1987; Bennett and Snyder, 1976; Ganten et al., 1981). Our additional finding of ANG I1 receptors in specific brain areas inside the bloodbrain barrier (Mendelsohn et al., 1984; Saavedra et al., 1986e) supported this hypothesis. Circulating ANG I1 does not have access to these receptors, which should be stimulated by endogenous peptide of brain origin. It is tempting to speculate that the peripheral and central peptide systems could be somehow linked, and that the places for such an association are located in the circumventricular organs. We have found anatomical evidence that this is indeed the case for ANG 11. A continuous forebrain ANG I1 receptor band extends from the subfornical organ along the wall of the third ventricle, including the median preoptic nucleus (Shigematsu et al., 1986), to reach the OVLT and extends laterally to the paraventricular nucleus, another site of high ANG I1 receptor concentration (Saavedra et al., 1986e). Neurons within this band contain immunoreactive ANG I1 (Lind et al., 1985b), as well as high levels of angiotensin-converting enzyme (Saavedra and Chevillard, 1982a). These neurons respond to iontophoretically applied ANG I1 (Lind and Johnson, 1982) and increase their metabolic activity after peripheral administration of ANG I1 (Gross et al., 1985a). This indicates the presence of an integrated ANG I1 forebrain circuit, responding to both peripheral and central ANG 11.

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JUAN M. SAAVEDRA el al.

The brainstem may contain another important site for peripheral and central ANG I1 interactions, the area postrema-nucleus of the solitary tract complex. This area contains not only ANG I1 receptors, but also ANG II-positive fibers and ANG II-sensitive neurons (Lind et al., 1985b; Casto and Phillips, 1985; Mendelsohn et al., 1984; Saavedra et al., 1986a). T h e analysis of the brain ANG I1 receptor distribution indicated that the peptide was most probably involved in the central regulation of cardiovascular function and fluid metabolism. Stimulation of ANG I1 receptors in the subfornical organ and other areas of the forebrain ANG I1 receptor band resulted in increased blood pressure, drinking behavior, vasopressin secretion, and salt appetite (Phillips, 1987). The nucleus of the solitary tract integrates and modulates the baroreceptor reflex (Palkovits and Zaborszky, 1977). In addition, the localization of ANG I1 receptors indicated a possible multiple role in the regulation of anterior pituitary function. Peptide receptors were highly concentrated in the parvocellular zone of the paraventricular nucleus (Castren and Saavedra, 1988),the site of formation of the corticotropin releasing factor (Swanson and Sawchenko, 1980), and in the median eminence, where they could regulate releasing factor release. In the periphery, ANG 11 regulates the formation and release of aldosterone (Vallotton, 1987). It is thus possible that the central regulation of corticotropin releasing factor, and therefore adrenocorticotropic hormone (ACTH) (Spinedi and Negro-Vilar, 1983), could be integrated with the peripheral regulation of mineralocorticoid secretion. Another likely action of central ANG I1 receptors relates to the modulation of the central sympathetic system. Many sites of high ANG I1 receptor concentration coincide with brain areas rich in sympathetic terminals (para- and periventricular hypothalamic nucleus, area postrema) or are located in areas rich in cell bodies of sympathetic neurons (locus coeruleus, nucleus of the solitary tract). This postulated role of central ANG I1 has also its counterpart in the periphery. ANG I1 has been shown to stimulate norepinephrine release from sympathetic nerves (Zimmerman et al., 1972) and catecholamine release from adrenal medulla (Peach and Ackerly, 1976). In addition, very high concentrations of ANG I1 receptors (Israel et al., 1985c; Cajtren et al., 1987) exist both in the adrenal medula and in peripheral sympathetic ganglia. I t is quite possible that central and circulating ANG 11, in conjunction with ANG I1 locally formed in chromaffin cells and sympathetic ganglion cells, modulates sympathetic activity at multiple levels. T h e suprachiasmatic nucleus was another area containing very high concentrations of ANG I1 receptors. This suggested an unsuspected role

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283

for central ANG I1 in the regulation of circadian rhythms (Mosko and Moore, 1979). It is of interest to note that, in the rat, drinking behavior has a characteristic circadian rhythm (Stephan and Zucker, 1972). In addition to the areas mentioned above, high concentrations of ANG I1 receptors were located in olfactory structures and other parts of the limbic system. The possible role of central ANG I1 in limbic system function has not been explored. ANP receptors are also discretely distributed in rat brain areas (Quirion et al., 1984, 1986) including the circumventricular organs (Bianchi et al., 1985; Quirion et al., 1984, 1986; Kurihara et al., 1987), which may represent the central sites of action for the peripherally formed peptide. T h e central effects of ANP are antithetical to those of ANG 11. This suggests that in the brain, as in the periphery, the two peptide systems could act as physiological antagonists. This hypothesis is supported by the fact that ANG 11-stimulated drinking behavior in rats is inhibited by ANP (Masotto and Negro-War, 1985), and it has its neuroanatomical basis on the colocalization of ANG I1 and ANP receptors in all circumventricular organs and in the localization of the largest accumulation of ANP-irnmunoreactive cell bodies in forebrain areas overlapping with the forebrain ANG 11 receptor band (Jacobowitz et al., 1985; Saper et al., 1985). ANP receptors are also located in brain areas inside the blood-brain barrier, (Quirion et al., 1984, 1986; Kurihara et al., 1987; Saavedra, 1987). It is now well established that ANP, or closely related peptides, can be formed and released in the brain, concentrated in discrete neuronal pathways (Clembotski et al., 1985; Tanaka and Inagarni, 1986; Jacobowitz et al., 1985; Kawata et al., 1985; Saper et al., 1985; Skofitsch et al., 1985; Standaert et al., 1986), and selectively stimulate specific neurons (Wong et al., 1986). These observations indicate that there is an endogenous brain ANP system. Since many of the ANP receptors and neurons are located in areas related to cardiovascular and fluid regulation, brain ANP may be involved in the modulation of similar functions as brain ANG 11. The presence of ANP receptors in the choroid plexus (Lynch et al., 1986; von Schroeder et al., 1985; Quirion et al., 1984, 1986) is of special interest. T h e choroid plexus shows not only ANP binding but also a second messenger response to ANP, the stimulation of cyclic GMP formation (Steardo and Nathanson, 1987), and ANP administration can alter the rate of cerebrospinal fluid formation. This suggested a role for ANP in the control of fluid regulation in the central nervous system. The discrete localization of ANG I1 and ANP receptors within the brain prompted a series of studies designed to clarify the role of these

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JUAN M. SAAVEDRA

PL

d.

peptides in the regulation of cardiovasculai function, fluid metabolism, and pituitary control. Alterations in ANG I1 and ANP receptors occurred in a model of genetic hypertension, SHR rats (Stamler et al., 1980b; Saavedra, 1986a,b, 1988; Saavedra et al., 1986 a,c-f; Gutkind et al., 1988a). These changes were in opposite directions, ANG I1 receptors increasing in number in SHR animals, while ANP receptors were decreased in number. ANG I1 receptors were high in the circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the paraventricular nucleus of SHK rats. Conversely, ANP receptors were lower in the subfornical organ, nucleus of the solitary tract, and paraventricular nucleus of SHR animals. Increased ANG I1 receptor concentrations occurred in a model of experimental, mineralocorticoid-salt excess hypertension, DOCA-salt hypertensive rats (Gutkind et al., 1988b). Higher receptor numbers in DOCA-salt hypertensive rats were present in the subfornical organ, median preoptic nucleus, paraventricular nucleus, nucleus of the solitary tract, and area postrema. With the exception of the area postrema, the increase in ANG I1 receptors occurred in the same areas as that observed in SHR rats. T h e changes observed were more widespread and of a higher magnitude than those observed in rats treated with salt o r DOCA only, which did not develop hypertension. The alterations in the median preoptic nucleus, a part of the forebrain ANG I1 receptor band, are of special interest, since lesions of this area prevent the development of DOCA-salt hypertension (Brody and Johnson, 1980; Songu-Mize et al., 1982). Salt-treated rats showed increased ANG I1 receptor concentrations limited to the median preoptic nucleus and the subfornical organ. In DOCA-treated rats, increased ANG I1 binding was present not only in the median preoptic nucleus and subfornical organ, but also in the paraventricular nucleus. Alterations in brain stem ANG I1 receptors occurred only in DOCA-salt hypertensive rats. Conversely, alterations in ANP subfornical organ receptors occurred only in rats treated with excess salt administration, which remained normotensive. T h e combined treatment with salt and DOCA resulting in hypertension, however, prevented the increase in AN P receptor number in the subfornical organ. Thus, in two models of hypertension, genetic and mineralocorticoid -salt induced, receptors for a prohypertensive, fluid conservative peptide, ANG 11, were increased in key areas of the brain related to cardiovascular control and fluid regulation. Conversely, receptors for an antihypertensive, diuretic, and natriuretic peptide, ANP, were greatly

ATRIAL NATRIURETIC PEPTIDE AND ANGIOTENSIN RECEPTORS

285

decreased in similar areas of genetically hypertensive rats and did not increase after salt load in DOCA-salt hypertensive animals. Such imbalance in receptor numbers of mutually antagonistic peptides may be reflected as an increased central response to even normal concentrations of ANG I1 in the blood of SHR rats, a model presenting normal or even lower plasma renin concentrations (Shiono and Sokage, 1976). However, there are other evidences of alterations of the peripheral ANG I1 and ANP systems in SHR animals. Genetically hypertensive rats have low plasma and tissue angiotensin-converting enzyme activity (Niwa et al., 1985; Chevillard and Saavedra, 1983), and decreased cardiac content and increased blood ANP levels (Morii et al., 1986a; Sonnenberg et al., 1983; Gutkowska et al., 1986). Thus, our findings could be interpreted as suggesting an upregulation of central ANG I1 receptors and a downregulation of central ANP receptors, subsequent to primary alterations in peripheral peptide systems. However, there is increased brain ANP content in SHR rats (Morii et al., 1986b), and this could also be related to the alterations in brain ANP receptors. While the changes in ANG I1 and ANP receptors in genetically hypertensive rats are already present before the full expression of the hypertension, and they are probably not the result of chronic alterations in blood pressure, alterations in peptide receptors appear in chronically hypertensive DOCA-salt rats. This may indicate that the mechanisms for receptor changes could be different in each model. T h e physiological significance of the decreased brain ANP receptor number in SHR rats, however, is an open question. ANP receptors are also decreased in sympathetic ganglia from SHR rats (Gutkind et al., 1987) but in these tissues the second messenger, cyclic GMP increase after ANP, was unchanged. This indicated a discrepancy between the number of ANP receptors and the ANP-mediated biochemical response in SHR. T h e increased number of ANG I1 receptors may be well integrated into the well-documented complex system of increased central ANG I1 activity in SHR rats (Chevillard et al., 1984; Cole et al., 1978; Gutkind et al., 1988a; Schelling et al., 1982; Ganten et al., 1983; McDonald et al., 1980; Phillips et al., 1977; Weyhenmeyer and Phillips, 1982; Gehlert et al., 1986; Saavedra et al., 1987b). Similarly, despite suppression of the peripheral angiotensin system by chronic administration of mineralocorticoids, DOCA-salt hypertensive rats also show evidence of increased central ANG I1 system activity (Itaya et al., 1986; Weyhenmeyer and Meyer, 1985). Increased activity of the brain ANG I1 system in hypertension could explain the therapeutic effects of angiotensin converting enzyme inhibitors (Stamler et al., 1980a; Unger et al., 1987).

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Alterations in peptide receptors in specific brain areas could help to explain certain autonomic and endocrine abnormalities in hypertension. Changes in receptor concentrations in the subfornical organ and the paraventricular nucleus may be associated with alterations in vasopressin formation and release. Both SHR (Crofton et al., 1978) and DOCA-salt hypertensive rats (Chen et al., 1986; Crofton et al., 1979) show increased vasopressin release, normalized in DOCA-salt rats by inhibition of peripheral angiotensin converting enzyme (Igarashi et al., 1985). Circulating ANG I1 plays a role in the release of vasopressin, and ANG II-induced vasopressin release is enhanced by mineralocorticoid treatment (Wilson et al., 1986). Since the posterior pituitary is devoid of ANG I1 receptors (Israel et al., 1985c, 1986), it is most likely that this is a central effect. T h e first step in this chain of events is probably the stimulation of ANG I1 receptors in the subfornical organ, followed by stimulation of the forebrain ANG I1 band and stimulation of the posterior pituitary, with the end result of increased vasopressin release. However, the exact anatomy and biochemistry of this pathway is still an open question. T h e significance of the changes in ANG I1 receptors in the paraventricular nucleus may be related to those in the subfornical organ. When administered by iontophoresis in the paraventricular nucleus, ANG I1 stimulates neurosecretory cells (Akaishi et aE., 1981). Lesions of the paraventricular nucleus decrease the hypertension produced by subfornical organ stimulation (Ferguson and Kenaud, 1984). Endogenous ANG I I is colocalized with vasopressin in paraventricular magnocellular neurons (Killcoyne et al., 1980). However most, if not all, ANG I1 receptors are located in the parvocellular zone, site of formation of the corticotropin releasing factor (Castren and Saavedra, 1988). Thus, the observed increase in ANG 11 receptors in the parvocellular paraventricular nucleus may be related to other endocrine alterations in SHR animals, namely, increased ACTH release (Okanioto, 1972). We have also found increased numbers of ANG 11 receptors in the paraventricular nucleus of DOCA-salt treated rats. Since these receptors are high during administration of DOCA alone, it is conceivable that part of this increase could be related to a local effect of mineralocorticoids in this nucleus. Increased number of ANG I1 receptors in the median preoptic nucleus of SHR rats can be explained if we consider the stimulation of the complete forebrain ANG I1 receptor band, of which this nucleus is a part, as necessary for increased vasopressin release and blood pressure in SHR rats. The median preoptic nucleus may play an important role in the integration of fluid and cardiovascular regulation, as shown by the

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deficits in drinking and vasopressin secretion that follow its lesion (Mangiapane et al., 1983). The situation may be different, however, in DOCA-salt rats, since the increase in ANG I1 receptors in this nucleus occurs to the same extent as in DOCA-treated o r salt-treated normotensive animals. Thus the changes may be related to alterations in fluid homeostasis and to effects secondary to mineralocorticoid administration in this model. In the nucleus of the solitary tract, ANG I1 receptors were increased in both SHR and DOCA-salt rats. Both models have impaired baroreflex function and that of DOCA-salt rats returns to normal after treatment of the hypertension with angiotensin converting enzyme inhibitors (Matsuguchi and Schmid, 1982). T h e evidence for a role of this structure in blood pressure regulation is compelling (Palkovits and Zaborszky, 1977), and the area may represent still another site for a central interaction between ANG I1 and ANP systems, since ANP receptors are decreased in the nucleus of the solitary tract and area postrema of SHR. As to the pathophysiological significance of the alterations described in this area, they may be related to the increased peripheral sympathetic drive in SHR (Grobecker et al., 1975, 1982). Whether the changes in peptidergic systems are related to those observed in catecholamines in this nucleus, and especially those in epinephrine neurons (Saavedra et al., 1978), is an open question. In the area postrema, however, the number of ANP receptors in SHR rats is low, but ANG I1 receptor concentration is normal. These findings are consistent with the proposed lack of a significant role of the ANG I1 system in rats’ area postrema in blood pressure control (Haywood et al., 1980). However, the area postrema has been implicated in the hypertensive action of chronic ANG I1 administration (Fink et al., 1987; Joy, 1971) and ANG I1 receptors are high in the area postrema of DOCA-salt hypertensive rats. Still another area of interest is the choroid plexus. This structure is devoid of ANG I1 receptors but contains high numbers of ANP receptors. The number of ANP receptors was low in the choroid plexus of SHR rats and did not increase in DOCA-salt animals even though their numbers were higher after administration of salt alone. In addition to increased number of ANG I1 receptors in the subfornical organ of salt-treated rats, a model of extracellular dehydration, similar changes in ANG I1 receptors are present in another model of dehydration, the acutely dehydrated, water-deprived rat. In dehydration due to water deprivation, vasopressin formation and release is significantly altered. Water-deprived rats increase their vasopressin

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release to increase water conservation by the kidneys and show signs of a great increase in the metabolic activity of the subfornical organposterior pituitary axis (Gross et al., 1985b). The peripheral ANG I1 system is greatly stimulated (Mann et al., 1980). The increase in ANG I1 receptors (Hwang et al., 1986; Mendelsohn et al., 1983) is probably the result of upregulation triggered by incresed circulating ANG I1 concentrations (Mann et al., 1980) and may represent an amplification mechanism to ensure the central biochemical and behavioral responses necessary for the maintenance of homeostasis. Of all circumventricular structures, the subfornical organ may play the most important role in the central actions of circulating ANG I1 during dehydration, induction of thirst and vasopressin release (Gross, 1985; Phillips, 1987; Simpson, 1981; Miselis, 1981; Mangiapane et al., 1984). The rest of the forebrain ANG I1 receptor band, however, may play a role during dehydration, since increased ANG I1 receptors were found in the median preoptic nucleus and OVLT of water-deprived rats, although the changes did not attain statistical significance (Nazarali et al., 1987). Alteration in subfornical organ peptide receptors are not limited to ANG 11; both water deprivation of normal rats and analysis of homozygous Brattleboro rats revealed increased ANP receptors in this structure (Saavedra et al., 1986b, 1987a). Decreased extracellular volume during acute or chronic dehydration should result in decreased release and plasma ANP levels as indicated by increased heart ANP concentrations, probably the result of reduced release; increased subfornical organ ANP receptors could thus be interpreted as part of a compensatory increase or upregulation secondary to changes in concentrations of the peripheral peptide. ANP receptor changes in the subfornical organ can also be related to the changes in ANG I1 receptor concentrations in this structure. ANP inhibits both spontaneous and ANG II-stimulated water intake, probably through its subfornical organ receptors (Masotto and Negro-Vilar, 1985; Antunes-Rodrigues et al., 1985). In any case, the study of dehydration models reveals another case for possible peripheral and/or central interactions between the ANG I1 and ANP systems. Moreover, administration of vasopressin reverses the alterations in angiotensin converting enzyme activity present in hypothalamic nuclei of Brattleboro rats (Saavedra and Chevillard, 1982b). It is of interest to note that, in turn, dehydration results in profound alterations in brain content of vasopressin and ANP (Samson, 1985b; Januszewicz et al., 1986a,b). This indicates the possibility of still another set of interactions between the peripheral and central peptide systems.

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Large increases in ANP receptor number were found in the choroid plexus of dehydrated rats, suggesting a role for this peptide in the control of ion fluxes across the brain-cerebrospinal fluid barrier. Other brain areas containing high ANG I1 o r ANP receptor concentrations did not show alterations in their number after dehydration. Of particular interrest is the lack of changes in the paraventricular nucleus, which again indicates that peptide receptors in this area are more probably related to regulation of anterior pituitary function than to vasopressin release. Our investigations on ANG I1 receptors during stress further suggests this hypothesis. During repeated immobilization stress, the number of ANG I1 receptors in the paraventricular nucleus, and those in the subfornical organ, were greatly increased. This kind of stress increases plasma renin activity (Jindra et al., 1980),ACTH release, and peripheral sympathetic activity (Kvetnansky et al., 1970). In addition to its role in the regulation of ACTH release, the paraventricular nucleus is involved in the control of peripheral sympathetic activity (Swanson and Sawchenko, 1983). If plasma ANG I1 concentrations, as indicated by the increased plasma renin activity, are increased during stress, our results may indicate the presence of an enhanced central effect of circulating ANG 11, probably through sensitization of neuronal connections between the subfornical organ and the paraventricular nucleus. Whether such a mechanism involves the participation of the forebrain ANG I1 receptor band has not yet been determined (Murakami and Ganong, 1987). It is also possible that corticoids, highly elevated in blood during stress, could play a more direct role in the regulation of ANG I1 receptors at the subfornical-paraventricular nucleus axis. Steroid hormones may alter ANG I1 receptor properties in peripheral organs (Douglas, 1987). ANG II-like immunoreactivity is increased in the parvocellular paraventricular nucleus after adrenalectomy, and in this model ANG I1 has been shown to coexist with corticotropin releasing factor and vasopressin in parvocellular neurons (Lind et al., 1984). These data, together with the described alterations in ANG I1 receptors in the paraventricular nucleus of DOCA-treated rats, may indicate an influence of steroid hormones in the local regulatory mechanisms for ANG I1 receptors in paraventricular parvocellular neurons. If circulating ANG I1 is involved, directly or indirectly, in the increased ACTH secretion during stress, this is probably through central, rather than pituitary, mechanisms, since the ANG I1 receptors in anterior pituitary are not different in number in stressed animals (Castren and Saavedra, 1988).

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V. Conclusions

1. Both ANG 11 and ANP receptors are located in selected brain areas, some accessible, like the circumventricular organs, and some not accessible to circulating peptides. The circumventricular organs are connected to some other brain areas that belong to the endogenous brain ANG I1 and ANP systems. The ANG I1 and ANP receptor distributions overlap in many areas related to cardiovascular and fluid regulation as well as to the central control of sympathetic activity and pituitary function. 2. Changes in peripheral peptide systems influence the brain directly through their circumventricular organ receptors and influence the central peptide systems as well. Brain receptor numbers can be modulated by alterations in peripheral peptide metabolism. 3. Specific alterations in brain peptide receptor number occur in several animal models of disease. The imbalance between brain ANG I1 and ANP receptors in hypertension, with a relative increase in receptors for ANG 11, should favor central prohypertensive mechanisms and may have therapeutic implications. If similar alterations occur in human essential hypertension, that may explain the increased sympathetic drive in cases without increased plasma renin activity and the central component of the therapeutic effect of angiotensin converting enzyme inhibitors. 4. During dehydration, the brain upregulates its circumventricular ANG I1 receptors in the presence of increased peripheral ANG I1 concentrations. This is probably a mechanism to secure the necessary biochemical and behavioral responses to the loss of body fluids, of fundamental importance to survival. 5 . The alterations in brain ANG I1 receptors during stress provide the first concrete evidence for a role of the peptide in the central control of the pituitary-adrenal axis. This may be part of still another feedback mechanism, since adrenal cortical hormones and the peripheral ANG I1 system mutually influence each other. 6. We are impressed by the association, in all models studied, of alterations in peripheral and central peptide systems, and by the frequent association between changes in ANG I1 and ANP systems, both peripherally and centrally. The most logical explanation is that of a close balance and feedback mechanism between peripheral and central ANG I1 and ANP systems, a balance of importance for hormonal and sympathetic regulation and crucial for maintenance of homeostasis.

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SCHIZOPHRENIA, AFFECTIVE PSYCHOSES, AND OTHER DISORDERS TREATED WITH NEUROLEPTIC DRUGS: THE ENIGMA OF TARDIVE DYSKINESIA, ITS NEUROBIOLOGICAL DETERMINANTS, AND THE CONFLICT OF PARADIGMS By John L. Waddington Deportment of Clinical Pharmacology Roy01 College of Surgeons in Ireland

Dublin 2, Ireland

1. Introduction

11.

111.

IV. V. VI.

VII.

A. Historical Perspective B. Medicolegal Issues C. Methodological Issues Prevalence A. General Issues B. Prevalence of Involuntary Movements in Subjects without Medical or Neuropsychiatric Disorders C. Prevalence of Involuntary Movements in Patients with Medical but Not Neuropsychiatric Disorders D. Prevalence of Involuntary Movements in Patients with Neuropsychiatric Disorders Never Treated with Neuroleptics E. Prevalence of Involuntary Movements in Neuropsychiatric Patients Who Have and Have Not Been Treated with Neuroleptics Incidence Natural History Morbidity and Mortality Vulnerability Factors A. Age B. Sex C. Duration and Intensity of Past Exposure to Neuroleptics D. Diagnostic Composition and Extent of Current Exposure to Neuroleptics E. Cognitive Dysfunction F. Specific Aspects of Psychopathology and Clinical Course G. Neurological Features H. Structural Brain Pathology I. Age at Onset of Illness J. History of Typical Extrapyramidal Side Effects K. Dental Status L. Smoking Habits M. Familial-Genetic Factors N. Interim Summary Pathophysiological Mechanisms A. Topography of Involuntary Movements B. The Dopamine Receptor Supersensitivity Hypothesis

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Copyright D 1989 by Academic Press, lnc. All rights of reproduction in any form reserved.

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C. Nondopaminergic Neurochemical Hypotheses D. An Alternative Perspective VIII. Synthesis: The Conflict of Paradigms Keferences

1. Introduction

A. HISTOKICAL PERSPECTIVE By convention, the term tardive dyskinesia refers to a syndrome of abnormal, involuntary, choreoathetoid movements that emerges as a late-onset, adverse effect of long-term treatment with neuroleptic drugs. This syndrome is seen only in a proportion of patients so treated, and when present can affect the orofacial, limb, and trunk regions of the body and the respiratory musculature; classical buccal-lingualmasticatory dyskinesia is perhaps the most widely considered manifestation. The initial descriptions are usually credited to Schonecker (1957) and Sigwald et al. (1959). Over the subsequent decades, a voluminous literature on the phenomenon has evolved, the first 20 years of which have been exhaustively reviewed (Kane and Smith, 1982; Jeste and Wyatt, 1982). T h e aim of this review is not to update these earlier authoritative works, but rather to concentrate on a number of quite fundamental questions concerning the syndrome that remain to be clarified (Waddington, 1987) by focusing on the more recent studies. Over the past decade, a number of investigators have extended the concept of tardive dyskinesia to include phenomena such as tardive dystonia, tardive Tourette syndrome, and tardive akdthisia (Stahl, 1986). Such putative and less extensively researched variants will not be considered here. In stating the above aim, one problem is immediately apparent. Why, after more than 30 years of study, are fundamental issues concerning the syndrome yet to be resolved? The answer lies in an inability to apply conventional scientific methods to our clinical studies of the problem and in the absence of any homologous animal model. Tardive dyskinesia is one of the few topics of scientific enquiry concerning which studies are entertained when they fail to include features that would be deemed mandatory in virtually all other areas of neuroscience research (Waddington, 1988a). It is the contention of this reviewer that we will only advance our fundamental understanding of this potentially debilitating syndrome when we face these dilemmas. In so doing, our views on its neurobiological substrate(s) may require radical modification.

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B. MEDICOLEGAL ISSUES Though the topic of tardive dyskinesia is most commonly considered in a clinical context, it has become a matter of litigation in some countries. These medicolegal issues have been reviewed from the medical perspective by Davis et al. (1983) and Gualtieri and Sprague (1984), and from the legal perspective by Taub (1986). It is not the purpose of this review to comment on relevant issues of professional competence, such as judicious versus injudicious use of neuroleptics by clinicians. However, consideration of the nature of the putative causal relationship between neuroleptic exposure and presence of involuntary movement disorder, and the extent to which we can specify that relationship in an individual case, is an integral component of the scientific analysis offered below.

C. METHODOLOGICAL ISSUES As emphasized by Kane and Smith (1982), there are no established or universally accepted criteria either for defining a case of tardive dyskinesia or for specifying its severity, and a number of neurological o r neuromedical disorders may involve movement disorder(s) with a clinical picture that is similar. Methodological problems permeate every aspect of the syndrome; though they are considered within this section, they will have general applicability.

1. Differential Diagnosis Clearly, to investigate the syndrome of tardive dyskinesia, one must exclude patients with extrapyramidal disorders, such as Huntington’s disease, which are known to involve involuntary, choreoathetoid movements as an inherent feature of the illness independent of any possible exposure to neuroleptics; such issues have been reviewed by Granacher (1981) and Casey (1981).Other iatrogenic dyskinesias involving involuntary choreoathetoid movements, such as L-DOPA dyskinesia in some patients with Parkinson’s disease, must also be excluded. T h e total exclusion, rather than the independent consideration, of those cases of involuntary movements occuring in association with other organic brain disease(s) is conceptually more problematic; it is argued in later sections that such instances of spontaneous dyskinesia have much to reveal about tardive dyskinesia. A remaining problem is whether to exclude other neurolepticassociated movement disorders that are not choreoathetoid in nature; these may be conceived as variants of tardive dyskinesia in its broadest

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sense o r may represent alternative, phenomenologically and pathophysiologically distinct, syndromes of iatrogenic movement disorder (Munetz and Cornes, 1983; Stahl, 1986; Gardos et al., 1987). Perhaps until these issues are clarified, they should be seen as potential confounds in studies of classical tardive dyskinesia (see Section I,A) and should be separately considered. 2. Clinical Measurement Two studies (Munetz and Schulz, 1986; Weiden et al., 1987) have confirmed the prevailing view that many cases of tardive dyskinesia, especially those of mild severity, may go unrecognized in the absence of some form of systematic examination. The most widely used standardized examination procedures include the Abnormal Involuntary Movement Scale (AIMS) (Guy, 1976) and the Simpson-Rockland Scale (Simpson et al., 1979); factors influencing the reliability and repeatability of such assessments, including experience of the rater and use of video technology, have been widely investigated (Barnes and Trauer, 1982; Lane et al., 1985; Firth and Ardern, 1985; Bergen et al., 1988). Alternative approaches include determining frequency counts for specific movement patterns, which may particularly emphasize problems of intrapatient variability (Richardson et al., 1982). When using a standardized rating scale such as the AIMS, the presence or absence of abnormality can be dichotomized in a variety of ways, such as by the presence of at least mild but definitely abnormal involuntary movements of one or more body regions, by a global rating indicating at least mild involuntary movements, or by the application of Research Diagnoses for Tardive Dyskinesia (Schooler and Kane, 1982). Therefore, even when standardized rating scales are used, the adoption of different criteria to define an instance of tardive dyskinesia clearly contributes to variations between studies. At the very least, it is important that each investigation specifies its criterion of abnormality. Critical methodological issues relating to clinical measurement have been reviewed by Owens (1986). 3. Automated Measurement In light of the above, it is no surprise that investigators have sought electronic or electromechanical devices for more objective, automated evaluation of tardive dyskinesia. Various studies have utilized the following technologies: ultrasound, with the transducer directed from spectacles or a headband onto the face, for the detection of buccallingual-masticatory dyskinesia (Resek et al., 1981; May et al., 1983; McClelland et al., 1987); Doppler radar directed over the whole body

NEUROLEPTICS AND TARDIVE DYSKINESIA

30 1

(Buruma et al., 1982); electromyographic recordings of multiple regions (Bathien et al., 1984); and accelerometric recordings (Tryon and Pologe, 1987) for detection of limb movements. T h e ultrasound and Doppler radar techniques have shown acceptable degrees of concordance with visual assessments, and electromyographic profiles of typical involuntary movements can be distinguished from those of tremor. However, these technologies have not achieved a wide usage in the investigation of tardive dyskinesia and should perhaps be regarded as promising experimental procedures. It should be emphasized that both standardized rating scales and such automated techniques can only assess involuntary movements; they are not diagnostic instruments and neither is able, in itself, to distinguish the involuntary movements of tardive dyskinesia from those of, say, Huntington’s disease.

II. Prevalence

A. GENERAL ISSUES In their authoritative review of 20 years of studies, Kane and Smith (1982) summarized 56 reports on the prevalence of tardive dyskinesia in neuroleptic-treated populations totaling some 34,555 patients; the mean prevalence was 20.0%,but the range encountered (0.5-56.4%) indicated a highly inhomogeneous group of studies. Such marked heterogeneity between studies is commonly held to reflect differences in how an individual case of tardive dyskinesia is defined in treatment practice and in the demographic and diagnostic composition of the patient populations. This latter variable is of particular importance as it has implications for, inter alia, the critical issue of what baseline level of involuntary movements would be encountered in comparable populations of untreated patients; in the absence of this information, we cannot be sure of the extent to which such a baseline of spontaneous dyskinesia might contaminate and inflate the “true” neuroleptic-associated component of the overall level of involuntary movements. At a theoretical level, a conventional approach to clarifying the extent of such a drug effect would involve the long-term evaluation of a large, homogeneous group of patients receiving a given neuroleptic in a specified manner and its comparison with a group of otherwise indistinguishable patients who remain unmedicated. I n this point lies the root of much of our relative ignorance concerning the syndrome of tardive

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JOHN L. WADDINGTON

dyskinesia and its neurobiological substrate; the great majority of patients receiving neuroleptic drugs do so for serious psychotic disorders, most commonly schizophrenia, making it usually unethical to withold such medication from patients so as to derive the appropriate control group. Clearly, determination of the prevalence of indistinguishable involuntary movements in normal subjects never exposed to neuroleptics is an important first step to confronting this impasse. However, it is not a valid control group for patient populations; critically, it fails to control for the factor of the disease process, presumably itself involving cerebral dysfunction, that resulted in their requirement for treatment. A second approach is to study involuntary movements in patients with neuropsychiatric disorders for which neuroleptics are not invariably prescribed; for example, one might compare the prevalence of involuntary movements in groups of patients with diagnoses such as Alzheimer’s disease or bipolar affective disorder who have and have not received long-term neuroleptic therapy. However, a second critical problem is apparent. I f we presume that neuroleptics are given to patients for specific clinical reasons, it must follow that a patient with a given diagnosis who is deemed to require neuroleptics is different in some way from one with the same diagnosis who is not deemed to require such treatment; again, the approach does not control for the influence of some element of the relevant disease process. A third approach is to study involuntary movements in individuals without psychotic or neurological disorder but who have been prescribed neuroleptics or pharmacologically related agents on a long-term basis for other reasons; such putative indications have in thF past included anxiety and gastrointestinal or labyrinthine disorders. However, the number of patients who have received neuroleptics for such reasons is extremely small; also, they are rarely prescribed continuously for periods of time or at doses comparable with neuroleptic regimens adopted for more typical indications. Critically, the limited conclusions that might be drawn from the few studies of this type (Klawans et al., 1974; Wiholm et al., 1984) would once again fail to control for the disease process(es) inherent to those major psychiatric disorders for which neuroleptics are much more commonly prescribed. This is not simply a matter of seeking to specify the prevalence of tardive dyskinesia with greater mathematical precision. As will be argued below, lack of attention to these issues may have led us astray as to how we should view this syndrome and made more difficult the search for its neurobiological determinants.

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B. PREVALENCE OF INVOLUNTARY MOVEMENTS I N SUBJECTS WITHOUT MEDICALOR NEUROPSYCHIATRIC DISORDERS There are many studies that have investigated the prevalence of involuntary movements in populations never exposed to neuroleptic drugs (Kane and Smith, 1982; Casey, 1984). However, to develop the thesis to be offered in this review, the more recent and/or those with the most detailed descriptions of the populations studied have been subdivided along specific lines. In this first group, information has been collated to address the issue of the baseline prevalence of involuntary movements in healthy, normal individuals, that is, those without significant medical disorder(s) in addition to the absence both of a neuropsychiatric history and of exposure to neuroleptics. They focus on the elderly because of robust evidence that the prevalence of tardive dyskinesia in neuroleptic-treated populations increases reliably with advancing age (Smith and Baldessarini, 1980; Kane and Smith, 1982). Table 1 lists five studies on the prevalence of involuntary movements in such elderly populations. They were selected for their specific references to subjects being “in good physical and mental health” (Villeneuve et al., 1974), “healthy” and without either a neurological or a significant psychiatric history (Kane et al., 1982a), or “well-functioning and essentially without serious medical or psychiatric disease” (Lieberman et al., 1984). Though some unspecified degree of cognitive deficit appears to have been tolerated by Koller (1982), his population was specifically stated to be free from neurological or serious medical disease. That of D’Alessandro et al. (1986) involved a large, unselected population of the community elderly. The prevalence of involuntary movements was very low, and three of the six instances of spontaneous TABLE 1 PREVALENCE OF INVOLUNTARY MOVEMENTS I N SUBJECTS WITHOUT MEDICAL OR NEUROPSYCHIATRIC DISORDERS Prevalence‘ Study Villeneuve et al. (1974) Kane et al. (1982a) Koller (1982) Lieberman et al. (1984) D’Alessandro et al. (1986)

Age” 73-93 73 f 7 69 ? 60-99 67-87

*

N

Status

(%I

32 127 75 400 398

Physically and mentally well Healthy community elderly No medical or neurological disease Normal community elderly Unselected community elderly

O.Ob

Age range or mean t SD. Buccal-lingual-masticatory d yskinesia specified. Weighted prevalence over 1032 subjects: 1.6% (unweighted: 1.3 t 1.6%).

3.9 O.Ob

1.2 1.5b

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JOHN I.. WADDINGTON

dyskinesia were noted to occur in association with primary neurodegenerative disease; thus, the very low prevalence reported would, if anything, overestimate that relating to the healthy elderly and was therefore considered to provide relevant information. It can be seen that involuntary movements indistinguishable from those of tardive dyskinesia are exceptionally rare events in the normal elderly, free of medical or neuropsychiatric disorder(s) and without exposure to neuroleptics, even up to the tenth decade of life.

C. PREVALENCE OF INVOLUNTARY MOVEMENTS IN PATIENTS WITH MEDICALBUT NOTNEUROPSYCHIATRIC DISORDERS Listed in Table I1 are studies of the prevalence of involuntary movements in elderly populations without neuropsychiatric disorder or exposure to neuroleptics, but including subjects with various illnesses and a history of exposure to appropriate nonpsychotropic medications. For a number of these studies, particular illnesses were mentioned (Delwaide and Desseilles, 1977; Klawans and Barr, 1982; Campbell et al., 1983). In others, they were implied by the inclusion of nonpsychiatric nursing home patients with medication histories (Varga et al., 1982) or of otherwise unspecified surgical cases (Barnes el al., 19832).'lhe studies

TABLE 11 MOVEMENTSIN PATIENTSWITH MEDICALRUT N o r NEUROPSYCHIATRIC DISORDERS

PREVALENCE OF INVOLUNTARY

Prevalence' Study

Age"

Delwaide and Desseilles (1977) Varga et nl. (1982) Klawans and Barr (1982)

Koller (1982) Barnes et al. (1983a) Campbell et al. (1983)

Age range or mean 2

60-99 >65 50-59 60-69 70-79 62 2 ? 17-49 50-8 1 <30->80

N

Status

(%I

55 365 238 233 190 75 56 29 186 157

Medical disorders included Nursing homes included Medical referals Medical referals Medical referals All edentulous Surgical cases Surgical cases Rheumatology cases included Obstructive pulmonary disease

29.1' >10.3'

SD.

' Buccal-lingual-masticatory

dyskinesia specified. Weighted prevalence over 1584 subjects: 8.9%(unweighted: 13.1 t 8.2%).

0.8h 6.0' 7.8' 16.0' 3.6' 20.7' >6.8

>15.1

NEUROLEPTICS AND TARDIVE DYSKINESIA

305

of Varga and colleagues (1982) and of Campbell and colleagues (1983) allowed determination only of the minimum prevalence estimates given, which have been used in relevant calculations. It is apparent that the inclusion of medical disorders in such elderly “control” populations is associated with an increased prevalence of involuntary movements similar to those of tardive dyskinesia, even in the absence of neuropsychiatric disorders and of exposure to neuroleptics. If we assume that involuntary movements do not have a major peripheral origin, then these illnesses or the medications used to treat them must sometimes result in a change (or changes) in central nervous system function that is reflected in the emergence of such movement disorder. Many of the illnesses included in these studies are far from uncommon and may be particularly common in long-term neuropsychiatric populations. D. PREVALENCE OF INVOLUNTARY MOVEMENTSIN PATIENTS WITH NEUROPSYCHIATRIC DISORDERS NEVERTREATED WITH NEUROLEPTICS Having established that involuntary movements are extremely rare occurences in normal healthy individuals, including the elderly, it appears that they are somewhat more likely to occur in association with general medical conditions (and/or resultant nonpsychotropic medications) that might have some secondary effect on cerebral function. This makes it important to clarify the relative extent to which various untreated disorders that directly impair cerebral function might themselves be associated with involuntary movements; this is imperative in relation to those neuropsychiatric disorders for which neuroleptics are commonly prescribed on a long-term basis. Listed in Table I11 is the prevalence of involuntary movements in patients with various neuropsychiatric disorders but never exposed to neuroleptics; for a number of these studies, the populations appeared to be of some diagnostic homogeneity, while for others a variety of neuropsychiatric disorders were referred to, as indicated. In the study of Bourgeois et al. (1980), no diagnostic composition is specified for this retirement home population, but points of discussion clearly imply the inclusion of an appreciable number of cases of senile dementia. Blowers (198 1) also does not specify the diagnostic composition of this nursing home o r day care population, but personal conversation with the author confirmed that senile dementia and other neurological and psychiatric histories were included.

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J O l l N L. WADDINGTON

TABLE 111 NEUROPSYCHIATKIC DISORDERS NEVERTREATED WITH NEUROLEPTICS ~~

Prevalence' Study

Age"

Brandon et al. (197 1)

Villeneuve et al. (1974) Delwaide and Desseilles (1977) Bourgeois et ul. (1980) Blowet-s (1981)

30-50 51-70 >70 62-96 60-99 78 k ? 59-102

Maninelli and Gabellini, (1982) 8-84 Owens et 41.(1982) 29-90 Molsa et al. ( 1 982) 45-85 Molsa et al. (1984) 75 t 12 Lieberman et al. (1984) 59-99 2 1-40 Stone et al. (1988) 41-60 >60 Nods ct al. (1988) ?

" Age range or mean

N

Status

(%)

26 124 135 16 185

Psychoses and organic disorders Psychoses and organic disorders Psychoses and organic disorders Chronic schizophrenia Senile dementia Senile dementia included Neurological and psychiatric disorders included Essential tremor Chronic schizophrenia Arteriosclerotic dementia Alzheimer's disease Organic disorders included Marked mental handicap Marked mental handicap Marked mental handicap Metabolic encephalopathy

11.5' 17.7' 23.0h 3 1.2b 38.gh 18.0' 24.3b

21 1 378 104 47 91 143 29 1 367 66 41 127

7.76 53.2' 12.1b 16.8' 4.8 38.1 51.5 58.5 6.3

2 SD.

' Buccal-lingual-masticatory

dyskinesk specified. Weighted prevalence over 2372 subjects: 24.9% (unweighted: 23.4

?

16.2%).

It is evident that several serious neuropsychiatric disorders are associated with the emergence of appreciable numbers of cases of involuntary movements similar to those of tardive dyskinesia in the absence of treatment with neuroleptic drugs. This appears less so for acute conditions and those without major psychological impairments, such as metabolic encephalopathy or essential tremor. However, it was particularly so in relation to major psychosis and the atrophic or neurodevelopmental disorders such as senile dementia of Alzheimer type or marked mental handicap. In support of the results of' Stone et al. (1988), we have also noted a similar prevalence of orofacial dyskinesia in a much smaller sample of the adult mentally handicapped who had not been exposed to neuroleptics (Youssef and Waddington, 1988a). Such findings are of particular importance when they concern populations with chronic schizophrenia, the disorder for which neuroleptics are now almost universally prescribed. The study of Villeneuve et al. (1974) involved a small population of 16 elderly, female chronic schizophrenic patients who had never received neuroleptics or other biological treatment; the reason for their lack of treatment was not

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stated. Five of these (31%) showed typical orofacial dyskinesia. More recently, Owens et al. (1982) were able to study a larger group of 47 predominantly male, schizophrenic inpatients who had remained free of neuroleptics because of the therapeutic community orientation of the consultant in charge of their care. Twenty-five of these (53%) were found to have at least mild involuntary movements of one or more body regions, with the orofacial area being involved in virtually all instances. We ourselves have found at least mild involuntary movements of one or more orofacial areas in three of four elderly schizophrenic inpatients who were never exposed to neuroleptics because of a lack of florid psychotic symptoms in old age (Waddington and Youssef, 1989a). T h e significance of such studies is predicated on the reliability of the purported absence of any history of treatment with neuroleptic drugs, and it is always advisable to be skeptical of categorical assurances in this regard; the provocative but controversial reports from the preneuroleptic era have been reviewed (Waddington and Crow, 1988). Also, a variety of methodological procedures and criteria of abnormality have been employed, and in some instances they are not described at all. The issue of severity of involuntary movements is rarely mentioned. Despite these caveats, the extent of the data in Table I11 leads to the conclusion that involuntary movements, particularly those of the orofacial region, which typify tardive dyskinesia, can be seen to an appreciable extent in patient populations with major psychosis or neurodegenerative and neurodevelopmental disorders that have not been exposed to neuroleptics. This must be taken into account in any analysis of tardive dyskinesia, and in the opinion of this reviewer must be an integral part of any future hypothesizing in this area.

E. PREVALENCE OF INVOLUNTARY MOVEMENTS I N NEUROPSYCHIATRIC PATIENTS WHO HAVEAND HAVENOT BEENTREATED WITH NEUROLEPTICS Reviewing more than two decades of literature on tardive dyskinesia in patient populations who have received long-term treatment with neuroleptics (Kane and Smith, 1982) indicates that this movement disorder can emerge in all diagnostic groups with a mean prevalence of 20.0% (25.7% for years 1976-1980) (Jeste and Wyatt, 1981). Systematic studies confirm that tardive dyskinesia is evident in carefully diagnosed, homogeneous neuroleptic-treated populations with schizophrenia (Owens et al., 1982; Waddington and Youssef, 1985), bipolar affective disorder (Mukherjee et al., 1986; Waddington and Youssef, 1988a),

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Alzheimer's disease (Molsa et al., 1987), mental handicap (Gualtieri et al., 1986; Richardson et al., 1986; Youssef and Waddington, 1988a), and childhood autism (Perry et aZ., 1985). Inspection of Tables I1 and 111, relating to untreated populations, clearly suggests that overall estimates of the prevalence of tardive dyskinesia in treated populations are inflated by an unknown but not insignificant number of cases of involuntary movements with origins other than in long-term exposure to neuroleptics. We can only begin to estimate these proportions from studies in which the same investigators have applied the same assessment procedures to populations that have and have not been treated with neuroleptic drugs. Such studies are listed in Table IV, and they present a rather inconsistent picture; some appear to indicate a substantial effect of neuroleptic medication, whereas others appear to indicate little or no such effect. However, it should be emphasized that these are not studies

TABLE IV PREVALENCE OF INVOLUNTARYMOVEMENTS I N NEUROPSYCHIATRIC PATIENTSWHO HAVE AND HAVENOT BEENTREATED WITH NEUROLEPTICS

Disorder and study Mixed neuropsychiatric disorders Brandon et al. (1971)

Bourgeois et al. (1980) Blowers (1981) Lieberman et al. (1984) Schizophrenia Villeneuve et al. (1974) Owens el al. (1982)

Age"

N

u ntredted Prevalence (%)

3 1-50 51-70 >70 78 % ? 59- 102 59-99

26 124 135 21 1 378 29 1

11.5 17.7 23.0 18.0 24.3 4.8

202 256 59 122 79

6.9' 31.2' 37.1' 42.4' 39.3b 16.5

16 47

31.2 53.2

51 364

31.4' 67.0'

62-96

21-91

N

167

Treated Prcvalcncc (%)

Alzheimer's disease Molsa et al. (1984, 1987)

75 f 12

143

16.8

34

52.9b

Mental handicap Stone et al. (1988)

2 1-40

310 66 41

45.2 51.5 58.5

367 113 40

43.6 58.4 67.5

4 1 -60 >60

Age range or mean f SD. Buccal-lingual-masticatory d yskinesia specified.

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of matched groups of patients randomly allocated to long-term neuroleptic treatment or to no such treatment; each is a retrospective analysis of those who have and have not been so medicated. They cannot exclude an influence of one o r more of a large number of uncontrolled factors. T h e study of Brandon et al. (197 1) typifies these problems. Initial and subsequent (Casey, 1984) analyses of this large and important database indicate only modest differences in the prevalence of involuntary movements between neuroleptic-treated and untreated patients. However, these two groups of movement-disordered patients were not matched for diagnosis, 57% of the latter having an organic disorder while the majority of the former had a psychotic disorder; an overrepresentation of organic brain disease in the untreated group may have inflated the prevalence of apparently non-drug-related involuntary movements in these patients. In the studies of Bourgeois et al. (1980), Blowers (1981), and Stone et al. (1988), few details on specific diagnostic composition were given, and the fundamental question must be, Why had some patients, but not others, received long-term treatment with neuroleptics? Features of the relevant illness that are associated with a requirement for such treatment may also be associated with differing vulnerabilities to the emergence of spontaneous o r of drug-induced involuntary movements. T h e untreated group of Villeneuve et al. (1974) was of elderly female schizophrenic patients, whereas the majority of their treated group were male, with schizophrenia only a predominant diagnosis. Conversely, for the studies of Molsa et al. (1984, 1987) and of Lieberman et al. (1984), which reported the most substantial differences in prevalence between treated and untreated populations, both treated groups had an excess presence or severity of organic brain disease; this may have inflated the “true” difference between these populations in terms of the effect of treatment. The study of Owens et al. (1982) best approaches the requirements for addressing these quantitative issues. Here, neuroleptic treatment or nontreatment was in some sense random, being determined by the therapeutic orientation of particular consultant psychiatrists, and the study focused on a diagnostically homogeneous group within the limits of current operational definitions of schizophrenia. The study might appear to indicate at most a relatively minor role for neuroleptics in the emergence of involuntary movements. However, the untreated patients were significantly older and more likely to be male than their treated counterparts; when this difference in age was taken into account, there was a further excess of such movement disorder in those that had been exposed to neuroleptics (Crow et al. 1982).

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A further complication, which applies to all of the studies in Table IV and which is most difficult to address, is that of current treatment with neuroleptics when movement disorder is assessed. Acute neuroleptic treatment may suppress existing involuntary movements, and may thus mask their true prevalence in treated populations. This will be considered in more detail below in relation to the issue of neuropsychiatric diagnoses as vulnerability factors (see Section V1,D). For the reasons discussed above, the evidence of Table IV does not allow us to estimate with any accuracy the neuroleptic-associated component of the totality of involuntary movements within a neuropsychiatric population that has been exposed to such medication. A parsimoneous interim conclusion would be that the prevalence of involuntary movements in untreated neuropsychiatric disorders appears to have been underestimated and that long-term treatment with neuroleptic drugs makes it more likely that such movements will emerge, rather than creating them de novo.

111. Incidence

The above studies on the prevalence of tardive dyskinesia have concerned the proportion of a study population showing the syndrome at a given time (cross-sectional, point prevalence estimate) and will include both recently emerged cases and those persistent cases that have emerged in the past; it will exclude cases that have emerged previously but have now remitted. The incidence of the syndrome is the number of new cases that arise during a given period of time. This longitudinal element provides important information on the rate of emergence of the syndrome, but prospective studies of a relatively infrequent, late-onset disorder such as tardive dyskinesia are difficult to sustain, in terms both of logistics and of finance. The only study to adopt this strategy satisfactorily is that of Kane et al. (198213). Its critical element is that several hundred patients have been followed longitudinally, with periodic assessment of their movement status; the patients are in the main relatively young and have been followed from early in their treatment course. In the most recent update on this study (Kane et al. 1986a), the overall incidence of tardive dyskinesia was 18.5 4.5% and 40 2 7% following 4 and 8 years, respectively, of cumulative exposure to neuroleptics, defined as the percentage of patients who ever showed the emergence of involuntary movements.

*

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Included in this study have been 28 schizophrenic or schizophreniform patients with either no o r less than 2 months of exposure to neuroleptics on entry, and none of these have shown the emergence of involuntary movements (Kane et al. 1986b). This would be consistent with the data of Yarden and DiScipio (1971), who described 18 cases of involuntary movements in younger, poor-prognosis schizophrenic patients who had received little or no exposure to neuroleptics; the authors did not indicate the number of patients screened to locate these 18 cases, but personal correspondence revealed that they were derived from a population of approximately 275 (a prevalence of 6.5%) (see Waddington and Crow, 1988). T h e studies included in Table I11 indicate that involuntary movements in untreated schizophrenia are most evident in older, chronically ill patients with the most severe form of the illness (see also Rogers, 1985; Waddington, 1987; Waddington and Crow, 1988; Waddington and Youssef, 1989a). In a much smaller study of initially 58 autistic children who have been followed prospectively during long-term neuroleptic treatment (Perry et al. 1985), 22% have shown the.emergence of involuntary movements after 0.3-3.5 years of cummulative exposure; the buccallingual-masticatory region was most commonly affected.

IV. Natural History

In addition to considerations of the prevalence and incidence of tardive dyskinesia, a third important issue is the natural history of the disorder once it has emerged. Though in the past tardive dyskinesia has been considered by some to be an irreversible disorder, authoritative reviews on the first two decades of studies have indicated that the syndrome can, in many instances, remit at various times both after continued exposure to neuroleptics and (more commonly) after reduction o r withdrawal of medication; however, the likelihood of remission may decline with advancing age, and the disorder can in some instances prove to be at least persistent (Smith and Baldessarini, 1980; Jeste and Wyatt, 1982) Subsequent to these extensive reviews, studies have examined more elaborately the natural history of tardive dyskinesia within defined populations over specified follow-up periods of varying length. These include the follow-up of patients with tardive dyskinesia after neuroleptic withdrawal, dose reduction or contin’uation of treatment (Seeman, 1981; Yassa et al., 1984c; Glazer et al., 1984) or the follow-up of a

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population including patients both with and without tardive dyskinesia on initial assessment (Barnes et al., 1983b; Chouinard et al., 1986; Robinson and McCreadie, 1986; Gardos et al., 1988). Such studies confirm that cases of tardive dyskinesia clearly can remit and that the likelihood of such remission does appear to diminish with increasing age. They also indicate that in many instances enduring tardive dyskinesia is not a progressive disorder and may continue to be manifested in a mild form even during continuing neuroleptic treatment; there remains, however, a small but particularly problematic group of patients who develop, often rapidly, a very severe form of the disorder (Gardos et al., 1987). In our own long-term follow-up study of 74 schizophrenic inpatients with long-term exposure to neuroleptics who were reassessed 5 years later, 27 (36%) did not show tardive buccal-lingual-masticatory dyskinesia on either occasion, 22 (30%) exhibited the syndrome on each occasion, and 20 (27%of the total group; 43% of those initially without evidence of tardive d yskinesia) exhibited the syndrome at follow-up but not on initial assessment; for only five patients (7% of the total group; 19%of those initially showing tardive dyskinesia) were those involuntary movements evident on initial assessment no longer manifested at follow-up; these five were all younger females who initially showed only mild involuntary movements of a single buccal-lingual-masticatory region (Waddington and Youssef, 1989b). Many of the above studies contain such a pseudoprospective element, in that cross-sectional evaluations are repeated several years later on the same patient population. This approach can yield useful information but lacks the temporal resolution of a true prospective study in relation to changes in movement status. T w o studies have investigated any progressive change in tardive dyskinesia over very prolonged periods after reduction or discontinuation of neuroleptic treatment to address the issue of whether involuntary movements that persist for a year or more after such manipulations in therapy can be said to be irreversible. Klawans et al. (1984) have described six patients, aged 20-57, whose tardive dyskinesia persisted for 2 years after neuroleptic withdrawal but who had shown late remission of their predominantly buccal-lingual-masticatory dyskinesia over subsequent periods of from 0.5-3 years. This indicates that persistence of involuntary movements for 2 or more years after neuroleptic withdrawal need not imply permanence in all patients. Similarly, Casey (1985) was able to follow 25 patients (mean age 55) over a 5-year period following neuroleptic withdrawal or dose reduction. T h e majority of these patients showed a meaningful reduction in the severity of their involuntary movements; improvement was most prominent in those for whom drug discon-

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tinuation was possible, but it was also evident for a number of those who continued on a reduced dosage. In both groups, improvement was noted between 2-5 years after the respective changes in treatment, with younger patients being more likely to show such amelioration of their movement disorder. Continuing neuroleptic treatment led only infrequently to progression of tardive dyskinesia, and even modest dose reduction could be associated with improvement over several years. In a prospective study of younger psychiatric patients with little or no neuroleptic drug exposure on entry (Kane et al., 1986a,b), the majority of patients who developed tardive dyskinesia at some point over an %year period did not show progression in the severity of their involuntary movements. Incidence figures for the development of tardive dyskinesia over 8 years reduced substantially from 40 ? 7% on the basis of those ever showing involuntary movements to 22 ? 6% on the basis of those showing involuntary movements persisting for at least 6 months. Thus, many cases of tardive dyskinesia remitted, and the likelihood of remission was associated with its emergence following shorter rather than longer prior exposure to neuroleptics, use of lower modal doses of neuroleptics following its emergence, and younger age of the patient. There remained a small subgroup of patients who appeared to develop rapidly a very severe form of the disorder. In a prospective study of children with autism (Perry et al., 1985), the majority of cases of tardive dyskinesia which emerged during 0.3-3.5 years of neuroleptic treatment did not progress and were found to remit within 0.5-9 months after emergence. Few data are available on the persistence or otherwise of involuntary movements that emerge in association with untreated neuropsychiatric disorder. We have been able to study the involuntary buccal-lingual-masticatory movements evident in a small group of elderly, chronically ill schizophrenic inpatients who had never been treated with neuroleptics; these movements did not remit but progressed very little over a 5-year period (Waddington and Youssef, 1989a). T h e above data indicate that tardive dyskinesia does not commonly progress and is often mild but can occasionally be very severe; this suggests some element of an all-or-none process. In those who are vulnerable to the disorder, it may develop to some “preset” severity that in part reflects the extent of their vulnerability. Those involuntary movements that persist after several years of drug discontinuation, where this is feasible, may be related to the baseline level of such movements that is associated with the relevant neuropsychiatric or other disorder, were it not to have been treated (Marsden, 1985; Waddington and Crow, 1988).

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V. Morbidity and Mortality

Accumulating evidence suggests that tardive dyskinesia is more than an iatrogenic, choreoathetoid movement disorder that affects the buccal-lingual-masticatory, limb, and trunk regions. In some instances the syndrome may include a variety of complications that can present serious health care problems in a much more general sense. A review of the earlier literature by Yassa and Jones (1985) considered a number of impairments associated with tardive dyskineska, both medical (impaired gait and posture, altered gastrointestinal function, disrupted speech, and disturbed respiration) and psychosocial (personal and occupational) problems. Gerratt et al. (1984) have studied further the specific speech abnormalities that can be associated with tardive dyskinesia and found them to be associated with more severe trunk rather than buccal-lingual-masticatory movements. 'The literature on the possible respiratory component of tardive dyskineska has been reviewed by Yassa and La1 (1986); we have found patients with such respiratory movement disorder to show more severe buccallingual-masticatory movements and higher blood pressure (Youssef and Waddington, 1989). l'ardive dyskinesia may be associated with low body weight when the respiratory-esophageal musculature is involved (Yassa and Nair, 1987) and with low serum calcium levels (Youssef and Waddington, 1989b). Two studies have found patients with tardive dyskinesia to show a greater susceptibility to infections (McClelland et al., 1986), especially those affecting the respiratory tract (Youssef and Waddington, 1987). Such general medical morbidity, in addition to the more typical disabilities associated with involuntary movement disorder, raises the question of whether tardive dyskinesia might be associated with increased mortality. Casey and Rabins (1978) have discussed whether tardive dyskinesia can be a life-threatening disorder, particularly when the ventilatory and gastrointestinal musculatures are affected. If morbidity in tardive dyskinesia can also be reflected in nonmotoric factors such as increased susceptibility to infection, the issue of mortality requires some careful consideration. In the first study to address this issue systematically, Mehta et al. (1978) reported that patients with tardive dyskinesia showed a higher mortality rate over a 5-year period than did patients without such movement disorder. Subsequently, Kucharski et al. (1979), adopting a different approach, found no difference in prevalence or severity of tardive dyskinesia between small

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groups of deceased and living patients who were assessed 16-18 months previously; similar results were obtained with this same approach, over a 2-year period, by Yassa et al. (1984a). Three studies have reported an elevated mortality rate in patients with tardive dyskinesia over periods of 2.7-18 years. I n the first (McClelland et al., 1986), buccal-lingual-masticatory dyskinesia was associated with a shorter survival time for inpatients with functional psychiatric disorders, but not for those with organic brain disease. We subsequently reported an elevated mortality rate for chronic schizophrenic inpatients with, as opposed to those without, buccal-lingualmasticatory dyskinesia (Youssef and Waddington, 1987), and in both studies this relationship was most robust for patients with the more evident movement disorder. Takamiya et al. (1988) have also reported a higher mortality rate for schizophrenic patients with tardive dyskinesia but did not note any specific relationship with the severity of involuntary movements. This issue requires further systematic study, focusing on the long-term follow-up of patients with and without buccal-lingualmasticatory movements in relation to the presence and absence of a functional psychiatric versus organic disorder. The above studies have at least two major implications: They suggest (1) that the emergence of tardive dyskinesia, especially in older patients, should be a cause of more general medical concern and (2) that theories of the disorder may need to invoke a broader, whole body concept.

VI. Vulnerability Factors

A critical issue for research into tardive dyskinesia is the identification of factors that distinguish, on an individual basis, those patients in whom such involuntary movements emerge from those in whom they do not. This section evaluates research on a series of such putative predisposing factors; some of these have been the subject of study for some decades, while others have only lately become a focus for systematic investigation. Though they are considered sequentially, each is not necessarily independent from one o r more other factor(s). Indeed, it will be argued that several of these putative predisposing factors may have their origin in a common neurological process associated with core vulnerability to the emergence of involuntary movements.

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JOHN L. WADDINGTON

A. AGE

There are few established facts concerning tardive dyskinesia, but perhaps the most robust general finding is that its prevalence (and severity) increases with advancing age. This has been apparent on review of the first 20 years of studies on the topic in heterogeneous populations (Smith and Baldessarini, 1980; Kane and Smith, 1982). A multitude of more recent studies, often in more homogeneous populations, generally (though not invariably) continue to report such a relationship; for example, using similar assessment techniques and criteria, we have found the prevalence of involuntary movements to increase reliably with age in patients with schizophrenia, both inpatients (Waddington and Youssef, 1985) and outpatients (Waddington and Youssef, 1986a), bipolar affective disorder (Waddington and Youssef, 1988a), and mental handicap (Youssef' and Waddington, 1988a). There are at least three potential artifactual explanations for this phenomenon that require careful consideration. First, it may reflect the likelihood that older patients may have been receiving neuroleptics for longer periods of time, with age being merely a proxy for increasing exposure to the presumed offending medication. Such an explanation is at variance with the absence of any consistent relationship between duration of neuroleptic exposure and likelihood of emergence of tardive dyskinesia, at least over the prolonged periods that are relevant to chronically ill populations (see Section V1,C). We have been able to compare the duration of exposure to neuroleptics for schizophrenic inpatients aged 51-70 years with that for patients aged >70 years, the latter showing a marked increase in the prevalence of buccal-lingualmasticatory dyskinesia; there were no differences in the number of years of neuroleptic treatment received by patients in these two age groups, either for those with or for those without such involuntary movements (Waddington and Youssef, 1985). Second, it may reflect at least in part a reduced rate of remission (i.e., greater persistence) of instances of the disorder which arose in preceding years, leading to an accumulation of cases in elderly populations. Analysis of early studies initially suggested such an effect (Smith and Baldessarini, 1980), and this has been supported by subsequent follow-up studies (Barnes et al., 1983b; Glazer et al., 1984; Casey, 1985). T h e prospective study of Kane et al. (1986a) indicates that the risk of tardive dyskinesia increases only moderately between 20-40 years of age but rises substantially thereafter; preliminary analyses from this study also support the notion of reduced remission of tardive dyskinesia with

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increasing age, which contributes to the increased prevalence in older patients. Third, it may reflect contamination by an increasing number of cases of idiopathic dyskinesia unrelated to neuroleptic treatment. Earlier analyses acknowledged the possibility of such an effect but in view of the generally low prevalence of involuntary movements in heterogeneous older, untreated populations it was suggested not to be a major confound (Smith and Baldessarini, 1980). On the basis of the relative rarity of such involuntary movements in the normal, healthy elderly (see Table I), Kane et al. (1986b) have expressed a similar view. However, the “control” populations on which such interpretations have been based are not valid for this purpose; the correct control population is not one of the normal, healthy elderly but rather one of older patients with the relevant neuropsychiatric disorder(s) who have not been treated with neuroleptics. Inspection of Tables 11-IV leads to a contrary conclusion, namely that at least in chronically ill, older populations, the contribution of idiopathic involuntary movements indistinguishable from those of tardive dyskinesia may have been seriously underestimated. Where d o these considerations leave the argument that the aging brain is more vulnerable to those long-term effects of neuroleptic drugs that are associated with the emergence of involuntary movements? We have reported that elderly patient groups with a high prevalence of such movement disorder have previously received lower average daily doses of neuroleptics than younger groups of otherwise similar patients with a lower prevalence of involuntary movements over indistinguishable durations of treatment (Waddington and Youssef, 1985; Waddington et al., 1987); this would be consistent with such an explanation. Similarly, Kane et al. (1986a) have reported that the incidence of tardive dyskinesia is higher among older than among younger patients, independent of previous level of neuroleptic exposure. There is also an emerging body of modern studies that indicates that patients who are first prescribed neuroleptics in old age for late-onset neuropsychiatric disorders may be at particular risk for the development of tardive dyskinesia (Section VIJ). An interaction between general pathophysiological effects of aging and brain dysfunction associated with neuropsychiatric disorder may enhance some neurological process that determines the likelihood that involuntary movements, both idiopathic and neuroleptic-associated, will emerge and persist. This synthesis of the above arguments on the age dependency of tardive dyskinesia has two corollaries. First, involuntary movements associated with neuropsychiatric disorder and those associated with long-term neuroleptic treatment may not be mechanistically

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distinct; indeed, it will be argued that the latter are drug-precipitated variants of the former. Second, attention must be focused not only on neuroleptic treatment, but also on features of the neuropsychiatric disorder for which that treatment was prescribed.

It is often asserted that tardive dyskinesia is more likely to emerge in females than in males. However, as Kane and Smith (1982) have argued, on the basis of the first 20 years of studies, the evidence available to support such an assertion is far from robust statistically. They reviewed evidence that female preponderance appeared to be most evident in patients aged over 70 years and to increase with the criterion of severity adopted to define a case. Subsequent studies have also noted similar patterns. Thus, Richardson et al. (1984) reported females to demonstrate an increase in prevalence of tardive dyskinesia through all age groupings to beyond 75 years, while males showed an increase in prevalence only up until the 65-75-year-old age group and a decline thereafter; there was very little difference between the sexes in the prevalence of tardive dyskinesia up to age 64 using mild severity to define a case, but there was evidence for female preponderance when using a criterion of at least moderate severity. In our own studies, the prevalence of involuntary movements tended to be higher in females, but in each age range (<50, 51-70, and >70 years) this failed to attain statistical significance (Waddington and Youssef, 1985); however, no males aged over 80 years showed involuntary movements whereas 83% of females aged over 80 showed such movement disorder. Similarly, Kane et al. (19864 have not found sex to be a significant vulnerability factor in their prospective study of a generally younger cohort but have noted in a prevalence study that sex is a more important risk factor in older than in younger patients. It appears that females do tend to have a slightly higher overall prevalence of tardive dyskinesia than do males, but whether or not such an effect is seen in a given study will depend upon the age range of the population and the severity criterion adopted to define a case. Sex is clearly a much weaker vulnerability factor than is age, with which it appears to interact.

C. DURATION AND INTENSITY OF PASTEXPOSURE TO NEUROLEPTICS

In general terms, there has been little consistent evidence that patients with tardive dyskinesia have received longer or more intensive

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treatment with neuroleptics than have patients without such movement disorder in terms of data from predominantly chronically ill populations. Review of the first 20 years of studies (Kane and Smith, 1982) suggested that the majority of those (few) studies that reported a significant positive relationship between an index of prior neuroleptic exposure and tardive dyskinesia involved populations with relatively modest cumulative drug exposure; this led to the notion that for patients with a high vulnerability to develop tardive dyskinesia, such a relationship may be evident over the lower range of indices of exposure; conversely, patients with little or no vulnerability may fail to demonstrate any such relationship no matter how long or intensive is their treatment. Some prevalence studies continue to point in this direction (Toenniessen et al., 1985; Waddington et al., 1987). However, differing relationships may be seen according to the severity criterion adopted to define a case (Richardson et al., 1984; Waddington et al., 1987). There is evidence from prevalence studies that for younger patients there is only a small rise in the probability of developing tardive dyskinesia with longer exposure to neuroleptics, while for older patients this probability rises more sharply with similar increases in drug exposure (Toenniessen et al., 1985; Kane et al., 1986a). It must be noted that in chronically ill populations with long-term exposure to neuroleptics, duration of treatment can be highly correlated with age (which itself is a vulnerability factor of some robustness); also, duration of neuroleptic treatment can, like age, influence the persistence of tardive dyskinesia (see Section IV), and so may increase its prevalence independent of any increase in incidence. In this area there is the potential for a multiplicity of confounding factors in terms of the accuracy of quantitative estimates of neuroleptic exposure and intercorrelations with other demographic and clinical variables. One of the most contentious issues in the area of tardive dyskinesia research is whether individual neuroleptic drugs differ in their propensity to induce this disorder. Although there have been many such assertions, they stem almost invariably from anecdotal, uncontrolled, o r unrefereed reports, or from animal studies of questionable relevance to the clinical syndrome they purport to model. There is little work, either prospective or retrospective, on patients who have been treated only with a single neuroleptic drug, and no amount of epidemiological sophistication can compensate for such a dearth of appropriate data. This is not to say that differences between neuroleptics in liability to induce tardive dyskinesia may not exist; it is the contention of this reviewer only that there are as yet few or no data to substantiate any such assertion(s). In the absence of such data, no review is possible. If neuroleptics are associated with the emergence of tardive dys-

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kinesia, then one would expect some form of relationship, however coarse, between extent of exposure to such drugs and incidence of the disorder. The data of Kane et al. (1986a) from their prospective study provide such information: The incidence of tardive dyskinesia increased with duration of neuroleptic exposure over a (so far) 8-year period. However, there remains a critical issue in defining those factors that distinguish patients in whom involuntary movements emerge over a given period of treatment from patients in whom they do not. Clearly, other vulnerability factors are operating. Indeed, their influence would appear to be more fundamental than any index of past neuroleptic exposure, as inspection of Tables II-IV suggests that they can predispose to the emergence of involuntary movements even in the absence of a history of treatment with such drugs.

D. DIAGNOSTIC COMPOSITION AND E X T E N T OF EXPOSURE TO NEUROLEPTICS

CURRENT

As previously discussed above (Section II,A and II,E), the emergence of tardive dyskinesia has been reported in all disorders for which neuroleptics and pharmacologically related agents have been prescribed, be it for schizophrenia, affective disorders, mental handicap, neurodegenerative disease, childhood autism, or for gastrointestinal and labyrinthine dysfunction. N o comment is made here on the merits or faults of the use of neuroleptic drugs in each of these disorders (see Section I,B), only on published data relating to the consequences of such use. T h e question is whether any particular diagnostic group(s) might show an elevated risk of developing tardive dyskinesia on prescription of neuroleptics. T h e one patient grouping posited to be at particular risk, other than those with organic brain dysfunction, is those with affective disorders. Initial anecdotal reports and case studies to this effect have been reviewed by Gardos and Casey (1984). Subsequent and more systematic prevalence and incidence studies have continued to suggest such a relationship. Thus, Yassa et al. (1984b) reported the prevalence of tardive dyskinesia to be higher in patients with bipolar affective disorder or organic mental syndromes than in those with schizophrenia, in a manner independent of age and duration of treatment with neuroleptics and not totally accounted for by the potential masking effects of differences in current neuroleptic dose. Similarly, Glazer et al. (1988) have reported the presence of buccal-lingual-masticatory dyskinesia to be particularly associated with affective or schizoaffective rather than

NEUROLEPTICS A N D TARDIVE DYSKINESIA

32 1

schizophrenic diagnoses, in a manner independent of age. Attention has recently been drawn to the number of instances in which patients with very severe tardive dyskinesia have an affective diagnosis (Gardos et al., 1987). In the prospective studies of Kane et al. (1986a,b), the incidence of tardive dyskinesia was significantly higher among patients with an affective diagnosis, the most significant distinction being between the pure affective disorders in comparison with schizophrenia; this difference remained robust even after controlling for sex, age, and electroconvulsive therapy. In comparing the prevalence and incidence of tardive dyskinesia between populations of differing diagnostic composition, each with a history of prior exposure to neuroleptics, it is necessary to consider a potential confounding factor: Might any such differences merely reflect the influence of differing doses of current neuroleptic treatment, which is known to have some action to attenuate the expression of involuntary movements? There is evidence from several studies that the manifestation of tardive dyskinesia may be inversely related to the current daily dose of neuroleptics administered (usually expressed as chlorpromazine equivalents). In cases of schizophrenia and mental handicap, Richardson et al. (1984, 1986) have reported negative relationships between current neuroleptic dose and both the presence and severity of involuntary movements; in schizophrenia, current daily dose was also negatively correlated with age and duration, as we ourselves have previously noted (Waddington and Youssef, 1985). In the prevalence study of Kane et al. ( 1985) in heterogeneous populations, the severity of tardive dyskinesia among positive cases was correlated with being free of neuroleptics at the time of motor evaluation. In our own studies (Waddington et al., 1987), we found current daily dose of neuroleptics to be negatively correlated with the presence of mild but not of more prominent buccal-lingual-masticatory movements; thus, the dose of neuroleptic administered on the day of evaluation may be more likely to mask mild signs and less likely to mask the more prominent movements. Of course, it might be considered why patients with tardive dyskinesia might be receiving lower current neuroleptic doses: Was dosage lower because of recognition of tardive dyskinesia or, more likely, as a reflection of different clinical characteristics in those with such movement disorder? When actual neuroleptic serum levels, as distinct from daily dose administered, are determined in patients with and without tardive dyskinesia, the results may not only reveal differences in the pharmacokinetics of such current treatment but also suggest whether neuroleptics have been absorbed, metabolized, and eliminated differently over previous treatment. T h e literature on this potentially important issue is

322

J O H N L. WADDINGTON

neither extensive nor consistent and has been reviewed in negative (Csernansky Pt al., 1983) and positive (Yesavage et al., 1987) studies. These issues of current neuroleptic dose in relation to diagnostic composition are relevant not only to comparisons between schizophrenic, affective, and organic populations, but also to comparisons between more homogeneous populations differing in chronicity of illness. In a comparison of the prevalence of tardive dyskinesia in three distinct hospital facilities (Kane et al., 1985, 1986b), prevalence was found to be lowest in the hospital serving the less chronic schizophrenic and affective-schizoaffective patients and to be highest in that serving the most chronically ill schizophrenic patients. Because of concern that even this high prevalence in those most chronically ill might be an underestimate because of masking by current neuroleptic treatment, drug discontinuation was attempted to unmask covert dyskinesia; among patients without evident tardive dyskinesia on initial evaluation when medicated, the rate of covert dyskinesia was also greatest (67%)in the most chronically ill when compared to the less chronic populations (17%). Age and duration of prior neuroleptic treatment were related to the emergence of covert dyskinesia, but age alone did not account for these differences and duration of treatment appeared to contribute only modestly; current dose of neuroleptic prior to discontinuation was not related to the emergence of covert dyskinesia. There appear to be differences in the prevalence (and incidence) of tardive dyskinesia both between populations bearing different neuropsychiatric diagnoses and between more homogeneous populations differing in chronicity of illness, independent of extent of current neuroleptic treatment. As Kane et al. (1986b) remind us, even after controls are instituted for age, sex, age X sex interaction, and duration of prior neuroleptic treatment, considerable differences in the prevalence of tardive dyskinesia still endure between populations. This indicates that other important and possibly more fundamental vulnerability factors are acting and that they appear to involve the nature and course of the illness for which neuroleptic treatment was prescribed.

E. COGNITIVE DYSFUNCTION There has been a long-standing debate over whether patients with preexisting forms of organic brain dysfunction might be more likely to develop involuntary movements if prescribed neuroleptics. In an early, more systematic study, Edwards (1970) reported that signs of organicity, defined in terms of both neuropsychological and clinical variables,

NEUROLEPTICS AlVD TARDIVE DYSKINESIA

323

occured more frequently in a group of elderly, chronically ill female inpatients with persistent oral dyskinesia than in a group of otherwise similar patients without such movement disorder. However, an overview of the first 20 years of studies on tardive dyskinesia, composed of studies on heterogeneous patient groups and with varying indices of organicity, is far from conclusive on this issue (Kane and Smith, 1982). In Table V are listed systematic studies of neuropsychological test performance in schizophrenic patients with and without tardive dyskinesia. Of the 21 studies located, 17 have reported those patients manifesting involuntary movements to show greater cognitive dysfunction. This is apparent using a very broad range of neuropsychological test procedures and in a wide variety of patient populations in terms of age range and chronicity. The association remains robust in those

TABLE V I N SCHIZOPHRENIC PATIENTS W I T H COGNITIvE FUNCTION INVOLUNTARY MOVEMENTS Study

Association with involuntary movementsh

Age"

49 2 5 Famuyiwa et al. (1 979) Owens and Johnstone (1980) 60 -t- ? Donnelly el al. (198 1) 21-72 Itil et al. (1981) 52-88 Struve and Willner (1983a) 32 5 ? 66 f 10 Bartels and Themelis (1983) Wolf et al. (1983a) 54 8 Waddington et al. (1985a) 1 - 6 8 rt 14 Waddington and Youssef (1986b) Wegner et al. (1985a) 27 5 4

*

31 f ?

Richardson et al. (1985) Spohn et al. (1985) Waddington and Youssef (1986a) Waddington and Youssef ( 1 9 8 6 ~ ) Thomas and McGuire (1986) Kolakowska et al. (1986) Wade et al. (1987) Hoffman et al. (1987) Waddington et al. (1988) Sorokin et al. (1988) DeWolfe et al. (1988) ~

18-55

63 -t 5 61 5 8 51 +- 10 3 7 2 12 4 5 * 12 62 ? 6 36 rt 5 4 7 5 11 5 5 * 11 ~~

Age range or mean f SD * NS, no significant association found. ' Relationship with buccal-lingual-masticatory

A N D WITHOUT

& & &

Paired associate learning Function of the sensorium Perceptual function .1 Orientation and memory & Abstracting ability .1 Visual retention' NS Memory and verbal learning

.1 Orientation and memory' & Abstraction and visuomotor tracking NS Orientation and memory .1 Eyetracking and reaction time' & Orientation and memory' & Orientation and memory' $ Memory quotient NS Neuropsychological test battery 1 Neuropsychological test battery NS Neuropsychological test battery .1 Visuomotor tracking' & Visual memory' & Memory function' ~

~~~~

dyskinesia specihed.

324

JOHN L. WADDINGTON

studies that have been able to control for possible confounding influences of age, duration of illness, extent of past and current treatment with neuroleptics and anticholinergics, and potential performance deficits associated with such movement disorder. It appears particularly robust in relation to buccal-lingual-masticatory rather than limb-trunk dyskinesia (Waddington et al., 1987; DeWolfe et al., 1988) and to persistent rather than transient dyskinesia (Struve and Willner, 1983a). Indeed, the association is most reliable in, but by no means restricted to, older chronically ill inpatient populations with a mean age >55 years and specified as showing involuntary movements with a buccal-lingualmasticatory topography. All four negative studies in Table V involved patients without these characteristics; by their inclusion of cases defined solely by the presence of limb-trunk dyskinesia, any association between buccal-lingual-masticatory dyskinesia and cognitive dysfunction may have been diluted. Two studies of specialized cognitive functions in schizophrenic patients with and without tardive dyskinesia (Collerton et al., 1985; Myslobodsky et al., 1985) are not included in Table V, as both utilized a screening procedure to exclude patients with these very forms of generalized cognitive deficit that were associated with the presence of involuntary movements in other studies. If such associations in schizophrenia are of any general significance in relation to vulnerability to tardive dyskinesia, they should be evident in populations with other diagnoses for which neuroleptics are prescribed and the emergence of involuntary movements reported. Listed in Table VI are studies of neuropsychological test performance in neuroleptic-exposed patients with affective diagnoses (most commonly bipolar affective disorder) or mental handicap. The association between cognitive dysfunction and tardive dyskinesia appears robust in affective patients and was evident in two of three studies of the mentally handicapped. In our own studies (Waddington and Youssef, 1988a; Youssef and Waddington, 1988a), we were able to control for possible confounding influences of age and of extent of past and current treatment with neuroleptics and anticholinergics. This association between cognitive dysfunction and the presence of tardive dyskinesia might be explained in a number of ways: Does cognitive impairment precede and predispose to the emergence of involuntary movements, or is the association an artifactual one through the temporally related induction of a syndrome of “tardive dementia” (Myslobodsky, 1986) by long-term neuroleptic treatment? The answer is to be found in longitudinal rather than cross-sectional studies. Thus, it has been reported from the prospective study of relatively young schizophrenic and affective patients ( Wegner et aE.,

NEUROLEPTICS AND TARDIVE DYSKINESIA

TABLE VI COGNITIVE FUNCTION IN AFFECTIVE AND MENTALLY HANDICAPPED PATIENTS WITH WITHOUT INVOLUNTARY MOVEMENTS

325 AND

Association with involuntary movementsb

Disorder and study

Age"

Affective disorder Wolf et al. (1983a) Wade et al. (1987) Waddington and Youssef (1988a) DeWolfe et al. (1988) Waddington et al. (1989a)

49 k 14 47 f 15 64 -1- 9 52 ? 9 46 f 15

J Memory and verbal learning

Mental handicap Gualtieri et al. (1986) Richardson et al. (1986) Youssef and Waddington (1988a)

5-47 41 '. 12 51 2 10

t Severity of mental handicap NS Severity of mental handicap t Severity of mental handicapc

a

Age range or mean f SD. NS, no significant association found. Relationship with buccal-lingual-masticatory

.1 Neuropsychological test battery Orientation and memoryc

.1 Memory function' .1 Visuomotor tracking"

dyskinesia specified.

1985b) that poor neuropsychological test performance among those initially without evident tardive dyskinesia was indeed associated with the subsequent emergence of such movement disorder a mean of 1.6 years later. Though this was a finding of general applicability to all groups in the study, it appeared particularly robust in relation to persistent rather than transient dyskinesia and in patients with affective diagnoses. In our own pseudoprospective study in older, chronically ill schizophrenic inpatients (Waddington and Youssef, 1989b), initial cognitive function in those without such movement disorder was not associated with the manifestation of involuntary movements 5 years later. However, those patients in whom such involuntary movements emerged over this 5-year period showed a significant deterioration in their cognitive function, while no such deterioration was evident in those in whom this movement disorder did not emerge. Thus, there appeared to be some close relationship between cognitive dysfunction and emergence of involuntary movements whose precise temporal characteristics could only be resolved by a true prospective protocol.

F.

SPECIFIC

ASPECTS OF

PSYCHOPATHOLOGY AND CLINICAL COURSE

If there is a generally robust association between tardive dyskinesia and cognitive dysfunction, might this extend to other psychological

326

JOHN L. WADDINGTON

features having a putative association with organic cerebral disorder? Negative symptoms are one aspect of schizophrenic psychopathology that has been equated by some with structural brain pathology (Crow, 1980; Andreasen et al., 1986). While the evidence for this may be less robust than originally perceived (Walker and Lewine, 1988; Waddington, 1989a), it is of some interest to compare measures of negative symptoms in schizophrenic patients with and without tardive dyskinesia. Table VII lists 16 such studies, 11 of' which report a positive association between one or more negative symptoms (most commonly flattening of affect and poverty of speech) and some measure of involuntary movements. This association remains robust in those studies that have been able to control for possible confounding effects of age, duration of TABLE VII NE(:ArIvE SYMPTOMS I N SCHIZoPHKENIC PATIENTS W I T H INVOLUNTARYMOVEMENTS Study

Association with involuntary mnvemen t s h

Age"

Owens and Johnstone (1980)

60 2 ?

Itil et al. (1981)

52-88

McCreadie rt al. ( 1 982)

75 t ?

Csernansky et al. (1983) Lindenmayer et al. (1984)

28-60

Opler el al. (1984)

3 7 2 13

Glazer et al. (1984)

50 k ?

Jeste et 01. (1984) Waddington et al. ( I 985a) Karson et al. (1985) Richardson et al. (1985) Waddington and Youssef (1986b) Iager el al. (1986) Hoffman et al. ( 1 987)

35 k 6 6 8 2 14 32 f 6 18-44 68 2 14 18-55 62 6

Waddington et al. (1987) Monteleone et al. ( 1 988)

6 3 * 13

18-34

*

57 2 7

AND W I T I I O U T

r

Flattened affect and poverty of speech t Flattened affect and emotional withdrawal T Flattened affcct and social withdrawal f Negative symptom constellation NS Negative symptom constellation NS Negative symptom constellation t Flattened affect and emotional withdrawal t Negative symptom constellation T Poverty of speech' t Negative symptom constellation NS Flattened affect t Flattened affect' f Poverty of' speech NS Negative symptom constellation t Flattened affectc NS Negative symptom constellation

~

Age range or mean 5 SD. NS, no significaht association found. ' Relationship with buccal-lingual-masticatory

dyskinesia specified.

NEUROLEPTICS AND TARDIVE DYSKINESIA

327

illness, and extent of past and current drug treatment. It may be most robust in relation to persistent rather than intermittent dyskinesia (Karson et al., 1985) and to buccal-lingual-masticatory rather than limb-trunk movements (Waddington et al., 1987). Indeed, the association is most reliable in, but by no means restricted to, older chronically ill populations; three of the five negative studies in Table VII involved patients without these characteristics. Regarding other aspects of psychopathology in schizophrenia, tardive dyskinesia has been reported to be unrelated to any of 17 items of the Brief Psychiatric Rating Scale by Lindenmayer et al. (1984) and by Opler et al. (1984). It was found by Richardson et al. (1985) to be associated with somatic concern, conceptual disorganization, tension, mannerisms and posturing, unusual thought content, and hostility; there were also associations with manic symptoms and inappropriate affect on additional rating scales. An association between tardive dyskinesia and inappropriate affect has also been noted by Itil et al. (1981) and an association between tardive dyskinesia and both depressive symptoms and reduced anxiety by Karson et al. (1985). Thus, with the possible exception of some form of affective dysregulation, the inconsistency of the above reports contrasts with the greater reliability of an association between tardive dyskinesia and negative symptoms. Regarding clinical course, schizophrenic patients with involuntary movements have been reported to show greater premorbid associality (Wegner et al., 1985a) and both earlier onset of illness and poorer prognosis (Yarden and DiScipio, 197 1) than similar patients without such movement disorder. In patients with affective diagnoses who have been exposed to neuroleptics, tardive dyskinesia has been reported to be associated both with less depressed mood (Glazer et al., 1984) and a history of fewer depressive episodes in groups matched for age, age at onset, and duration of illness (Waddington and Youssef, 1988a). Conversely, Yassa and Schwartz (1984) have reported affective patients with tardive dyskinesia to have more commonly experienced depression at first hospitalization and to have more subsequent depressive episodes; however, their patients with tardive dyskinesia were older than those without such movement disorder and had experienced the onset of their affective illness at a later age. There is an emerging body of evidence that among neuroleptic-treated patients with affective diagnoses, the likelihood of emergence of tardive dyskinesia is inversely related to the extent of exposure to lithium (Mukherjee et al., 1986; Kane et al., 1986a; Waddington and Youssef, 1988a; Waddington et al., 1989a; but see Perenyi et al., 1984). One must ask why prescription of lithium might

328

JOHN L. WADDINGTON

differ between such patients; presumably this reflects, at least in part, differences in psychopathology, for which it may be a proxy. We have found bipolar patients with involuntary movements to have more commonly received antidepressants (Waddington et al., 1989a), and this may also be a proxy for differences in psychopathology; there is no reliable body of evidence that antidepressants play any direct causal role in the emergence of involuntary movements (Yassa et al., 1987a). On a longitudinal basis, tardive dyskinesia in bipolar affective disorder has been variously reported to be most evident in depressive and least evident in manic phases (Cutler et al., 1981; Weiner and Werner, 1982; de Potter et al., 1983) or least evident in euthymic phases (Goswami et al., 1985). While this emphasizes some relationship between tardive dyskinesia and affective state, more experimental evidence is needed to substantiate the proposal of Wolf et al. (1982) that organic factors underlie the association.

G. NEUROLOGICAL FEATURES Measures of cognitive dysfunction and specific aspects of psychopathology are, of course, only indirect indices of organic brain dysfunction. If they are revealing important facets of vulnerability to tardive dyskinesia, they should be complemented by more direct assessment techniques. Listed in Table VIII are studies comparing a wide range of neurological features in patients with and without tardive dyskinesia. Seven of nine such studies have reported a positive association between neurological abnormality and tardive dyskinesia, in terms of B-mitten EEG pattern, brainstem auditory evoked potentials, neurological soft signs, and developmental reflexes; Villeneuve et al. (1974) also refer to release phenomena (developmental reflexes) being associated with oral dyskinesia, though no quantitative data were given. Such associations have been reported in both schizophrenia and bipolar affective disorder and have proved robust in those studies that have been able to control for age, duration of illness, and drug treatment. T h e association with abnormal 13-mitten patterning on EEG is of particular interest as, first, it may be most robust in relation to persistent rather than transient dyskinesia (Struve and Willner, 1983b). Also, there remains the problem of specifying the temporal relationship of such abnormalities to tardive dyskinesia. In a prospective study (Struve and Willner, 1983b), an abnormal 13-mitten pattern among those initially without evident tardive dyskinesia was associated with the subsequent emergence of such movement disorder.

NEUROLEPTICS AND TARDIVE DYSKINESIA

329

TABLE VIII NEUROLOGICAL FEATURES IN SCHIZOPHRENIC AND BIPOLAR AFFECTIVEPATIENTS WITH WITHOUT INVOLUNTARY MOVEMENTS

Association with involuntary movementsb

Disorder and study

Age"

Schizophrenia Wegner et al. (1979) Zeitlhofer et al. (1984) Wegner et al. (1985a) Kolakowska et al. (1986) Wilson et al. (1986) Gureje (1987) Youssef and Waddington (198%)

2 9 T 10 29-60 27 i 4 3 7 ? 12 25-67 44 It 12 50-86

Abnormal evoked potentials Soft signs NS Soft signs t Soft signs NS Soft signs t Developmental reflexes'

Bipolar affective disorder Mukherjee et al. (1984) Youssef and Waddington (1988~)

41 f 10 40-77

t t

Age range or mean f SD. NS, no significant association found. Relationship with buccal-lingual-masticatory

AND

r EEG dysrhythmiac t t

Soft signs Developmental reflexes'

d yskinesia specified.

H. STRUCTURAL BRAINPATHOLOGY The most direct way of investigating further these putative associations is actually to examine the brain in patients with and without tardive dyskinesia, either through in vivo imaging technology or at postmortem. Listed in Table IX are 12 imaging studies in schizophrenic or predominantly schizophrenic populations, 8 of which report a positive association between one or more indices of structural brain pathology and some aspect of involuntary movement disorder; the results of Jeste et al. (1980a,b) are not included as they appear to be subsumed within the subsequent and more extensive report of Kaufmann et al. (1986). These findings appear most robust in relation to buccal-lingualmasticatory rather than limb-trunk dyskinesia and to severe rather than mild-moderate involuntary movements; each of the four negative studies in Table IX did not appear to have examined such distinctions. The study of Pandurangi el al. (1980) suggests that abnormalities of cerebral structure may be associated more with persistent than with reversible dyskinesia. In our own preliminary study (Waddington et al. 1985b), significantly increased ventricular size was found in a small group of age-matched schizophrenic patients with involuntary movements of at least two orofacial areas; no significant effect was evident if patients exhibiting mild movements of only one orofacial area were

330

J O H N L. WADDINGTON

STRIJ(:.IURALBRAIN

TABLE IX SCHIZOPHRENIC PAIIENTS INVOLUNTARY MOVEMENTS

PAI‘HOLOGY I N

Study

Association with involuntary movementsb

Age“ ~

Famuyiwa et al. (1979) Pandurangi et al. (1980) Bartels and Themelis (1983)

49 2 5 42-69 6 6 % 10

Brainin et al. (1983)

20-59

Owens et al. (1985); Owens (1985)

56 2 14

Waddington et al. (1985b, 1989b) Albus et al. (1985)

25-67 51 -+ 10

Gimenez-Roldan et al. (1985)

62

Kolakowska el al. ( 1986) Kaufmann et al. (1986) Hoffman et al. (1987) Sorokin et al. (1988)

37 2 52 -+ 62 2 472

2

8

12 23 6 11

WITH A N D W I T H O U I

~~

T

Ventricular abnormality; CT t Caudate atrophy: PEG t Ventricular size arid caudate atrophy‘; CT t Cerebral atrophy with severe dyskinesia‘; C T T Ventricular size with severe dyskinesia‘; C T T Ventricular size‘; CT t Coriical atrophy with severe dyskinesia; C T t Cerebral atrophy with severe dyskinesia‘; C T and PEG NS ventricular size; CT NS ventricular size; C T NS ventricular size; (2‘1’ NS ventricular size: CT

~~

‘ Age range or mean

2 SD. NS, no signifirant association found; CT, computed tomography; PEG, pneumoencephalography . Relationship with buccal-lingual-masticatory dyskinesia specified.

included in the movement-disordered group or if limb-trunk movements were considered (Waddington et ul. 1989b). There is no consistent evidence for an effect of long-term exposure to neuroleptics on such indices of cerebral structure or for extent of exposure to neuroleptics to distinguish schizophrenic subgroups differing in ventricular size (Owens et al., 1985; Waddington et al., 1985b). It should be emphasized that although the term “atrophy” is used in Table IX, there is evidence that abnormalities of cerebral morphology in schizophrenia may reflect static, developmentally determined anomalies rather than active disease (Weinberger, 1987; Murray and Lewis, 1987; Waddington, 1988b, 1989a). Using magnetic resonance imaging, Besson et ul. (1987) have reported not structural brain changes but, rather, increased T1 values in the basal ganglia of schizophrenic patients with tardive dyskinesia. Postmortem studies of patients with tardive dyskinesia have been undertaken infrequently and with inconsistent results; the early studies have been briefly reviewed by Arai et al. (1987). Molsa et ul. (1987) have

NEUROLEPTICS A N D TARDIVE DYSKINESIA

33 1

studied postmortem brains collected from patients with Alzheimer’s disease who had been followed up from contact until death and rated for severity of involuntary movements; all but one had been treated with neuroleptics. The severity of involuntary movements was negatively correlated with brain weight; more specifically, this was positively correlated with plaque and tangle counts in several cortical and subcortical regions and negatively correlated with axonal torpedoes in the cerebellum. These relationships were much more robust for buccallingual-masticatory than for limb-trunk movements. Among brains collected from elderly, institutionalized chronic schizophrenic inpatients with generally long-term exposure to neuroleptics (Pakkenberg, 1987), there were no differences in brain weight or ventricular size between those noted to have and not to have shown buccal-lingual-masticatory dyskinesia. However, Arai et al. (1987) have reported brains from neuroleptic-treated schizophrenic patients with buccal-lingualmasticatory dyskinesia to show markedly inflated neurones in the cerebellar dentate nucleus in the absence of prominent neuronal loss or gliosis.

I. AGEAT ONSETOF ILLNESS On the basis of the incidence of tardive dyskinesia apparent in prospective studies of younger schizophrenic patients (Kane et al., 1986a) and the high prevalence and rapid emergence in elderly populations with much smaller cummulative exposure to neuroleptics (Toenniessen et al., 1985; Kane et al., 1986b), it has been suggested that the incidence of tardive dyskinesia may be much higher among patients who are already elderly when prescribed neuroleptics. Subsequently, two studies have provided more direct evidence that patients with late-onset psychosis are particularly vulnerable to such movement disorder when prescribed neuroleptics. Yassa et al. (1986) have reported patients with late-onset psychosis, that is, at ages greater than 45 years, were more likely to develop tardive dyskinesia with more severe involuntary movements, did so after a shorter period of neuroleptic treatment, and showed more organic factors than did patients with an early onset of psychosis, i.e. at ages less than 25 years. In our own study (Waddington and Youssef, 1986a), we found that among a group of older, neuroleptic-treated outpatients who in general developed psychosis relatively late in life, those with buccal-lingual-masticatory dyskinesia experienced the onset of their psychosis at an older age, were more likely to be female, and were more cognitively impaired than similar patients

332

JOHN 1.. WADDINGTON

without such movement disorder. It should be emphasized that certain individual vulnerability factors may differ in patients with a more typical presentation of schizophrenic psychosis, in whom an earlier age at onset and treatment may be associated with involuntary movements (Yarden and DiScipio, 1971; Waddington et al., 1987). However, an association with cognitive dysfunction appears common to both forms of presentation of psychosis. Among patients with affective psychosis, females with tardive dyskinesia were noted to have developed their affective disorder later in life than had similar patients without such movements, with a trend in that direction being noted in males (Yassa et al., 1983). Similarly, in patients of unspecified neuropsychiatric status who were prescribed metoclopramide for gastrointestinal disorders (Wiholm el al., 1984), those noted to have developed involuntary movements showed buccal-lingualmasticatory dyskinesia and were all elderly females who had received a median of 14 months of treatment before onset of such movement disorder. It is known from recent computerized tomography studies that after 60 years of age there begins an exponential increase in cerebral atrophy with advancing age in normal individuals (Waddington, 1988b) and that patients with late-onset psychosis are particularly likely to show abnorma1itie.s of cerebral morphology in comparison with age-matched patients without such psychiatric disorder (Naguib and Levy, 1987; Rabins et al., 1987). J. HISTORY OF TYPICAL EXTRAPYRAMIDAL SIDEEFFECTS

It has been speculated that patients who show a greater sensitivity to early extrapyramidal side effects of neuroleptics, such as parkinsonism, may be at a greater risk of subsequently developing tardive dyskinesia. However, review of the first 20 years of studies (Kane and Smith, 1982) does not indicate any consistent body of evidence to this effect. It should be noted that use of anticholinergic (antiparkinsonian) drugs might be in theory related to the emergence or expression of tardive dyskinesia through their intrinsic neuropharmacological actions or as a proxy for such extrapyramidal side effects. Again, there was no consistent evidence for differences in extent of exposure to anticholinergics between patients with and without tardive dyskinesia (Kane and Smith, 1982). More recently, it has proved possible to readdress these issues in a more systematic manner. Thus, in prospective studies, Kane et al. (1986a) reported that the incidence of tardive dyskinesia was markedly higher in a group of patients who had initially shown severe extrapy-

NEUROLEPTICS A N D TARDIVE DYSKINESIA

333

ramidal side effects; following 4 years of exposure to neuroleptics, the incidence was 37% in those with severe initial extrapyramidal problems and 15% in those without such disorder. Similarly, Chouinard et al. (1986) have reported, from studying a cohort of patients without evidence of tardive dyskinesia, that initial parkinsonism score was the best predictor of manifest involuntary movements 5 years later; additionally, there was an association with an interaction term that indicated that brain-damaged patients who received an increase in dose of antiparkinsonian medication were further vulnerable to subsequently manifesting tardive dyskinesia. T h e presence of drug-induced parkinsonism has been reported to be associated with tardive dyskinesia both in schizophrenia (Kolakowska et al., 1986) and in affective disorder (Wolf et al., 1985). In our own cross-sectional study (Waddington et al., 1987), we found that the severity of buccal-lingual-masticatory dyskinesia among those prominently affected, but not the presence or absence of such movement disorder, was negatively associated with current use of anticholinergics and positively associated with extent of prior exposure to such drugs. While this would be consistent with earlier parkinsonism being a vulnerability factor for tardive dyskinesia, our data might alternatively suggest that this may relate more to the severity of orofacial dyskinesia within those patients already tending toward vulnerability rather than to the overall likelihood of emergence of involuntary movement disorder. Interestingly, cognitive dysfunction, negative symptoms, and structural brain pathology have also been reported to be associated with the presence and severity of neuroleptic-induced parkinsonism, whether inferred from a history of antiparkinsonian drug treatment o r evaluated directly (Luchins et al., 1983; Hoffman et al., 1987).

K. DENTAL STATUS There is a long-standing literature that dental problems, particularly edentulousness, may be more common in those patients with as opposed to those without buccal-lingual-masticatory dyskinesia, both in patients who have and those who have not been exposed to neuroleptics (Brandon et d.,1971; Sutcher et al., 1971; Koller, 1982). However, a debate has centred on the nature of the relationship between these two factors: Does edentulousness alter sensorimotor feedback within the buccal cavity and thus contribute to the promotion and/or exacerbation of oral dyskinesia, or do such involuntary oral movements contribute to poor dental hygiene and difficulty in retaining dentures in the mouth and thus contribute to a state of edentulousness?

334

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Our own results point towards a different explanation. We have found edentulousness to be reliably associated with both the presence and the severity of buccal-lingual-masticatory dyskinesia in chronic schizophrenia (Waddington et al., 1987); however, edentulousness was the only variable found also to predict the presence of limb-trunk dyskinesia. Such an unexpected finding suggests that edentulousness may be an epiphenomenon of a more fundamental process that is of‘ some importance for the emergence of involuntary movements. It may be a proxy for a process of ‘progressive deterioration (or the late consequences of early developmental anomaly) characterized by global physical, mental, and behavioral dysfunction; it is such features that characterize so many older patients with chronic schizophrenia or Alzheimer’s disease who show involuntary movements (Waddington et al., 1987; Molsa et al., 1987). This would suggest some association between involuntary movement disorder and “organicity” in a more whole body sense, as outlined in Section V. L. SMOKING HABITS

Two studies (Yassa et al., 1987b; Binder et al., 1987) have reported that the prevalence of tardive dyskinesia is higher in neuroleptic-treated psychiatric patients who are cigarette smokers than in those who are not. Such an association might have its basis in one or more of several processes: the intrinsic neuropharmacological properties of nicotine (or other tobacco constituents), an action of nicotine to influence the pharmacokinetics of neuroleptic drugs, or some association with another vulnerability factor. These differences in prevalence between smokers and nonsmokers could not be accounted for by conventional demographic characteristics; though smoking is known to have some effect on neuroleptic drug metabolism and elimination, in neither study did this appear to account for the overall pattern of results when current daily dosages were compared. In our own studies (Youssefand Waddington, 1987), we did not find smoking to be either more common or heavier in patients with as opposed to those without buccal-lingual-masticatory dyskinesia. However, over a 32-month follow-up period, those with such movement disorder were more likely to show morbid signs or have a cause of death suggestive of smoking-related pathology, such as respiratory and cardiovascular problems or a vascular demise. Thus, there appeared to be an association between buccal-lingual-masticatory dyskinesia and a greater sensitivity to the deleterious respiratory and vascular effects of

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smoking. Campbell et al. (1983) have previously reported the prevalence of involuntary movements in medical outpatients with chronic obstructive pulmonary disease to be higher than that in a control group of rheumatology patients and normal elderly. This relationship was independent of history of exposure to neuroleptics, with the overall severity of involuntary movements shifted upward in those who had received such treatment; a positive correlation between mean arterial pC02 and severity of such movement disorder indicated an association with hypoxia. The above data would suggest that chronic subclinical hypoxic states can lead to an increased vulnerability to involuntary movements both in patients who have and have not been treated with neuroleptics. Smoking would be one factor capable of inducing or exacerbating such a state, and this would be further consistent with an association between involuntary movements and “organicity” in a whole-body sense.

M. FAMILIAL-GENETIC FACTORS Regarding familial aspects of neuroleptic-associated involuntary movements, concordance for such movement disorder has been reported in a schizophrenic sibling pair and concordance for its absence noted in four similar sibling pairs, including one set of twins (Yassa and Ananth, 1981). There is a report that older, chronically ill schizophrenic inpatients with tardive dyskinesia appeared less likely to have any family history of schizophrenia (Bartels et al., 1985), but no such relationship was evident among the first-degree relatives of younger outpatients with a diagnosis of schizophrenia or schizoaffective disorder; indeed, in these younger outpatients, there was an excess of affective disorder in the first-degree relatives of those with tardive dyskinesia (Wegner et al., 1985a). Concordance for involuntary movements in two schizophrenic brothers has been reported (Weinhold et al., 1981); both showed some degree of cognitive impairment. In our own studies, we have reported concordance for buccallingual-masticatory dyskinesia and cognitive dysfunction in each of a rare group of four schizophrenic inpatient siblings (Waddington and Youssef, 1988b). Additionally, there was an excess of schizophrenia (but not of affective disorder) in the first-degree relatives of those younger schizophrenic outpatients with, as opposed to those without buccallingual-masticatory dyskinesia, in addition to an association between such movement disorder and cognitive dysfunction (Waddington et al., 1988).

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Such data raise the issue of whether the greater cognitive and neurological impairment that appears evident in many patients with involuntary movement disorder might be in part related to a familial component to their illness. Using a variety of neuropsychological tests, cognitive dysfunction and soft signs have been reported to be more evident in schizophrenic patients with as opposed to those without a family history of such illness (Orzack and Kornetsky, 1971; Asarnow et al., 1978; Walker and Shaye, 1982). There is evidence that manifestation of such deficits is a familially transmitted abnormality of neurological state in schizophrenia (Kinney et al., 1986). If this is to influence the consequences of subsequent long-term neuroleptic treatment, an interaction with neurodevelopmentally determined processes is implied. N. INTERIM SUMMARY In Sections VI,A-D, evidence was reviewed to indicate that among conventional demographic, clinical and treatment variables, only increasing age appears a statistically robust, general vulnerability factor for the emergence of tardive dyskinesia. The paucity of consistent associations with a range of indices of duration and intensity of treatment with neuroleptic (and other) drugs is evident, while preliminary evidence for the greater vulnerability of particular diagnostic groups is provocative and requiring of further systematic study. This picture must be contrasted with the evidence reviewed in Sections VI,E-H, which appears to indicate a consistent (though by no means invariable) overrepresentation of both indirect and direct signs of organic brain dysfunction in patients with as opposed to those without tardive dyskinesia. ‘The evidence to this effect is diffuse, often subtle but occasionally gross, sometimes absent, and may depend upon the topography and severity of those involuntary movements considered; however, it appears generally reliable across modalities and diagnostic groupings. While it points towards no specific pathophysiological process, such heterogeneity may in itself be of fundamental significance. Additionally, other putative vulnerability factors reviewed in Sections VI,I-M appear to be compatible with, and often further supportive of, an association between tardive dyskineska and otherwise unspecified organic brain dysfunction. Critically, the limited available evidence from prospective studies at both the neuropsychological and electroencephalographic levels suggests that putative organic signs are overrepresented amongst those patients not yet manifesting involuntary movements but in whom such movement disorder will subsequently emerge.

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VII. PathophysiologicalMechanisms

A. TOPOGRAPHY OF INVOLUNTARY MOVEMENTS Before the issue of pathophysiology can be considered, it is first necessary to clarify whether tardive dyskinesia is a homogeneous syndrome. Even when one has excluded putative variants such as tardive dystonia, tardive Tourette syndrome, and tardive akathisia (see Section I,A), there still remains the critical issue of heterogeneity in the topography of classical choreoathetoid movements. Lateralization of such involuntary movements has been studied by several investigators. Waziri (1980) reported that both schizophrenic and affective patients with tardive dyskinesia, all right-handed, showed more prominent right-sided movements of both orofacial and limb regions; this suggested greater pathophysiology in the left hemisphere. In a subsequent study, Myslobodsky et al. (1984) reported primarily on tremor; therefore the implications for tardive dyskinesia of their negative findings are unclear. A trend towards more right-sided involuntary movements of the limbs in right-handed patients was noted by Wilson et al. (1984), but orofacial movements were not analyzed. Conversely, Altshuler et al. (1988) found more severe involuntary movements on the left side of the face with no asymmetry in limb dyskinesia. It should be noted that movements of the right side of the mouth appear to be more rapid and more excursive in normal individuals (Wolf and Goodale, 1987), and normal motor asymmetries may be programmed at the very earliest stages of neurodevelopment (Hopkins et al., 1987). However, in relation to tardive dyskinesia, the data on this issue are as yet too inconclusive to have clear pathophysiological implications. In contrast, there is some weight of evidence to suggest that the involuntary buccal-lingual-masticatory and limb-trunk movements of tardive dyskinesia d o not share a common pathophysiology. Among patients with tardive dyskinesia, orofacial movements show a much greater age dependency than does limb-trunk dyskinesia (Kidger et al., 1980; Glazer et al., 1988). Similarly, involuntary orofacial and limbtrunk movements can be readily dissociated statistically in terms of a wide range of correlates (Spohn et al., 1985; Waddington et al., 1987; Glazer et al., 1988). More specifically, buccal-lingual-masticatory dyskinesia is much more reliably associated with cognitive dysfunction, negative symptoms, and neuropathological changes than are limbtrunk movements (Waddington et al., 1987; Molsa et al., 1987; DeWolfe et al., 1988). Such a profile of results strongly implies that these two

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regional distributions of involuntary movement are pathophysiologically distinct. When the relative prevalence of these two topographies of movement disorder is additionally considered, it appears that buccallingual-masticatory movements constitute the “core” symptomatology of tardive dyskinesia.

B. THEDOPAMINE RECEPTOR SUPERSENSITIVITY HYPOTHESIS T h e hypothesis that tardive dyskinesia has its basis in striatal (caudate-putamen) dopaminergic hyperfunction, and more specifically in striatal dopamine receptor supersensitivity, has long dominated pathophysiological theorizing. It has its origins in animal studies (Klawans and Rubovitz, 1972), whereby the treatment of rodents with neuroleptic drugs for u p to a few weeks, followed by their subsequent withdrawal, renders them supersensitive to the induction of perioral stereotyped behaviors when challenged with dopamine receptor agonists; such dopamine receptor supersensitivity, occuring as an adaptive response to their chronic blockade by neuroleptics, can be demonstrated directly in the striatum of such animals using radioligand binding techniques (Muller and Seeman, 1978). The endurance and influence of this hypothesis (Klawans et al., 1980) should be considered in relation to the following points (Waddington, 1984; Waddington et al., 1985b): 1. Such perioral and neurochemical responses are associated with abrupt withdrawal from relatively brief periods of neuroleptic treatment in relation to the animal’s lifespan, while the clinical syndrome commonly emerges only after years of treatment, often during such treatment. 2. Such perioral stereotypies are not manifested spontaneously and require challenge with a dopamine receptor agonist, while the clinical syndrome is a spontaneously occuring disorder. 3. Dopamine receptor supersensitivity phenomena decline relatively rapidly after neuroleptic withdrawal, while the clinical syndrome can persist for prolonged periods of time in comparable circumstances. 4. Such responses can be reliably and consistently induced in animals, while the clinical syndrome is not readily predicted and is of variable prevalence. 5 . Both dopaminergic neurotransmission and the ability of dopamine receptors to develop such supersensitivity decline with advancing age, whereas the clinical syndrome becomes more evident in the elderly.

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Studies in rodents using much more prolonged treatment periods of 6 months or more have consistently failed to demonstrate spontaneous orofacial dyskinesia that has the characteristics of a dopamine receptor supersensitivity phenomenon (Waddington et al., 1983; Waddington and Molloy, 1987). There is an emerging body of evidence that the role of dopaminergic mechanisms in the control of motor behavior may not be a unitary phenomenon but may involve functional interactions between D- 1 and D-2 dopamine receptor systems (Waddington and O’Boyle, 1987, 1989). On the basis of rodent studies, it appears that acute perioral dyskinesias are particularly associated with D- 1 hyperfunction during concurrent D-2 hypofunction (Waddington et al., 1989~).As there is a report that D-1 receptor density is increased and D-2 receptor density decreased in the human brain with advancing age (Morgan et al., 1987) and that D-1 agonist-induced perioral dyskinesia may be more evident in aged animals (Molloy and Waddington, 1988), it has been speculated (Gerlach and Casey, 1988) that such processes may be involved in the pathogenesis of tardive dyskinesia. However, the significance of such readily reproducible, acute pharmacological studies in rodents for a clinical syndrome of late onset associated with both individual vulnerability and wide interpatient variability remains unclear. Also, there is evidence that D-1-D-2 interactions in both nonhuman and human primates may differ functionally from those evident in rodents (Waddington, 198913). Clinical support for the dopamine hyperfunction-receptor supersensitivity hypothesis might appear to be found in the clinical pharmacological profile of tardive dyskinesia: for example, the abilities of dopaminergic drugs such as L-dihydroxyphenylalanine (L-DOPA)to exacerbate involuntary movements acutely and to induce them as an adverse effect in the treatment of Parkinson’s disease or the ability of acute challenges with neuroleptic (dopamine receptor-blocking) or dopamine-depleting drugs to attenuate at least temporarily the expression of dyskinesia (Jenner and Marsden, 1986). However, such a clinical pharmacological profile may simply be revealing the important role of dopamine as a modulator of extrapyramidal function; this is well established and clearly need not indicate that tardive dyskinesia has a dopaminergic pathophysiology ( Waddington, 1986). Indeed, as L-DOPA dyskinesia has been shown to have a topography distinct from the classical buccal-lingual-masticatory features of tardive dyskinesia (Karson et al., 1983), this would be contrary to the dopaminergic hyperfunction hypothesis. A further problem for the hypothesis is the extent of evidence that

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tardive dyskinesia can coexist with neuroleptic-induced parkinsonism (Richardson and Craig, 1982; Wolf et al., 1983b; Jankovic and Casabona, 1987). Parkinsonism is a presumed dopaminergic hypofunction syndrome; therefore, if tardive dyskinesia has its basis in dopaminergic hyperfunction, their coexistence would appear to be pathophysiologically incompatible. While the clinical pharmacological profiles of these two syndromes have been presumed to be opposite, systematic studies of tardive dyskinesia do not indicate that it shows the profile expected of a pathophysiological inverse of parkinsonism (Gardos et al., 1984; Lieberman et al., 1988). Regarding more direct tests of the dopamine receptor supersensitivity hypothesis, it has been possible to apply radioligand binding techniques to measure dopamine receptors in postmortem brain tissue from schizophrenic patients rated in life for the presence and severity of involuntary movements (Waddington, 1985). In the first such study (Crow et al., 1982), there were no differences in either D-1 or D-2 receptor binding in the putamen between patients with and without such movement disorder, and there were no relationships between binding values and the severity of involuntary movements. A further study (Cross et al., 1985) confirmed these negative findings and extended them to D-1 and D-2receptors in the nucleus accumbens also; there were no differences in the concentrations of dopamine and its metabolite dihydroxyphenylacetic acid (DOPAC) but some increase in homovanillic acid (HVA) in the putamen and nucleus accumbens of schizophrenic patients with involuntary movements. Imaging of D-1 and D-2 receptors in the living human brain by positron emission tomography has the potential to clarify dopamine receptor status in patients with and without tardive dyskinesia (Waddington, 1989~). The above studies, when considered together with the insubstantial nature of the indirect evidence, indicates that the dopamine receptor supersensitivity hypothesis has major problems. This hypothesis has dominated research for more than a decade and a half, and there is still an evident reluctance in some quarters to accept its demise. While it may be of relevance for readily reversible dyskinesias that emerge when neuroleptic treatment is withdrawn or reduced (Baldessarini, 1979), it appears unable to account for typical, persistent buccal-lingualmasticatory dyskinesia that emerges during the long-term administration of neuroleptics. It has channeled both studies and theorizing down a rather narrow path and may have impeded consideration of alternative hypotheses and of other important clinical issues concerning tardive dyskinesia.

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C. NONDOPAMINERGIC NEUROCHEMICAL HYPOTHESES Although the dopamine receptor supersensitivity hypothesis has held center stage, other hypotheses have been proposed and investigated. T h e neurochemical data consistent with a noradrenergic hyperfunction hypothesis for tardive dyskinesia have been reviewed by Kaufmann et al. ( 1986); interestingly, among patients with tardive dyskinesia there was evidence for an inverse relationship between noradrenergic neurochemical and structural (i.e., CT) abnormalities. However, contrary negative findings have been reported (Glazer et al., 1987). More studies are iherefore required to evaluate the hypothesis systematically and to derive from it a putative pathophysiological process. There is currently much interest in a y-aminobutyric acid (GABA) hypothesis of tardive dyskinesia (Fibiger and Lloyd, 1984). I n the brains of monkeys who had developed dyskinesia during several years of treatment with neuroleptic drugs, there were reduced concentrations of GABA and its synthesizing enzyme glutamic acid decarboxylase in the globus pallidus, substantia nigra, and subthalamic nucleus; these changes were not evident in a control group of monkeys who had received similar neuroleptic treatment without the emergence of dyskinesia (Gunne et al., 1984). Also, levels of the dopamine metabolites HVA and DOPAC were reduced in the caudate nucleus, though not in the putamen. On the basis of the ability of some GABAergic drugs to attenuate the symptoms of tardive dyskinesia, Thaker et al. (1987) studied GABA concentration in the cerebrospinal fluid of schizophrenic patients and found it to be reduced in those with tardive dyskinesia. CABAergic neurons in the extrapyramidal motor system can both modulate activity in the dopaminergic system and be modulated by such activity. Additionally, GABAergic neurons appear to be part of the effector system for striatal function and are therefore capable of influencing motor function independent of dopaminergic activity. ‘These complex interrelated and independent roles of GABAergic a d dopaminergic neurons in the extrapyramidal motor system have been reviewed in the context of tardive dyskinesia by Gerlach and Casey (1988). However, two caveats must be mentioned. In the primate studies of Gunne et al. (1984), all dyskinetic monkeys manifested limb-trunk movements, with only one additionally manifesting a buccal-lingualmasticatory syndrome. Also, GABA concentrations have been found not to l..c reduced in the globus pallidus, substantia nigra, and nucleus accumbeils of postmortem brains from schizophrenic patients with

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involuntary movements in life, in comparison with normal individuals and schizophrenic patients who had not shown such movement disorder (Cross et al., 1985); while schizophrenic patients with involuntary movements showed reduced GABA concentrations in the caudate nucleus, this arose through elevated values in similar patients without movement disorder, and there were no differences on comparison with normal subjects. There is no consistent relationship between involuntary movements and concentrations of choline acetyltransferase, cholinergic and GABA receptors, and the neuropeptides cholecystokinin, substance P, neurotensin, somatostatin, and vasoactive intestinal polypeptide in the postmortem brains of schizophrenic patients rated in life for such movement disorder (Cross et al., 1985). Conversely, there are other putative neurochemical correlates of tardive dyskinesia (see, e.g., Mukherjee et al., 1985; Richardson et al., 1989) that await to be developed into heuristic pathophysiological models. For these and most of the preceding hypotheses, the mechanism(s) by which long-term neuroleptic treatment might induce such pathophysiology is not clear. Also, many authors have maintained that the inconsistency of those neurochemical studies addressing the several hypotheses outlined above may reflect a variety of causal mechanisms, that is, that tardive dyskinesia is heterogeneous with regard to pathophysiology. It is likely that buccal-lingual-masticatory and limb-trunk dyskinesias are at least in part pathophysiologically distinct, and it is possible that transient withdrawal-emergent dyskinesias may involve a dopaminergic mechanism. However, it is the contention of this reviewer that there is likely to be a different, but unitary, basis for the core syndrome of persistent buccal-lingual-masticatory dyskinesia.

D. AN ALTERNATIVE PERSPECTIVE Most pathophysiological theories of tardive dyskinesia center on neurochemical dysfunction in the basal ganglia. The extrapyramidal motor system clearly influences the expression of buccal-lingualmasticatory (and other) dyskinesias, but need it be the locus of the fundamental abnormality? A n alternative perspective might consider how and where such buccal-lingual-masticatory motor patterns are generated. I n the pontine reticular formation there is evidence for a substrate in a central pattern generator capable of elaborating the basic pseudocyclic pattern of activity that is expressed in these masticatory muscle regions

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(Dubner et al., 1978; Luschei and Goldberg, 1981); related patterns of lingual and buccal-facial movements also appear to be elaborated in this general region (Dubner et al., 1978; Rinn, 1984). Additionally, this region appears to be a locus for the origin of respiratory rhythms (Onimaru et al., 1988), which appear to be disrupted in more instances of tardive dyskinesia than may previously have been appreciated (Section V). Pontine reticular pattern generator function can be influenced by some sensory and psychological mechanisms; by cortical activity, though this seems more complex than originally envisaged; and by limbic activity (Dubner et al., 1978; Rinn, 1984; Takeuchi et al., 1988). It should be emphasized that although tardive dyskinesia is commonly refered to as a disorder of abnormal involuntary movements, the buccal-lingual-masticatory syndrome is not abnormal per se, but rather is an inappropriate and overelaborated manifestation of innate and even fundamental motor patterns. Thus, the core pathophysiology of tardive buccal-lingual-masticatory dyskinesia may reside in an effect of longterm exposure to neuroleptic drugs on a center generating and elaborating such motor patterns and/or on associated regions or centres influencing its function. However, an equally fundamental question is the mechanism of any such putative change in neuronal function induced by neuroleptics. There is no direct evidence that these are related to the classical dopamine receptor antagonist actions of such drugs, and a number of recent studies have indicated them to have effects on more general aspects of cellular function. Whether long-term treatment with neuroleptics can result in cell loss has long been a contentious issue (Nielsen and Lyon, 1978). Some evidence, at least for haloperidol, is more suggestive of an effect on neuronal cytoarchitecture by way of synaptic rearrangements (Benes et al., 1985a,b). Two in vitro studies have reported that relatively high concentrations of neuroleptics can have cytotoxic effects. This is particularly true of the phenothiazines and thioxanthenes, less so of haloperidol and loxapine, and least of molindone; interestingly, these neurotoxic effects of the phenothiazines were accentuated in hypoxic cells (Lehnert, 1987; Munyon et al., 1987). It would be important to determine whether similar effects might be induced in vivo by prolonged treatments that result in lower drug concentrations. While there is inconsistent evidence on whether long-term neuroleptic treatment does or does not effect lipid peroxidation processes in animals (Roy et al., 1984; Cohen et al., 1985; Dexter et al., 1987), there is recent clinical evidence that levels of lipid peroxidation products are found in higher concentrations in the cerebrospinal fluid of patients receiving phenothiazines, especially those experiencing “ill-effects” thereof (Pall et al., 1987). This would be

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consistent with a free-radical toxicity hypothesis of tardive dyskinesia (Cadet et al., 1986), and there is preliminary evidence that the lipidsoluble antioxidant a-tocopherol (vitamin E) may have some therapeutic effect on this movement disorder (Lohr et al., 1987). Using an alternative approach, Cohen and Zubenko (1985) have reported that acute treatment with phenothiazines, but not haloperidol or imipramine, induced marked changes in the biophysical properties of rat brain membranes; they have also reported similar changes to occur to a greater extent in the blood platelet membranes of patients with, as opposed to those without, tardive dyskinesia (Zubenko and Cohen, 1986). These putative influences of neuroleptic drugs on such fundamental neurobiological processes require much further systematic study. However, they are more readily compatible with the concept of vulnerability to tardive dyskinesia expressed in Section V1,E-H. If subtle disease- or age-related organic brain dysfunction is indeed a predisposing factor for the emergence of tardive dyskinesia during long-term neuroleptic therapy, and if neuroleptics are themselves associated with subtle neurotoxicological effects at the cellular level, one can conceive of these two deleterious processes interacting in those individuals in whom this movement disorder emerges. Thus, these two dimensions of causality need not be distinct but rather may synergize in the development of' buccal-lingual-masticatory dyskinesia through a unitary pathophysiological mechanism.

VIII. Synthesis: The Conflict of Paradigms

Reconsideration of perhaps subtle organic brain dysfunction as an important vulnerability factor for neuroleptic-associated buccallingual-masticatory dyskinesia requires that we address a critical conceptual issue: Can such vulnerability be present to such an extent as to lead to the emergence of overt involuntary movement disorder even in the absence of exposure to neuroleptics? T h e evidence reviewed in Tables I-IV clearly indicates that this appears to be the case; thus, the baseline level of such movement disorder in untreated illness has been underestimated, seriously so for patients with severe, chronic psychosis. These disease-related involuntary movements appear to have characteristics of topography, age dependency, and acute neuroleptic sensitivity that are indistinguishable from those of tardive dyskinesia (Waddington and Crow, 1988; Waddington and Youssef, 1989a). Also, putative organic vulnerability factors that appear to apply to

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the emergence of tardive dyskinesia seem also to apply to the emergence of involuntary movements in neuroleptic-naive populations; such relationships have been documented for cognitive dysfunction, negative symptoms, poor prognosis, and release of developmental reflexes in untreated schizophrenia (Yarden and DiScipio, 1971; Villeneuve et al., 1974; Owens and Johnstone, 1980; Waddington and Youssef, 1989a) and for cognitive dysfunction in untreated Alzheimer’s disease patients (Molsa et al., 1984). Thus, there is little evidence that they are fundamentally distinct. An important philosophical analysis of this critical but all too casually neglected issue has been made by Rogers (1985). He has identified what he terms the “conflict of paradigms”: Does motor disorder in (usually severe) untreated psychiatric illness such as schizophrenia imply the presence of some secondary neurological process or can mental and motor disorders both be an expression of a unitary cerebral disorder underlying the totality of the patient’s condition? That is, it may not be necessary to attribute such involuntary movement disorder in untreated populations to superimposed neurological disease if the more serious psychiatric illnesses are themselves regarded as a neurological disorder. Such psychiatric illness may per se be associated with an increased likelihood of neurological features such as cognitive dysfunction or structural brain pathology and with involuntary movements. Thus, long-term neuroleptic treatment may act in this situation as a form of catalyst in patients so disposed (Waddington et al., 1983; Owens, 1985). Described above (Section VI1,D) is a pathophysiological model that is conipatible with these notions. In conclusion, the available evidence supports the view (Waddir.gton, 1987, 1988a) that long-term treatment with neuroleptics does not “cause” tardive dyskinesia. Rather, their fundamental action in this regard may be (1) to interact with a neurological process that is (usually) an intrinsic neurodevelopmental o r atrophic component of the disorder for which thzt treatment was prescribed, and (2) to hasten the emergence of an inappropriate and overelaborated form of an innate buccal-lingual-masticatory motor pattern that has an unappreciatedly high likelihood of ultimately occurring spontaneously with increasing cerebral dysfunction.

Acknowledgments

The author is supported by the Health Research Board of Ireland and the Royal College of Surgeons in Ireland.

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Yassa, R., and Jones, B. D. (1985). Psychosomatics 26, 305-313. Yassa, R., and Lal, S. (1986). Acta Psychiatr. Scand. 73, 506-510. Yassa, R., and Nair, N. P. V. (1987). Acta Psychiatr. Scand. 75, 209-21 1. Yassa, R., and Schwartz, G. (1984). B i d . Psychiatry 19, 441-444. Yassa, R., Ghadirian, A. M., and Schwartz, G. (1983).J. Clin. Psychiatry 44,410-412. Yassa, R., Mohelsky, H., Dimitry, R., and Schwartz, G. (1984a). Am. J . Psychiatry 141, 1018- 1019. Yassa, R., Nair, V., and Schwartz, G. (1984b). Psychosomatics 25, 135-138. Yassa, R., Nair, V., and Schwartz, G. (1984~).Psychosomatics 25, 852-855. Yassa, R., Nair, V., and Schwartz, G. (1986). Bzol. Psychiatry 21, 1291-1297. Yassa, R., Camille, Y., and Belzile, L. (1987a).J. Clin. Psychophamacol. 7, 243-246. Yassa, R., Lal, S., Korpassy, A., and Ally, J. (1987b). Biol. Psychiatry 22, 67-72. Yesavage, J. A., Tanke, E. D., and Sheikh, J. I. (1987). Arch. Gen. Psychiatry 44,913-915. Youssef, H. A,, and Waddington, J. L. (1987). Acta Psychiatr. Scand. 75, 74-77. Youssef, H. A,, and Waddington, J. L. (1988a). J . Neurol., Neurosurg. Psychiatry 51, 863-865. Youssef, H. A., and Waddington, J. L. (1988b). Acta Psychiatr. Scand. 78, 523-525. B i d . Psychiatry 23, 791-796. Youssef, H. A., Waddington, J . L. (1988~). Youssef, H. A., and Waddington, J. L. (1989). Inl. Clzn. Psychophamacol. 4, 55-59. Zeitlhofer, J., Brainin, M., and Reisner, T. (1984).J. Neural. 231, 266-268. Zubenko, G. S., and Cohen, G. M. (1986). Psychophannacology 88, 230-236.

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NERVE BLOOD FLOW AND OXYGEN DELIVERY IN NORMAL, DIABETIC, AND ISCHEMIC NEUROPATHY By Phillip A. Low, Terrence D. Lagerlund, and Philip G. McManis Deporhnent of Neurology Mayo Clinic and Moyo Foundation

Rochester, Minnesota 55905

I. Special Anatomy of Nerve Microvasculature

11.

111.

IV.

V.

VI.

VII.

VIII.

A. Double Blood Supply B. Nerve Vascular Anatomy C. Capillary Density Special Physiology of Nerve Microvasculature A. Peripheral Nerve Autoregulates Poorly B. Peripheral Nerve Is a Nutritive Capacitance Microvascular System C. Peripheral Nerve Adapts to Hypoxic Stress Oxygen Delivery A. Normal Delivery and Critical Oxygen Tension B. Factors Affecting Oxygen Supply and Endoneurial Oxygen Tensions C. Factors Reducing Oxygen Delivery in Model Neuropathies D. Mathematical Modeling of Nerve Oxygen Supply Regulation of Blood Flow A. Intrinsic Mechanisms B. Extrinsic Mechanisms Nerve Blood Flow Measurements A. Methods B. Mathematical Modeling of Hydrogen Washout Diabetic Neuropathy A. Evidence of Endoneurial Ischemia in Diabetic Neuropathy B. T h e Hypoxic Hypothesis C. Evidence for the Hypoxic Hypothesis D. Mechanisms of Microvascular Ischemia E. Suggested Pathogenesis of Diabetic Neuropathy F. Results of Mathematical Modeling Ischemic Neuropathy A. Early Observations B. Experimental Models of Ischemic Neuropathy C. Pathology of Nerve Ischemia D. Nerve Conduction E. Physiology of Centrifascicular Infarction F. Molecular Mechanisms of Nerve Ischemia G. Results of Mathematical Modeling Edematous Neuropathy A. Pathophysiology B. Endoneurial Pressure

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Copyright 0 1989 by Academic Presa, Inc. All rights of reproduction in any form reserved.

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C. Blood Flow in Nerve Edema D. Results of Mathematical Modeling References

1. Special Anatomy of Nerve Microvasculature

A. DOUBLE BLOODSUPPLY Nerves are unique in having two separate vascular systems that have extensive anastomoses between them (Adams, 1943; Lundborg and Branemark, 1968; Blunt and Stratton, 1956). These are the extrinsic system, consisting of the regional nutritive arteries, arterioles, and venules together with the epineurial vessels, and the intrinsic system, which consists of the longitudinal microvessels within the fascicular endoneurium. Epineurial and perineurial vessels form the anastornotic link between these two systems. There is a rich network of interconnecting vessels within each of these systems. T h e plethora of anastomotic vessels within and between each source of blood supply confers a resistance to ischemia on peripheral nerve so that nerve suffers functional or structural changes only when there are widespread and diffuse vascular alterations. Other factors adding to the resistance to ischemia include the high basal nerve blood flow (NBF) relative to the metabolic needs of nerve (large safety margin), and the ability of peripheral nerve to utilize alternative sources of energy such as anaerobic metabolism. The presence of these anastomoses has been well documented by Lundborg and Branemark (1968), who identified vessels with intermittently stagnating and/or constantly reversing flow by means of vital microscopy, and by Bell and Weddell (1984b),who described erratic and patchy filling of longitudinal vessels using microradiography and injections of colored materials. These findings are consistent with the vessels acting as arteriovenous or arterioarterial anastomoses. The relative importance of these two vascular systems has been debated. Some authors believe that each is fully capable of maintaining the nutritive requirements of peripheral nerve because any reduction in NBF through either system is compensated by means of profuse anastomoses between them. Lundborg and Branemark (1968) report the consensus opinion that individual segmental nutrient arteries do not dominate the blood supply to nerve segments because the longitudinal anastomoses create overlapping between the territories of these segmental arteries. This is in keeping with the surgical experience that a nerve

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can be mobilized over a considerable length without impairing its function. Subsequent studies, however, have suggested that this resistance to ischemia is more a function of the low metabolic demand of nerve and the large safety margin between the baseline NBF and the minimal perfusion requirements than a reflection of the efficiency of the intrinsic system (see Section IV, B, 1). Others describe certain pathological changes in nerve as resulting from differential reductions in NBF in the extrinsic system (see, e.g., Sladky et al., 1985). It has been suggested that the centrifascicular infarct pattern commonly seen in severe nerve ischemia is due to focal defects in perfusion of the outer annulus (or subperineurial region). In a recent series of experiments, we examined these conflicting theories. We measured NBF after ligating individual component vessels of the extrinsic system and after producing a global reduction in tissue perfusion by making the experimental animals hypotensive. We found that ligation of individual arteries produced a reduction in NBF in segments of peripheral nerve supplied predominantly by that artery and that there are watershed areas between these arterial territories. However, in no instance did we find any difference in NBF between the subperineurial (outer) and the centrifascicular (inner) regions. Furthermore, measurements of NBF in hypotensive rats revealed reductions in NBF directly proportional to the blood pressure reduction, but there were again no differences between subperineurial and central regions. These findings support the concept that the extensive extraneural and endoneurial anastomoses are effective in maintaining maximal, even perfusion of the entire ischemic nerve within the limits of the available perfusion pressure. They provide no evidence to support the hypothesis that occlusion of single nutrient arteries causes relatively greater ischemia of the center of the nerve, nor do they support the alternative hypothesis that subperineurial axons are spared because the greater capillary density in this region has a protective effect. Thus, experimental work suggests that both systems are important in maintaining NBF and that reductions in flow through either vascular bed will reduce total nerve perfusion. When individual nutrient branches of the extrinsic system are occluded, there is a regional reduction in NBF in a longitudinal axis, but no radial differences in NBF occur. This suggests that longitudinal anastomoses are not as efficient as the regional (radial) anastomoses, at least in the nerve segments studied (M. Kihara and P. A. Low, 1989 unpublished observations). Blunt and Stratton (1956) made similar observations in distal segments but felt that the converse is true proximally, with the longitudinal system making a relatively greater contribution to nerve perfusion. This may be ex-

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plained by the greater abundance of nutrient vessels in the proximal segments providing a head of pressure for the intrinsic system that has dissipated by the time it reaches the distal parts of the limb, causing an increased dependence on regional nutrient arterioles. In addition, the nutrient vessels are much sparser distally. This increases the relative importance of each individual vessel. The degree of ischemia that results from a loss of perfusion through either system individually is not great enough to cause nerve infarction because nerve energy requirements are low and there is a large safety margin in the resting NBF. Pathological changes can be produced in experimental nerve ischemia only with very severe reductions in NBF, such as with ligation of the aorta and multiple regional arteries, or with the injection of high doses of arachidonic acid into a nutrient artery. This is consistent with the observations that vasculitis in humans produces ischemic damage in nerves only when there is very extensive occlusion of vessels and that large vessel occlusions cause nerve infarction only if there is extensive associated microvascular disease or a complete cessation of blood flow in the affected limb.

B. NERVEVASCULAR ANATOMY Endoneurial vessels have unique characteristics that are functionally important in conferring on nerve its characteristic responses to ischemic and hypovolemic stresses. Bell and Weddell (1984a,b) described the unusually large diameter of the capillaries and the increased intercapillary distances in nerves when compared with other tissues such as brain or muscle. They pointed out that these large capillaries closely resemble postcapillary venules but concluded that they are true capillaries because there are no smaller vessels within the substance of nerve, because they stain for alkaline phosphatase (unlike conventional venules), and because they do not have the permeability or the susceptibility to injury usually associated with venules. These morphological differences in nerve capillaries create two disadvantages for peripheral nerve. First, the large capillaries make nerve susceptible to small changes in blood volume and perfusion pressure. Second, the increased intercapillary distances make perfusion of the endoneurium inefficient, since NBF is strongly affected by changes in intercapillary distance (McManis et uZ., 1986; Lagerlund and Low, 1987). This makes nerve particularly vulnerable to endoneurial edema, which further increases intercapillary distance. This vulnerability to edema is accentuated by the relative lack of arteriolar smooth

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muscle and internal elastic lamina (Bell and Weddell, 1984b), which makes the arterioles less rigid and more likely to collapse when endoneurial pressure rises. The large volume of blood in capillaries, however, may act as a reservoir for the rapid equilibration of ionic species and osmotic loads. In addition, the structure of these vessels may be an adaptation to allow flow to occur in either direction as needed (Bell and Weddell, 1984b). Reversals in flow in the resting state and in response to changes in perfusion pressure through various nutrient arterioles can readily be observed during nerve microsurgery and have been well described by Lundborg (1975). A third morphological difference between peripheral nerve and conventional vasculature is the poorly developed smooth muscle around endoneurial arterioles (Bell and Weddell, 1984b). This finding suggests that arteriolar caliber is not likely to vary with humoral o r sympathetic nervous input and is consistent with the observations of several authors that nerve autoregulates poorly (Low and Tuck, 1984; Rundqvist et al., 1985; Sundqvist et al., 1985). T h e epineurial vessels, however, have dense perivascular plexes of serotoninergic and peptidergic nerves, as well as noradrenergic nerve (Appenzeller et al., 1984; Rechthand et al., 1986). These nerves may originate from the nerve trunk supplied by these vessels, suggesting the possibility that peripheral nerves may have the ability to control their blood supply. Rechthand et al. (1986) showed that epineurial and perineurial vessels have adrenergic plexes around them but that endoneurial vessels do not. Lundborg (1970; also cited in Rechthand et al., 1986) demonstrated perineurial arteriolar vasoconstriction with stimulation of the lumbar sympathetic chain. These findings could be interpreted to indicate that there is neural control of vessels at the arterial and large arteriolar levels and possibly at the small arteriolar level, but not at the level of endoneurial vessels. Whether this potential source of control of NBF has any effects or not is unclear, although the relative absence of measurable autoregulation makes it unlikely. It will be of interest to measure NBF during sympathetic stimulation and after sympathetic denervation. C. CAPILLARY DENSITY

The density of capillaries and the distances between them are critical determinants of tissue perfusion in nerve. An increase in intercapillary distance has a greater impact on NBF than an adverse change in any other variable (McManis and Low, 1986). This suggests that the normal

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intercapillary distance is close to the upper limits for efficient diffusion since any increases in this distance reduced NBF and nerve oxygerl tension (McManis and Low, 1988; Low et al., 1985a). This concept is consistent with the findings of Bell and Weddell (1984a,b) that intercapillary distances are greater in nerve than in other tissues such as muscle. Nukada et al. (1985) found that capillary density is greater in the subperineurial region in the sciatic nerve of rats and, to a lesser degree, in the proximal tibia1 nerve, although intercapillary distances were different in the two regions only in the sciatic nerve. They suggested that this morphological difference between the center and the periphery of nerve accounts for the relative resistance to ischemia of the subperineurial region because the shorter diffusion distances provide a greater safety margin when perfusion pressure is reduced. In subsequent experiments, we were unable to document any differences in NBF between the center and the periphery of rat nerves at rest or under ischemic stress, casting doubt on the functional significance of Nukada’s observation. It is unknown whether the same capillary distribution variability applies to human nerves.

II. Special Physiology of Nerve Microvasculature

Because the peripheral nerve axon is extremely long relative to its diameter and is a great distance from its parent cell body, it is exquisitely dependent on nerve microenvironment for its blood supply, oxygenation, nutrition, and the removal of toxic metabolic products. The physiology of nerve microenvironment has been the primary research focus of our laboratory for over 10 years and since 1983 we have focused on the physiology of nerve ischemia. Prior to this time there was considerable information available on the pathology of peripheral nerve ischemia but only scant information on nerve microvascular physiology. Since then we have demonstrated that peripheral nerve is physiologically unique in several ways, information that has an important bearing on any study of nerve ischemia or diabetic neuropathy. A. PERIPHERAL NERVEAUTOREGULATES POORLY Autoregulation is the maintenance of constant blood flow when blood pressure (BP) is changed. The range of BP over which this occurs is

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known as the autoregulated range. Autoregulation is achieved by varying arteriolar tone and is myogenic rather than neurogenic in most tissues. There is a characteristic blood flow-BP curve, which is flat within the autoregulated range and sloping at either end. At the time we studied this question, such curves had not been reported in peripheral nerve. Smith et al. (1977) studied cat sciatic nerve and found an incremental NBF-BP relationship to 90 Torr, followed by a decline in NBF at higher BPs. Their observation was likely due to the effect of infused angiotensin in increasing arteriolar tone and reducing NBF. These earlier methods changed BP (the independent variable) by infusing an agent that directly changed arteriolar caliber (the dependent variable). We avoided this problem by using the gravity-regulated method of BP control (Low and Tuck, 1984). We inserted a large-bore cannula into the common carotid artery, which in turn was connected to a large-diameter reservoir containing rat blood. The animal’s BP was controlled by adjusting the height of the column of blood. We found a curvilinear relationship of NBF-BP, a relationship that is explainable on the basis of a passive system. Thus mammalian peripheral nerve autoregulates poorly if at all (Low and Tuck, 1984), a finding that has subsequently been confirmed in anesthetized (Rundqvist et al., 1985) and nonanesthetized rats (Sundqvist et al., 1985). When the rate of exsanguination is altered, the shape of the NBF-BP curve also changes. With steady-state recordings of blood flow using HBpolarography, a curvilinear NBF-BP relationship occurs (Low and Tuck, 1984). When exsanguination is rapid and blood flow is monitored in real time using laser Doppler velocimetry, a linear NBFBP relationship occurs (Takeuchi and Low, 1987). The difference may relate to the time-dependent response of arterioles to intravascular pressure alterations (Borgstrom et al., 1981). T h e concept of autoregulating and nonautoregulating systems is artificial since different organs autoregulate to different degrees. Furthermore, for intensively studied tissues like brain, there is regional, temporal, and segmental heterogeneity of autoregulation. This range of autoregulatory capacities means that there is a moment-to-moment distribution and redistribution of blood in such a way that tissues with the greatest metabolic needs will have relatively the most constant blood flow while tissues with the least needs will have blood flow that varies the most. T h e response to the question as to whether a tissue is autoregulated or not should be, Relative to what tissue?

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B. PERIPHERAL NERVEIs A NUTRITIVECAPACITANCE MICROVASCULAR SYSTEM Nerve microvasculature is physiologically a nutritive capacitance system (Takeuchi and Low, 1987), a system that is disadvantaged since a small change in blood volume results in a disproportionate change in NBF. The morphologic basis of this system appears to be the largediameter capillaries, which have a median diameter of 8-9 pm (Bell and Weddell, 1984a) and poorly developed arteriolar smooth muscle (Bell and Weddell, 1984b). C. PERIPHERAL NERVEADAPTSTO HYPOXIC STRESS Peripheral nerve is metabolically unique, being able to function relatively well on anaerobic metabolism and having powerful adaptive mechanisms. In the rat, nerve has about 10% of brain’s oxygen requirements, but similar energy stores (Stewart et al., 1965; Low et d.,198513). When maximally active, nerve increases its energy demands by less than 10076, whereas energy requirements in brain increase several-fold (Low et al., 1985b). T h e relatively large energy stores and the low resting and maximal energy expenditure enable nerve to function quite well on anaerobically generated high-energy phosphates. We have demonstrated that ischemic nerve will conduct impulses for many additional minutes when energy substrates are increased, as in diabetes (Low et al., 1985b). Indeed Fink (Fink and Cairns, 1982) demonstrated that components of mammalian peripheral nerve will conduct impulses for hours when provided with a limitless supply of glucose. Another strategy of resistance to ischemic conduction failure is a further downregulation of energy-requiring enzymes, a situation that occurs in aging (Low et al., 1986a) and in chronic hypoxia (Low et al., 198613). There is also a suggestion that acute hypovolemic stress also results in adaptive mechanisms; peripheral nerve responds by reducing its oxygen consumption acutely (Takeuchi and Low, 1987). 111. Oxygen Delivery

A. NORMAL DELIVERY AND CRITICAL OXYGEN TENSION Peripheral nerve tissue consumes oxygen at the rate of about 0.01 cm3 02/cm3tissueimin (Ritchie, 1973; Low et al., 1986a).This amount of

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oxygen must be delivered by nutritive blood flow. A typical value of nutritive blood flow is 0.158 cm3 blood/cm3 tissue/min (Low and Tuck, 1984). Since the solubility of oxygen in blood is about 3.4 x lop5 cm3 O2/cm3blood/Torr (Thews, 1960), dissolved oxygen would not be able to meet the metabolic needs of nerve unless the arteriovenous oxygen tension difference were 0.01/(0.158 X 3.4 X Torr = 1860 Torr. Thus nerve, like all tissues, relies primarily on oxygen bound to hemoglobin in erythrocytes. Since 1 gm of saturated hemoglobin can bind 1.34 cm3 of oxygen, blood with 15 gm Hb/100 cm3 carries 0.20 cm3 02/cm3 blood, and with the above value of blood flow, 100% saturated arterial blood carries 0.032 cm3 02/cm3tissue/min. Thus, a 30% drop in saturation in traversing the capillary bed will supply the resting metabolic needs of nerve. Measurements have been made of endoneurial oxygen tensions at multiple sites in sciatic nerves of rats, and histograms of the relative frequency of oxygen tension values have been published (Low et al., 1984, 1985a). These data revealed a mean oxygen tension of 29-31 Torr, with oxygen tensions ranging from -10-60 Torr in normal animals. Measurements of oxygen consumption of a segment of nerve as a function of oxygen tension demonstrate that consumption first begins to decrease as oxygen tension falls below about 26 Torr (Low et al., 1985a). Portions of the nerve with local oxygen tensions below this critical oxygen tension can be thought of as relatively hypoxic, since the enzyme systems involved in aerobic metabolism are not saturated at lower oxygen tensions (Davis and Carlsson, 1973; Gibson and Duffy, 1981; Norberg and Siesjo, 1975).

B. FACTORS AFFECTING OXYGEN SUPPLY AND ENDONEURIAL TENSIONS OXYGEN T h e distribution of oxygen tensions in nerve depends on the size, length, and geometric arrangement of endoneurial capillaries, the arterial oxygen tension, the flow rate of blood in the capillaries, the oxyhemoglobin dissociation curve and hemoglobin concentration, the solubility and diffusivity of oxygen in blood and nerve, and the rate of consumption of oxygen. These factors will be considered in turn. 1. Capillary Size

T h e capillary radius r influences oxygen delivery in two ways. First, the blood flow F (volume per unit time) is equal to the product of the cross-sectional area of the vessel and the flow velocity v, F = T?V.

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Second, the area available for diffusion of oxygen out of the vessel is proportional to the circumference ( 2 n r ) of the vessel, and diffusion rate is proportional to this area.

2 . CapillaT Length The capillary length L, together with the velocity v of blood flow, determines the transit time T = L / v for erythrocytes. T h e transit time directly influences the arteriovenous difference in oxygen content of blood.

3 . Arrangement of Capillaries T h e oxygen tension at a given point depends on the distance away from the nearest capillary (which determines the distance over which oxygen must diffuse and the amount of intervening oxygen-consuming tissue) and the distance along the capillary from arterial to venous end. The lowest oxygen tension value thus occurs at the lethal corner (Reneau et al., 1967), which is the point farthest away from the capillary at the venous end (i.e., the point halfway between a capillary and its nearest neighbor). T h e highest oxygen tension is found at the wall of the arterial end of the capillary. The range of oxygen tension values in nerve therefore depends on the intercapillary distance. The above considerations are strictly true only for capillaries arranged as parallel, equally spaced tubes with flow in the same direction. For a countercurrent flow arrangement, in which flow in adjacent capillaries is in opposite directions, oxygen tensions show less variation with position, so the distribution of tensions is more sharply peaked (Metzger, 1971). 4 . Arterial Oxygen Tension

T h e arterial oxygen tension determines the saturation and oxygen content of blood entering the capillaries. There is some loss of oxygen from blood in passing through the arteriolar bed, so the capillary entrance tension is probably significantly less than the actual arterial oxygen tension (Pope1 and Gross, 1979). Tissue hypoxia that occurs as a consequence of arterial hypoxemia affects primarily the arterial end of the capillary bed; oxygen tensions near the venous end are much less affected. This type of hypoxia is, therefore, known as arterial hypoxia (Opitz and Schneider, 1950). 5. Blood Flow

The blood flow velocity determines the rate at which oxygen is transported in capillaries. Tissue hypoxia that occurs as a consequence of reduced blood flow affects primarily the venous end of the capillary

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bed, and is, therefore, known as venous hypoxia (Opitz and Schneider, 1950). Blood flow other than capillary flow, such as flow in shunt vessels, contributes much less to the oxygen supply of nerve tissue, expressed as a ratio of oxygen delivery rate per unit volume of tissue to blood flow per unit volume of tissue. This is because the larger diameter shunt vessels with greater blood flow velocities have much reduced oxygen extractions because of shorter transit times.

6. Hemoglobin on cent ration and Oxyhemoglobin Dissociation Curue T h e hemoglobin concentration determines the maximum oxygen capacity of the blood, and the oxyhemoglobin dissociation curve determines the release of bound oxygen to tissue. Like blood flow, these affect oxygen tensions primarily at the venous end of the capillary bed. T h e fractional saturation of hemoglobin can be approximated by the Hill formula (Hill, 1928), (PIp50)’ saturation = 1 + (P/&)‘ where P is the oxygen tension, P50 is the oxygen tension at 50% saturation, and 7) is the Hill constant (slope of the Hill plot). Smaller P50 values correspond to oxygen being more tightly bound to hemoglobin, thereby reducing the release of oxygen to tissue within the capillary bed (Duvelleroy et al., 1970) (a “left shift” of the dissociation curve). The value of P50 is increased by decreasing blood pH (the Bohr effect), decreasing erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration (Ditzel et al., 1975), and increasing hemoglobin Alc (Bunn and Briehl, 1970; Ditzel et al., 1979). Insulin per se has been reported to affect oxygen release from hemoglobin adversely (Ditzel et al., 1978). 7. Solubility of Oxygen The amount of oxygen dissolved in blood is proportional to the solubility coefficient; however, dissolved oxygen contributes a minor amount compared to oxygen bound to hemoglobin (about 1.5% in arterial blood). The solubility of oxygen in endoneurial tissue affects the oxygen tension distribution, since oxygen tension Po2 = [Oe]/Swhere [O,] is oxygen concentration and S is solubility. For example, in an isolated (nonperfused) piece of nerve consuming oxygen at 0.01 cm3 02/cm3tissue/min, the oxygen tension would drop at a rate of O.OU(3.0 X Torr/min = 330 Torrlmin, where the solubility S is 3.0 X cm3 02/cm3 tissue/Torr.

8. Diffusiuity of Oxygen Oxygen is a highly diffusible gas in both blood and tissue. Of course, oxygen must diffuse from erythrocytes, through capillary walls, and into

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the surrounding tissue. T h e maximum diffusion distance in a system of parallel, uniformly distributed capillaries is half the intercapillary distance. T o a first approximation (the Krogh-Erlang equation, which will be discussed later), the difference (AP)in oxygen tension between the wall of the capillary and a point at a given distance r from the capillary is inversely proportional to the product of the solubility S and diffusivity D of oxygen in nerve, and directly proportional to the oxygen consumption rate C:

where f(r ) is a function of distance r. 9. Oxygen Consumphon Rate As Eq. (2)shows, the oxygen consumption rate directly influences the distribution of oxygen tension values in nerve. The consumption rate of nerve does not increase during activity by nearly as much as a tissue such as muscle or even brain, though increases of as much as 50% may occur during rapid nerve impulse firing (Cranefield et al., 1957; Ritchie, 1973). Reductions in consumption of oxygen are brought about by a variety of conditions. a. Acu.te Hypoxia. There are immediate changes in consumption that result from reduced oxygen tension, as enzyme systems involved in aerobic metabolism became less than fully saturated. Various mathematical models have been put forward to represent the functional dependence of consumption C on oxygen tension P (Hudson and Cater, 1964). Examples include 1. Zero-order kinetics above a critical oxygen tension Pc, and first-order kinetics below this:

2. Michaelis-Menten kinetics

c = c,(

-) P

P

+ C50

(4)

where C50 is a constant representing the oxygen tension at which consumption is 50% of its maximum value CO(no relationship to the in the Hill formula for the hemoglobin dissociation curve). 3. Modified Michaelis-Menten kinetics incorporating a cooperative effect in oxygen binding to respiratory enzymes:

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where

r)

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is a constant (e.g., r) = 1.4).

4. Zero-order kinetics above a critical oxygen tension PC and an

inverse parabolic relationship below this:

where K is a constant. Other formulas have also been suggested, but there are no data on the rate of oxygen consumption in nerve at low oxygen concentrations to decide among these models. It is clear, however, that in many tissues (e.g., brain o r liver), cells die rapidly under conditions of low oxygen tension. The deleterious effects of hypoxia are significantly greater in tissues with large metabolic needs. Nerve, with its relatively low oxygen consumption compared to many other tissues (e.g., about 20% that of brain) has greater resistance to hypoxia. 6. Chronic Hypoxia. There is evidence that in states of chronic hypoxia, nerve oxygen consumption may be further reduced, perhaps due to adaptive changes in nerve metabolism. For example, shifts from aerobic to anaerobic (glycolytic) metabolic pathways may occur. This possibility is especially apt in diabetic neuropathy, in which nerve ischemia and consequent hypoxia is combined with increased glucose concentrations. c. Agzng. Evidence indicates that the oxygen consumption rate of nerve decreases with advancing age (Low et al., 1986a). This probably explains the increased ability of nerves in older animals to resist ischemic conduction failure.

C . FACTORS REDUCING OXYGEN DELIVERY IN MODELNEUROPATHIES 1. Diabetic Neuropathy

Experiments demonstrate a 33% reduction in nerve blood flow in experimental diabetic neuropathy (EDN). This is probably a major factor in reducing oxygen availability in diabetic nerves. Reduced blood flow may in part be due to closure of capillaries, which also increases

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intercapillary distance, further impairing oxygen delivery. However, a reduction in blood flow velocity in individual capillaries is probably a more important contributor to reduced blood flow. The oxyhemoglobin dissociation curve may also be changed in diabetic neuropathy, because of increased 2,3-DPG concentration and increased HbAlC concentration; however, these two factors have opposite effects on the dissociation curve and tend to cancel each other (Tuck et al., 1984). The chronic nature of the neuropathy also allows the development of an adaptive decrease in oxygen consumption in affected nerves.

2. Ischemic and Hypoxemic Neuropathies In experimental ischemic neuropathy caused by exsanguination, the reduction in blood flow velocity is the major factor reducing endoneurial oxygen delivery. In arterial hypoxemia caused by reduced oxygen content of inspired air, the effects of reduced arterial oxygen tension (arterial hypoxia) are supplemented by effects of reduced blood flow (venous hypoxia) caused by reduction in cardiac output due to cardiac muscle hypoxia. 3 . Edematous Neuropathy

In experimental edematous neuropathy (e.g., galactose neuropathy), the increased intercapillary distance is a major factor in reducing the oxygen supply. T h e adverse effects of increased intercapillary distance are partially offset, however, by a reduced oxygen consumption rate per unit volume of tissue that results from the effects of edema, since much of the increase in nerve volume is due to accumulation of extracellular fluid that is not metabolically active.

D. MATHEMATICAL MODELINGOF NERVEOXYGEN SUPPLY 1 . Previous Models Mathematical models of the release of oxygen from hemoglobin and its diffusion from capillaries to surrounding tissue have been applied to skeletal muscle (Krogh, 1919; Kety, 1957), cardiac muscle (Rakusan, 1971) and brain (Reneau et al., 1967). Although various assumptions and simplifications are made in these models (Baxley and Hellums, 1983; Kreuzer, 1982), their predictions, with appropriate choices of input parameters, are in good agreement with experimental data. These models may be applied also to peripheral nerve by using input parameters appropriate to the endoneurial microcirculation. Most models are based on a geometry first used by Krogh in 1919 for

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calculating oxygen tensions in skeletal muscle. Krogh assumed that capillaries are long, parallel tubes that are equidistant from each other and that the direction and velocity of flow in each capillary is the same. Then, each capillary can be assumed to supply oxygen to a cylinder of tissue surrounding it, whose radius is one-half the intercapillary distance. I n a given time interval, the difference between the amount of oxygen carried into the capillary by flowing blood at the arterial end and that carried out at the venous end is equal to the amount of oxygen consumed by the tissue cylinder around the capillary, since Krogh assumed that no oxygen diffuses into or out of the tissue cylinder except for the radial diffusion of oxygen out of the capillary and into the surrounding tissue where it is consumed. Krogh actually modeled only diffusion from the capillary into the tissue, ignoring intracapillary processes. This radial diffusion is governed by the differential equation D,r V2[02] = C, which in cylindrical coordinates takes the form

where DT is the diffusion constant of oxygen in the tissue, r is radial distance from the capillary center, [O,] is oxygen concentration (a function of r ) , and C is the oxygen consumption rate of the tissue per unit volume. This equation is independent of time and applies to steady-state (equilibrium) conditions. T h e differential operator, +$(r$),

is the form of the Laplacian operator V2 in cylindrical

coordinates, assuming radial symmetry. Using the fact that [O,]=Sr P where P is oxygen tension and S , , is tissue solubility of oxygen, this equation may be rewritten in terms of P as

c This equation may be solved if C, DT,and ST are taken to be constant, independent of P or r. T h e appropriate boundary conditions are: P = P Oa t r = Rc

and dP _ - 0 at r = RT

dr

(9)

370

PHILLIP A . LOW

el al.

where Rc is the capillary radius and RT is the tissue cylinder radius. T h e value of Po is the oxygen tension at the wall of the capillary. The second boundary condition states that there is no diffusion of oxygen through the outer boundary of the tissue cylinder. Integrating Eq. (8) over r from r to R . , gives

Using Eq. (lo), this becomes -r-

dP C = -(RT2- r2) dr ST

or

Integrating this over r from Rc to r gives

/&

dP dr

- dr =

c -

~DTST

(r -

$)dr

Using Eq. (S), this becomes

"

P - Po = - - (? - Rc2) - RT2(In r - In Rc) ~DTST2

or finally,

This is the Krogh-Erlang equation, which predicts a mixed parabolic and logarithmic dependence of P on distance r. Krogh's formulation requires that the oxygen tension Po at the wall of the capillary be known. Po is, in fact, a function of x, the axial distance along the capillary from the arterial to the venous end. Various methods have been used to calculate Po. Kety (1957) assumed arbitrarily that the oxygn concentration [ 0 2 ] of blood decreased linearly with distance x, from a maximum value [ 0 2 ] A at x = 0 (the arterial end) to a minimum [ 0 2 ] v at x = L (where L is capillary length):

Lo21= [ 0 2 1 A

- ([02]A

- [02]V)

(XlL)

(17)

BLOOD FLOW AND OXYGEN DELIVERY IN NEUROPATHY

37 1

The dissociation curve of hemoglobin relates oxygen concentration to partial pressure Pa. This curve may be expressed fairly accurately by the Hill relation given earlier:

[

'021= cB

(PO/p50)'

1

+ (Po/P5,,)']

(18) where CB is the oxygen concentration of fully saturated blood. The combination of Eqs. (17), (18), and (16) then allows the oxygen tension P to be calculated at any point in the tissue cylinder defined by radial distance r and axial distance x .

2 . Improved Model

of

Nerve Oxygen Supply

T h e Kety assumption is only approximate, and furthermore the above formulation neglects the radial variation of oxygen tension within the capillary and the necessity for oxygen to diffuse from its point of release in the blood to the wall of the capillary. A formulation by Reneau et al. (1967, 1969) provides a more accurate description of diffusion in a Krogh cylinder geometry. This formulation in its most general form takes into account axial diffusion of oxygen in both capillary and tissue, as well as axial flow of blood and radial diffusion of oxygen. We have applied both a steady-state and non-steady-state (time-dependent) version of this model to peripheral nerve and extended it by including a variable oxygen consumption term, specifically a Michaelis-Menten dependence of consumption on oxygen tension. In this model, the relevant differential equation for diffusion and flow in the capillary is:

where t is time, D B and SB are the diffusion and solubility constants of oxygen in blood, and v is the velocity of blood flow. T h e unitary term in braces represents the oxygen carried physically dissolved in blood, whereas the second term reflects the contribution (many times greater) of oxygen bound to hemoglobin. Note that this formulation ignores erythrocyte boundaries and assumes blood in capillaries to be a homogenous hemoglobin solution. The velocity u could be taken to vary with r [e.g., parabolically as in a viscous liquid, v = 2uaVg(1 - ? / R c 2 ) , where uaVgis the average velocity]. However, Reneau et al. showed that this leads to no significant difference in the tissue oxygen tension profile compared with the simpler assumption of constant v (plug flow).

372

PHILLIP A. LOW

el

al.

Similarly, the relevant differential equation for diffusion and consumption in the tissue is:

where DT is the diffusion constant and ST is the solubility of oxygen in tissue, and the last term represents Michaelis-Menten kinetics for oxygen consumption. These equations are solved simultaneously subject to the following boundary conditions: P=P*-(&)(Z-

I)(?-?)

(21)

forOsrsRc,x=O

-aP= o

for Rc

-ap _-0

at r = 0 , all x

(23)

P in capillary = P in tissue

at r = Rc, all x

(24)

at r = Rc, all x

(25)

ax

ar

I)*&

ap

-in capillary = I)-& ar

ap

- = S(r) ax

dP

-= ax

S(&)

ap ar

- in tissue

5

r

5

RT, x

for 0 5 r 5 Rc, x

=

0

=L

for Rc 5 r 5 RT, x

=

(22)

(27)

L

(28)

Equation (21) for the radial dependence of oxygen tension in the capillary at the arterial end was derived assuming radial diffusion outward from the center of the capillary. Equation (22) states that there is no axial diffusion into the tissue cylinder. Equation (23) states that there is no radial diffusion at r = 0. Equation (24) guarantees the continuity of tensions at the capillary wall. Equation (25)guarantees that the rate of diffusion out of the capillary equals the rate of diffusion into

BLOOD FLOW AND OXYGEN DELIVERY IN NEUROPATHY

3 73

the tissue at the capillary wall. Equation (26) states that there is no radial diffusion of oxygen out of the tissue cylinder. Equation (27) specifies the rate of egress of oxygen from the venous end of the capillary as a function of r. Equation (28) uses a nonzero value for axial diffusion of oxygen out of the venous end of the tissue cylinder since this seems to better represent the actual situation; the value of the axial derivative of P in the tissue is arbitrarily taken to be equal to its value at the capillary wall. For steady-state solutions, the dPldt terms are set to zero. For time-dependent solutions, an initial condition is used, as follows:

P = Pa( r,x) t = 0, all r and x

(29)

where Po ( r , x ) is the solution of the steady-state problem at specified initial values of parameters P A , v, and Co. One or more of these parameters is then assumed to vary with an exponential time course beginning at t = 0 to a new equilibrium value, according to the following formulas:

v = vf

+ ( vi - vf)exp(--t

IT?,)

(31)

where the subscripts i and f indicate initial and final values, respectively, and T is the time constant for each parameter. T h e above differential equations are solved numerically in the capillary and tissue in three distinct phases. In the first phase, axial diffusion and Michaelis-Menten variation of oxygen consumption are ignored. The Krogh equation is used to calculate oxygen tensions in the tissue after a numerical solution of the appropriate difference equations in the capillary has been determined. This phase requires a negligible amount of computer time. The second phase calculates initial, steadystate oxygen tensions including the effects of axial diffusion and Michaelis-Menten kinetics. This phase requires an iterative solution of difference equations in the capillary and surrounding tissue using, as starting values, the results of the first phase. T h e final phase involves solving the time-dependent differential equations using the results of the second phase as initial values. This is done via the alternatingdirection implicit method of Peaceman and Rachford as modified by Ananthakrishan, Gill, and Barduhn, with special estimation techniques used to calculate the nonlinear terms.

374

PHILLIP A. LOW et

01.

3. Parameters of' the Model In order to compare the predictions of the model with experimental data, we used the parameters appropriate for rat sciatic nerve shown in Table I. (See Lagerlund and Low, 1987, forjustification of these values.) The Michaelis-Menten constant C ~ has O not been measured in nerve, but values of 1-3 Torr have been found in other tissues. After obtaining a steady-state solution for these normal parameters, we varied individual parameters over a range of ?50% or more and determined the variation of the resulting calculated oxygen tensions. We also calculated time-dependent changes in oxygen tensions when one of the parameters P A , u, and COwere allowed to change, according to Eqs. (30)-(32), from normal to an abnormal value or vice versa. 4. Results $the Model Figure 1 shows the radial and axial dependence of the oxygen tension in the tissue and the capillary resulting from Phase 1 calculations (steady-state, no axial diffusion, zero-order oxygen consumption). The results from Phase 2 are qualitatively similar. Oxygen tension is highest at the arterial end of the capillary and falls off both with increasing distance along the capillary and with radial distance away from the capillary. The lowest oxygen tension is found at the lethal corner,

TABLE I NORMALVALUESOF PARAMETERS FOR OXYGEN SUPPLYI N RAT SCIATIC NERVE Parameter ~~

Sg,

Value ~

oxygen solubility coefficient in blood

S1,oxygen solubility coefficient in tissue oxygen diffusion coefficient in blood diffusion coefficient in tissue Rc, radius of capillary R.I.,radius of tissue cylinder L, capillary length C g , oxygen content of saturated blood P M ,oxygen tension for 50% hemoglobin saturation q. Hill coefficient for oxyhenioglobin CO,maximum oxygen consumption rate C ~ Ooxygen , tension for 50% consumption drop v , velocity of blood flow P A , oxygen arterial tension T ~ T,,, , rc, time constants for P A . v , C0 Dg,

D T , oxygen

3.4 X 10-' cm3 02/cm3blood/ Torr 3.0 x 10-' cm' 02/cm3tissue/ Torr 1.1 x lo-' cm'/sec 1.7 x 10-5 cm2/sec 4.5 pm 63 pni 1000 pm 0.2 cms 02/crn3blood 26 Torr 2.8 1.7 x lo-' cm3 02/cmS tissuelsec 3 Torr 420 pmlsec 90 Torr 0.1 sec

BLOOD FLOW AND OXYGEN DELIVERY IN NEUROPATHY 60

40

20

0

20

40

375

60

Radial distance, pm FIG. 1. Endoneurial oxygen tensions predicted by model using standard parameters, shown as a function of radial distance from the capillary center and axial distance from the arterial end (assuming no axial diffusion and zero-order kinetics of oxygen consumption). (From Lagerlund and Low, 1987.)

located at the venous end of the system and at maximum distance from the capillary. This point is most susceptible to hypoxia under conditions that adversely affect oxygen delivery. Figure 2 shows the effects of axial diffusion and Michaelis-Menten kinetics on the axial oxygen tension profile at maximum distance from the capillary. Axial diffusion lowers the oxygen tension at the arterial end of the Krogh cylinder by a large amount and raises the oxygen tension at the venous end by a small amount. Michaelis-Menten kinetics raise the oxygen tension everywhere, but especially at the venous end of the system where oxygen tensions are low. Figure 3 summarizes the effects on lethal corner oxygen tension of changing the six parameters GO, &, RT, PA, v, and C O .A change in intercapillary distance has the greatest effect on oxygen delivery, followed (in order) by changes in capillary radius, oxygen consumption, blood flow, arterial oxygen tension, and Michaelis-Menten constant. Figure 4 compares a histogram of oxygen tension values calculated from the model with an experimental histogram of oxygen tensions in

376

PHILLIP A. LOW et al. 120.

L L

100.

0 I-

!

80.

3

((I ((I

t LL

.-0

60.

40.

-Q L

0

a

20.

0.

0.0

200.0

4060 606 0 8 0 0 . 0 Axial Distance, V r n

ioooo

FIG. 2. Dependence of oxygen tension on axial distance for points at a maximum distance from the capillary. Results for models with axial diffusion and Michaelis-Menton kinetics (solid line); axial diffusion and zero-order kinetics (dashed line); and for no axial diffusion and zero-order kinetics (dotted line) are shown.

normal rats. The model distribution seems to have a larger number of oxygen tension values above the median compared with the experimental distribution. This difference might be due to greater losses of oxygen in the precapillary (arteriolar) vessels than we have assumed (resulting in a lower arterial oxygen tension), or it could reflect the presence of countercurrent flow arrangements in the endoneurial capillary bed. Figure 5 shows the time-dependent variation of oxygen tensions at the arterial end at various radial distances r from the capillary center, when the arterial oxygen tension varies from 90 to 235 Torr (corresponding to inspired oxygen fraction ranging from 20 to 40%) over 0.1 sec. These curves have been found to fit a biexponential function, P

= PO

+ CS exp( - Kst ) + Cpexp(- K F ~)

(33)

with the slow rate constants K S in the range 0.1-0.44 sec-' and the fast rate constant KF varying from as high as 19.6 sec-' at the wall of the vessel to as low as 0.12 sec-' at the maximum distance from the vessel.

BLOOD FLOW AND OXYGEK 1)ELIVERY IN NEUROPATHY

377

L

I-

0

?in

?!

a

fraction of Normal Conditions

FIG. 3. Lethal corner oxygen tensions as a function of six parameters: capillary radius (-+- ), arterial oxygen tension (a+.), blood flow velocity (-0-), consumption rate (-x-), and the Michaelis-Menten constant (MA.). (-0-),intercapillary distance

50.45.40.3 c

35.-

-

30.-

c

25.-

I-"

-

0

.---

0,

$

20.-

a

,

L

....

I

15.-

,

.~~~

10.-..~

5.,..,

0.7

,..-.

378

PHILLIP A. LOW r&al.

350~1I 300.

t b-0

50.1 0.0

6.0

12.0 18.0 Time, sec

24.0

30.0

FIG. 5. Time dependence o f oxygen tensions at the arterial end for several radial distances from the center of the capillary (wall of vessel = 4.5-prn radius corresponds to steepest curve). Curves are shown for wall of vessel (-), 9 pm (---), 13.5 pm (...), 18 pm (-.-), 22.5 prn (- -), and 27 p m (-).

At points away from the arterial end, a monoexponential curve generally describes the oxygen tension variation adequately, with rate constants in the range 0.017-0.3 sec-'. IV. Regulation of Blood Flow

A. INTRINSIC MECHANISMS Microvascular flow is predictable from the Poiseuille equation

Nerve blood flow depends on the fourth power of the capillary radius ( r ) ,on the viscosity of blood (v),on the capillary length ( l ) ,on the pressure gradient ( P I - P 2 ) , and on capillary density (a).Strictly speaking, the Poiseuille equation applies only to Newtonian fluids, and blood flow is non-Newtonian, so this formulation is semi-quantitative. 1. Viscosity of Blood

Hemorrheologic mechanisms that also have major influences on blood flow include the concentration of hemoglobin, serum proteins, the

BLOOD FLOW A N D OXYGEN DELIVERY IN NEUROPATHY

379

presence of abnormal hemoglobins, and factors affecting the coagulability of blood. Blood hematocrit is the major determinant of whole blood viscosity, but erythrocyte aggregability and deformability are also important contributors to blood viscosity (Dintenfass, 1979; Palinski et al., 1983). The influence of hemorrheologic factors on NBF in diabetes will be described in Section VI.

2 . Capillary Radius A small change in radius results in a major change in NBF, since NBF varies with the fourth power of the radius. Capillary diameter is altered in certain peripheral neuropathies. For instance, in diabetic neuropathy, there is endothelial cell hypertrophy and hyperplasia, intimal and smooth muscle cell proliferation leading to a reduction in lumen diameter (Johnson et al., 1986), and capillary thrombosis (Timperley et al., 1976) and closure (Dyck et al., 1985). 3 . Capillary Length

T h e length of the capillary influences the drop in hydrostatic pressure along the vessel,,although the largest dissipation of pressure is along small arteries and terminal arterioles (Joyner and Davis, 1987). In nearly all tissues studied a 40-60% reduction in the total pressure occurs before the first-order arterioles. A second major drop occurs across terminal arterioles; little evidence exists for precapillary sphincters in most tissues.

4. Pressure Gradient A fall in systemic BP will reduce P I and an increase in venous pressure will increase Ps. Particularly in a tissue that autoregulates poorly, P I is an important regulator of NBF. NBF has been found by all recent studies to be linearly related to systemic BP (Low and Tuck, 1984; Rundqvist et al., 1985; Sundqvist et al., 1985; Takeuchi and Low, 1987) in anesthetized and decerebrate nonanesthetized rats (Sundqvist et al., 1985). Since nerve microvasculature is a capacitive nutritive system, a small reduction in blood volume, such as might occur in hypovolemia or exsanguination, results in a disproportionate reduction in NBF (Takeuchi and Low, 1987) and precedes the reduction in systemic BP.

B. EXTRINSIC MECHANISMS 1. Perineurial Pinch and Endoneurial Edema Nerve is supplied by an intrinsic interconnecting system of microvessels. Feeding into the intrinsic system is an epineurial system, the

380

PHILLIP A. LOW et al.

extrinsic system. T h e two systems are interconnected via arterioles that traverse nerve perineurium. What is not known is whether the interconnections are so diffuse and uniform that endoneurial ischemia results only when many perforating arterioles are occluded, or whether each segment of nerve has a major controlling influence on its underlying intrinsic NBF. The former model would be one in which nerve ischemia occurs by a hemodynamic mechanism. T h e latter would be, at least in part, a model in which local ischeinia occurs when the regional supply is compromised. Myers et al. (1986) suggested that perineurial distortion, such as might occur in nerve edema, may result in regional ischemia (a perineurial pinch mechanism) and provided mathematical modeling data to demonstrate the feasibility of the hypothesis. Earlier investigators failed to produce fiber degeneration by undercutting peripheral nerve or by ligating the regional arterial supply, even when a long stretch of nerve was deprived of its blood supply by these maneuvers (Adams, 1943; Blunt and Stratton, 1956; Denny-Brown and Brenner, 1944; Lundborg, 1970). However, the focus of these studies was on the effect of arterial rather than arteriolar occlusion. Additionally, the endpoint of fiber degeneration may be too crude, since hypoxic-ischemic effects such as conduction failure occur well before fiber degeneration. We recently examined the effect of norepinephrineinduced epineurial vasoconstriction on the underlying endoneurial blood flow measured simultaneously in the subperineurial and centrifascicular sites using microelectrode-Hn polarography. T h e norepinephrine was applied externally to a short segment (2 cm) of nerve. Since noradrenergic innervation is confined to nerve epineurium and is absent in endoneurium, and since there are perineurial and blood-nerve barriers, the preparation is ideally suited to determine the effect of the regulation of regional endoneurial blood flow by overlying epineurial arterioles. We found a dramatic reduction in NBF in both sites at concentrations as low as lo-’ M. We excluded a systemic effect of norepinephrine by demonstrating that the local ischemia was unassociated with NBF reduction in the contralateral sciatic nerve or with an increase in plasma NE above baseline (M. Kihara and P. A. Low, 1989 unpublished observations). Our data indicate that there is regional vasoregulation of NBF by epineurial arterioles. However, the small residual NBF is sufficient to prevent fiber degeneration, presumably since the energy requirements of peripheral nerve are minimal. Endoneurial edema due to perineurial distortion may contribute to the NBF reduction demonstrated in experimental galactose neuropathy (see below).

BLOOD FLOW AND OXYGEN DELIVERY I N NEUROPATHY

38 1

2, Norepinephrine and Sympathetic Innemation T h e major neurotransmitter of mammalian peripheral nerve is norepinephrine (Appenzeller et al., 1984; Rechthand et al., 1986; Ward et al., 1989), which is confined to the microvasculature of epineurium and is essentially absent in endoneurium (Rechthand et al., 1987). We recently studied the role of a-adrenergic innervation in nerve vasoregulation (Zochodne and Low, 1989). Dynamic alteration in NBF was monitored using laser Doppler velocimetry, endoneurial NBF by microelectrode H2-polarography, and epineurial vasoreactivity by computerized videoangiology; a-agonists and antagonists were applied locally and systemically. a-Agonist-mediated vasoconstriction was regularly produced and was blocked by prior treatment with an a-antagonist. The videoangiologic recordings showed markedly heterogenous vasoreactivity to norepinephrine along segments of arterioles, suggesting a segregation of a-receptors. Additionally, there is also a local noradrenergic regulation of underlying endoneurial NBF by epineurial arterioles (see above). Sympathetic stimulation resulted in a reduction in NBF and chemical sympathectomy resulted in an increase in NBF (P. A. Low et al., unpublished observations). 3. Nonadrenergic Mechanisms

In addition to adrenergic innervation, there has also been the recent demonstration of prominent peptidergic innervation of vasa nervorum (Appenzeller et al., 1984). Prostacyclin (largely confined to endothelial cells) is the major microvascular vasodilator and inhibitor of platelet aggregation, and thromboxane A2 (largely confined to platelets) has the opposite effects. T h e ratio of prostacyclin to thromboxane A2 is considered to be important in the maintenance of vascular tone (Moncada and Vane, 1979). We recently demonstrated that nerve biosynthesis of 6-keto prostaglandin F,,, (GKPGF,,) the stable metabolite of prostacyclin, was largely confined to nerve sheath (Ward et al., 1989), suggesting another mechanism of epineurial regulation of endoneurial NBF. Similar observations to ours were made in diabetic rat aorta (Roth et al., 1983) and heart, in which altered regulation of phospholipase activity was suggested as the mechanism of reduced endogenous 6KPGF1, (Rosen et al., 1983). One possible mechanism of reduced prostacyclin biosynthesis is the reduction of nerve norepinephrine (NE). Norepinephrine release results in the increasd synthesis and release of prostaglandin I2 metabolites (Gilmore et al., 1968) by a-receptor-mediated and calcium-calmodulin-dependent mechanisms. Calmodulin is known to activate phospholipase A2 (Wong and Chung, 1979) resulting in a

382

PHILLIP A. LOW et al

breakdown of membrane phospholipids to generate arachidonic acid (Burton et al., 1986), whose availability is the rate-limiting step in prostaglandin synthesis (Irvine, 1982). NE may also be reduced by oxygen free radical (OFR) generation, which is thought to be increased in chronic experimental diabetes (Karpen at al., 1982) and may be generated from norepinephrine (Bovaris and Chance, 1973). The concept of a nonautoregulating NBF system needs to be reconciled with the demonstration of dense noradrenergic and peptidergic innervation of vasa nervorum (Appenzeller at al., 1984) and the known dense innervation of small arteries (Burnstock, 1975; Burnstock et al., 1984). Adrenergic innervation is reportedly increased in EDN (Dhital et al., 1986). These apparently disparate observations may relate to the regional and segmental heterogeneity of blood flow regulation described in other tissues (Bevan, 1984; Baumbach and Heistad, 1985). Regulation of blood flow in these tissues is largely neurogenic in vessels of diameter 50-200 p m and largely myogenic in vessels <50 pm in diameter (Folkow, 1964). In brain nutritive vessels, regulation appears to be metabolic, possibly mediated by adenosine (Bevan, 1984). There is also considerable variation from one vessel bed to another. For instance, the reactivity of carotid vessels is quite different from that of niesenteric vessels. Studies of endoneurial nerve blood flow and of whole nerve blood flow have been done, as have responses to alterations in BP, pop, and pC02 (Low and Tuck, 1984). What is lacking is a detailed evaluation of the reactivity of vessels of different diameters (capillaries, epineurial arterioles, and small supplying arteries) to standard physiological stimuli and relevant neurotransmitters.

V. Nerve Blood Flow Measurements

A. METHODS The ability to obtain accurate and reliable measurements of NBF is a major advance in the study of peripheral nerve physiology. NBF recordings have provided data that is critically important in the understanding of the pathogenesis of diabetic neuropathy as well as the physiology of edematous and ischemic neuropathies. Several different methods are available for the estimation of blood f l o w . T h e two methods best suited to and most commonly used in the measurement of perfusion in peripheral nerve are hydrogen polarography and iodoantipyrine

BLOOD FLOW AND OXYGEN DELIVERY I N NEUROPATHY

383

autoradiography. Each of these has specific merits and drawbacks. The maximum information can be obtained by combining the two methods to take advantage of the characteristics of each. Other methods are available and have been used to measure NBF but these techniques generally provide less reliable and less accurate data. 1. Hydrogen Polarography

Microelectrode hydrogen polarography is the standard technique used in our laboratory. It has proved to be reliable, accurate, and reproducible and gives values for NBF comparable to those obtained with other methods. It has several advantages. It can be used to make several consecutive estimations of NBF in a single animal. It provides a continuous readout of blood flow, making it possible to observe dynamic changes in NBF. T h e spatial resolution is better than other methods, so comparisons of flow in adjacent regions can be made with a separation of as little as 100 p m (Stosseck and Lubbers, 1971; von Kummer and Herold, 1986). It now appears likely that this technique can be used to separate nutritive from nonnutritive blood flow in peripheral nerve (Day et al., 1989). The disadvantages are that the equipment is expensive (microelectrode recordings produce small currents that require the use of sophisticated amplifiers), the microelectrodes are time-consuming and difficult to make, and recordings can be made from only a limited segment of nerve in each experiment. T h e physical characteristics of hydrogen make it eminently suitable as a tracer for NBF recordings. It is metabolically inert, lipid-soluble, and diffuses rapidly into nerve. It has a low blood-gas partition coefficient and is not normally present in the body. When the recording electrode is polarized at +0.25 volts, hydrogen is selectively ionized and the potential interference from other polarizable species such as ascorbate, oxygen, or blood proteins is minimal (Aukland et al., 1964). Some criticisms of hydrogen polarography have been made. Sugimoto et al. (1986) pointed out that the method requires the exposure of the nerve and suggested that this may alter blood flow. This argument was based on NBF recordings made with radiolabeled butanol. They found that NBF was increased by surgical exposure of the nerve in anesthetized but not in awake rats. However, they could proffer no explanation for this observation, which was puzzling in view of the lack of an effect of anesthesia on NBF in nonoperated rats in their preliminary experiments. I n addition, they used the similarity of the butanolderived values for NBF to those made with other methods to validate their new method, whereas all the other methods require anesthesia and exposure of the nerve. These inconsistencies suggest that there was a

384

PHII.I.IP A. LOW et al.

technical error in their recordings. It is possible that the delay in removing the nerve at the completion of the experiments in which the nerve had not been previously exposed made all their recordings under these conditions too low because of diffusion or efflux of butanol from the nerve. The use of electrodes to record NBF has been criticized on the grounds that they cause local tissue damage and thereby alter NBF (Young, 1980). This may be a problem when large-diameter electrodes are used to record from a large volume of tissue. Aukland et d ’ s (1964) early studies involved the use of electrodes with a diameter of 2 mm. We believe that several features of our electrodes including the very small diameter (one thousand times smaller than Aukland et ale’s),the sharp beveled tip, and the smooth glass coating that extends to the tip of the electrode minimize tissue trauma. It is unlikely that the simple puncture of a nerve with one of these electrodes causes any significant injury. Young (1980) also raises the objection that although the method assumes that arterial hydrogen concentrations fall to zero when the gas is removed from the inspired air this may not be the case in small vessels. Any significant recirculation of hydrogen would introduce an error into the calculation of blood flow. Mitigating against recirculation is the low liquid-gas partition coefficient of hydrogen, which ensures almost complete extraction of this gas during a single pass through the lungs. Early workers in this area demonstrated that the arterial hydrogen does indeed fall to zero very rapidly (Aukland et al., 1964; Feischi et al., 1969; Halsey et al., 1977)’ and this has recently been confirmed in studies from our laboratory (Day et al., 1989). Even if the hydrogen concentration in small arterioles does not fall as rapidly, this will not cause recirculation of the tracer. Persistence of hydrogen in small vessels would affect blood flow recordings in nonhomogeneous structures such as brain but not in a unicornpartmental tissue like peripheral nerve. Diffusion from regions of high tracer concentration to areas with lower concentration may occur in the nonhomogeneous organs, leading to measurement errors. The polarogrdphic technique depends on the ionization of hydrogen by a positively polarized electrode. Recording electrodes are made of tapered glass containing a fine platinum wire. The glass insulates the wire except at the tip where a 2-5 pm recording surface is exposed. When the animal is ventilated with a low concentration of hydrogen (usually lo%), the hydrogen is polarized at the tip of the active recording electrode into hydrogen ions and electrons. This causes current to flow through a circuit completed by a subcutaneous reference electrode, with the magnitude of current flow being directly proportional to the amount of hydrogen present at the recording electrode. Measurement of this

BLOOD FLOW AND OXYGEN DELIVERY IN NEUROPATHY

385

current requires a very sensitive amplifier because only nanoampere or picoampere currents are produced. When ventilation with hydrogen is discontinued, the gas is cleared from the tissues (nerve) at a rate dependent on blood flow and direct diffusion. Since diffusion is an insignificant component of the washout at any but the lowest blood flows, it can be ignored in most situations (Day et al., 1989). The washout curve can be analyzed by manual curve fitting or by computerized analysis. It is important to recognize that the curve is often biexponential. An initial fast component is seen in most well-perfused nerves and may be due to rapid intraneural diffusion into a large adjacent arteriole o r arteriovenous shunt vessel (Aukland et al., 1969), proximity of the electrode to a large capillary, or the presence of anatomical regions with much greater perfusion. The last possibility is very unlikely in peripheral nerve since this tissue, unlike brain o r kidney, is uniform in its metabolic requirements (Tuck et al., 1984) and NBF (McManis et al., 1986). In contrast, this is an important consideration in organs with nonhomogeneous perfusion, in which the boundary effect of diffusion from one compartment to an adjacent one greatly influences recordings made within 2 mm of the interface (Pearce and Adams, 1982). T h e degree to which intercompartmental diffusion affects the recordings depends primarily on the difference between the flow rates in the two areas (Pearce and Adams, 1982). Some of the observations made on the characteristics of the washout curves obtained when recording cerebral blood flow wirh diffusible gases as first described by Kety (1951) are very relevant to hydrogen polarography, particularly the stripping of the curves into individual components with different flow rates. Hoedt-Rassmussen and Skinhoj (1966) and Reivich et al. (1969) noted the biexponential nature of the washout curves and postulated that the fast and slow components are produced by different flows in different “compartments” in both experimental animals and humans. The faster flow rates are thought to be in grey matter, whereas white matter is less well perfused. This type of compartmentation is not present in peripheral nerve. Instead, the fast exponent may be the result of clearance of H2 in large vessels such as epineurial-endoneurial arteriovenous shunts (Tuck et al., 1984; Day et al., 1989). Microelectrodes are manufactured by heating a glass capillary tube containing a fine platinum wire in an electrode puller. With practice the wire can be made to break flush with the end of the glass pipette. The resulting tapered tip has a diameter of between 2 and 5 pm. The tip is then beveled to a 60” angle and the electrode is annealed in a 400°F oven. Microelectrodes made in this way are strong enough and sharp

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enough to be inserted into nerve and will record hydrogen clearance in a sphere of approximately 30 p m (Stossock and Lubbers, 1981). T h e recording of the washout curve has been described in detail (Low and Tuck, 1984; McManis et al., 1986). Briefly, the rat is weighed and anesthetized. A tracheostomy is performed and arterial lines are inserted for the monitoring of blood pressure and arterial blood gases. Microelectrodes are positioned in the nerve and the rat is ventilated with hydrogen. When the hydrogen has equilibrated with the nerve and a steady state has been reached, the hydrogen is turned off and the decay of the current produced at the polarizing electrode is recorded. The curve is resolved into a monoexponential or biexponential fit. When the curve is biexponential the slower component is taken to reflect tissue perfusion. This approach is justified by the similarity of the values obtained in this way to those obtained with other techniques, and by experimental evidence that suggests that the fast component is due to diffusion into shunt vessels, while the slow component reflects nutritive flow. The curves are then analyzed and the slope is converted into NBF. This can be done by linear regression of the logarithms of hydrogen current or by direct nonlinear regressions using a modification of the Marquardt algorithm.

2 . Iodoantipyrine Autoradiography Another useful technique for NBF measurement is iodoantipyrine autoradiography. This method has been widely used in measuring blood flow in other organs such as brain. It was adapted for the recording of NBF by Myers et al. (1982) and Sladky et al. (1985). The major advantages of this technique are its relative simplicity and the ability to measure blood flow in many nerve segments or other tissues simultaneously. Its drawbacks include its relatively low spatial resolution, the inability to make more than one recording per experimental animal, the need to use radioisotopes (tritium has very low radioactivity in the published studies), and the need to make autoradiographs, which require 2 or more weeks of exposure. This method is the best way to create a three-dimensional reconstruction of tissue perfusion along the entire length of the nerve. When the information from this threedimensional picture is combined with the high-resolution NBF data obtained with the polarographic technique, an extremely detailed image of nerve perfusion can be created. The iodoantipyrine method depends on tissue equilibration principles. The experimental animal is prepared by inserting a brachial arterial line and a femoral venous line in addition to the carotid arterial catheter needed for monitoring of blood pressure and arterial blood

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gases. When the animal is in a physiological state, a predetermined amount of radioactive isotope is infused rapidly into the femoral vein. Aliquots of blood are collected every 2 sec from the freely flowing brachial arterial catheter before, during, and for 15 sec after the infusion for the construction of an arterial concentration curve. At the completion of isotope administration the animal is killed and the relevant tissues are rapidly dissected out and frozen in isopentane in liquid nitrogen. This tissue is then sectioned on a cryostat and the sections are placed on X-ray film for varying periods. These autoradiographs are then analyzed on a computerized imaging system and compared with similarly exposed standards for the calculation of NBF. T h e arterial concentration curve is constructed by measuring the radioactivity in the blood samples collected during the infusion of isotope. Volumes of 20 61 are collected from the aliquots obtained every 2 sec. These are solubilized, decolorized, and adjusted for pH. A fluorescence cocktail is then added and emissions are counted on a @-counter(I4C emits @-radiationwith a half-life of about 5700 years). The resulting counts are integrated to produce the activity-time integral curve. Blood flow is determined by calculations based on the work of Kety (195 1). T h e same principles apply to tissue equilibration as to indicator dilution methods. Kety showed that the concentration of tracer in tissue at time T (the time of the arrest of the circulation) is determined by the tissue-blood partition coefficient of the tracer multiplied by a constant and by the integrated blood concentration of the tracer during the time between beginning the injection of the tracer and the time the circulation is stopped. T h e constant referred to is related to blood flow, the partition coefficient, and the equilibrium point for diffusion between blood and tissue. Thus, it becomes necessary to determine these variables for any substance being used to measure blood flow. Because radiolabeled volatile gases (as used by Kety) are difficult to measure autoradiographically, are not readily available commercially, and have short half-lives, Sakurada et al. (1978) selected 14Cfor its long half-life and superior autoradiographic performance. Initial experience with 14C-labeledantipyrine was disappointing because this tracer did not diffuse well though the blood-brain barrier, thereby giving falsely low blood flow values. These researchers (Sakurada et al., 1978) showed that '&-labeled iodoantipyrine was not subject to the same limitation and gave blood flow values similar to those made with volatile gases. Their method has become the standard way of measuring cerebral blood flow (Jay et al., 1988). This technique subsequently has been adapted for the measurement of nerve blood flow and found to be equally useful in this

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tissue (Myers et al., 1982; Sladky et al., 1985). Because the blood-brain partition coefficient for iodoantipyrine is 0.80 (Sakurada et al., 1978) and the blood-nerve partition coefficient is 0.82 (Sladky et al., 1985), determinations of blood flow using this tracer are underestimated by about 20% unless allowance is made for this in the calculations. The iodoantipyrine method can also be used to measure composite blood flow in a tissue by homogenizing the tissue and using liquid scintillation counting on the sample. This has the advantages of being simpler and avoiding the problems of autoradiography but has the major drawback of being unable to provide information on regional differences in perfusion. 3 . Other Methods

Other ways of recording blood flow include heat clearance, laser Doppler recordings, microsphere embolization, and indicator dilution (fractionation) using diffusible gases such as radiolabeled xenon or krypton or other substances. In general, these methods are not as satisfactory as the abovementioned methods. Some are not suitable for measuring NBF because of their physical characteristics and the limitations these impose, but other are widely used in various forms. a . Laser Doppler Velocimetry. Laser Doppler velocimetry can provide useful information on NBF. This technique is extremely sensitive to fluctuations in blood flow and therefore provides a way of looking at dynamic alterations in NBF with precision. It is, however, very sensitive to “noise” such as changes in ambient lighting and does not provide accurate quantitative NBF measurements. T h e method depends on the Doppler principle that moving objects cause a change in the frequency of signals produced by or reflected from them when recorded at a stationary site or a site moving relative to the object being studied. A laser signal emitted from a miniature source can be reflected from a moving column of red blood cells, permitting the recording of velocity-dependent frequency shifts. The Doppler shift is determined by the mass and velocity of the red cells. This technology has been adapted for the recording of NBF (Rundqvist et al., 1985; Takeuchi and Low, 1987). Laser Doppler recordings have specific advantages in addition to the very rapid responses to NBF alterations. They are noninvasive (although they do require exposure of the nerve) and can provide multiple and continuous recordings. However, they cannot distinguish regional flow differences and will measure a composite of epineurial and endoneurial flow unless the epineurium is stripped off the nerve, a maneuver that may adversely affect endoneurial perfusion.

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b. Indicator Dilution. Indicator dilution techniques fo the calculation of blood flow were developed by Kety (1951). He demonstrated that an extracranial detector could be used to record the arrival and clearance of a &emitting gas in brain and that the rates of arrival and clearance are determined by tissue perfusion. He used an exponential equation to calculate the blood flow, taking into account the arterial concentration of the gas, the blood-brain partition coefficient (calculated separately), the weight of tissue being recorded from, and the relative weighting of blood flow in different compartments per unit time. His methods have been modified and developed for the measurement of nerve blood flow (Myers et al., 1982; Sladky et al., 1985). Diffusible gases cannot be used effectively to measure NBF with Kety’s methodology because of the small volume of tissue involved and because the flow is recorded with an external counter. Radiolabeled gases could be employed with an autoradiographic technique as described above, but the tissue equilibration variation using iodoantipyrine has proved to be more convenient and practical. Indicator dilution methods use a bolus injection of indicator and the measurement of the flow rate in an artery to determine the input of indicator into the tissue being examined. The tissue equilibration methods employ the constant infusion of indicator at a known rate. Both are ways of estimating the amount of indicator “seen” by the organ under study. Both require that multiple blood samples be collected during the infusion up to and after the cessation of circulation. It is likely that the constant infusion method is more accurate since the measurement of arterial flow (used in the calculation of tissue perfusion in the dilution methods) can only be approximated by weighing the total amount of blood collected and dividing this by the duration of the collection to estimate the flow rate per unit time. Another problem is that the flow may not remain uniform during the collection; fluctuations or slow trends are not taken into account by this methodology. A third problem is that the weight of the blood is converted to volume by multiplying the weight by a standard correction factor for the specific gravity of blood, which introduces another potential source of error. The period of observation after infusion of the indicator is another important variable since recirculation may alter the flow calculations. The extraction ratio, the ratio of arteriovenous concentration difference to arterial concentration of the indicator, determines the tissue concentration when recirculation occurs. Tissues that have a high extraction ratio for the indicator being used will accumulate indicator during recirculation whereas those with a low extraction ratio will give up indicator. Therefore, it is necessary to calculate this ratio for the

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indicator being used and the tissue under study when injections or the period of observation after injection exceed the recirculation time, as is the case for all such studies. Salts of rubidium and potassium are useful in the measurement of blood How in tissues such as heart, kidney, and gut, since the extraction ratios in these tissues is close to unity. In contrast, brain has a very low extraction ratio for these salts, so blood flow is grossly underestimated with these indicators (Sapirstein, 1958). Iodoantipyrine, however, has an almost ideal extraction ratio in neural tissue, making it the preferred indicator in this situation (Sapirstein, 1958; Sakurada et al., 1978; Sladky et al., 1985). Sugimoto et al. (1986) has described the use of ''C-labeled butanol in a variation of the indicator dilution technique. They used standard Fick principles to calculate NBF based on the quantity of indicator in the tissue at time T (the time of death of the animal), the arterial concentration of the indicator at various times during the infusion of the indicator, and the flow rate in the artery used for sample collection. Butanol is highly diffusible, penetrates nervous tissues readily, has a high oil-water partition coefficient and a low molecular weight (Sugimoto et al., 1986). These characteristics are important in a tracer used for NBF measurements. c. Heat Clearunce. Another method for recording blood flow is to measure the amount of current required to maintain a defined increment in local tissue temperature (usually 1°C). When other factors such as environmental temperature and radiant heat loss are held constant, the only variable becomes blood flow. Moving blood acts as a heat sink since the inflowing blood is cooler than the heated tissue. Higher flow rates enable the blood to dissipate the heat faster, requiring more current to maintain the increase in temperature (Hayakawa et al., 1985). A related technique is the recording of the time taken for tissue to return to baseline temperature after a defined heat increment is applied: A more rapid return indicates higher flow. These methods employ very accurate heating and heat-sensing electrodes, and the technology is not available to permit this type of recording in peripheral nerve. In addition, there are other problems with the use of this system in nerve, primarily related to the small volume of tissue involved. T h e degree of local trauma caused by the electrodes would undoubtedly have a large effect on NBF and the application of the heat pulse would also likely damage nerve because it cannot conduct heat away as effectively as can an organ with greater bulk, such as brain. Nevertheless, this is an interesting field for future development if small, accurate, atraumatic electrodes can be made. Multiple recordings from multiple sites could be made during a single

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39 1

session, and the method has the considerable advantages of ease of use and the short time needed to make measurements. These features compare favorably with hydrogen polarography, which requires a great deal of expertise in the manufacture and use of the electrodes and in the analysis of the data, and with autoradiographic methods, in which only a single recording can be made in each animal and for which some weeks are necessary for the autoradiographs to develop prior to the calculation of NBF. d. Microspheres. The injection of graded microspheres has been used for NBF estimations but the most important contribution of this technique has been in the demonstration of shunt vessels within nerve in the separation of nutritive and nonnutritive flow (Hales, 1981). This technique has no other inherent advantages over the methods already described. As in the tracer methods, the animal must be killed to complete the recording, so only a single measurement of flow can be made.

MODELINGOF HYDROGEN WASHOUT B. MATHEMATICAL One of the salient features of experimental hydrogen clearance curves in a number of tissues including brain and peripheral nerve is their frequent biexponential character. In brain, the two components are thought to represent blood flow in the grey matter (fast component) and white matter (slow component). Two such discrete anatomical compartments are not present however, in peripheral nerve. Experimental data indicate that the slower component in nerve correlates well with nutritive blood flow, whereas the fast component may arise from diffusion into larger vessels such as arteriovenous shunts. In order to understand how the individual compartment rate constants may be related to such variables as the capillary flow, large vessel flow, average density of large shunt vessels, diameter of shunt vessels, and arterial clearance functions, a mathematical model can be used. We have modeled the situation of a large arteriovenous (AV) shunt vessel surrounded by a cylinder of tissue. The tissue cylinder is taken to contain capillaries with a great enough density that clearance by capillary flow can be assumed to occur diffusely from all points in the tissue rather than from discrete capillaries. T h e model is then similar to the one discussed previously in reference to oxygen delivery to nerve, in which the large vessel surrounded by a tissue cylinder corresponds to a capillary surrounded by a Krogh cylinder, and clearance of hydrogen by capillary flow corresponds to consumption of oxygen by metabolically

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active tissue. The same mathematical methods may thus be used to solve the hydrogen washout problem as were used to solve the timedependent oxygen supply model. T h e relevant differential equation for diffusion and flow in the large shunt vessel is

where t is the time, DB is the diffusion constant of hydrogen in blood, and v is the velocity of blood flow. Similarly, the relevant differential equation for diffusion and consumption in the tissue is:

dP = DT($

+

r ar ax2 where DT is the diffusion constant of hydrogen in tissue, CO is the clearance rate of hydrogen by capillary flow alone, and P f is the final arterial hydrogen tension (usually zero) for the washout phase. Boundary conditions are those given by Eqs. (21-28) in Section 111, D[ COin Eq. (2 1) is, however, set to zero.] T h e initial condition is P = Pi at t = 0 for all 7 and x. Also, P A (arterial hydrogen tension) and COvary according to Eqs. (30) and (32) in section III,D with Ci=O. Cf is the steady-state clearance rate; for example, a capillary flow of 10 ml/min/ 100 gm tissue = 0.1 ml H2/ml tissue/min corresponds to C ~ 0 . 1 min-'=0.00 16'7 sec-'. The numerical solution proceeds as previously described for the oxygen supply time-dependent model. T h e parameters applicable to rat sciatic nerve which were used in this model are shown in Table 11. We have studied the effects of varying at

TABLE I1 NORMAL VALUESOF PARAMETERS FOR HYDROGEN CLEARANCE I N RAT SCIATIC NERVE Parameter

SB,hydrogen solubility coefficient in blood ST,hydrogen solubility coefficient in tissue

DB,hydrogen diffusion coefficient in blood D.1, hydrogen diffusion coefficient in tissue Rc, radius of shunt vessel R 1 , radius of tissue cylinder L, vessel length CI, clearance rate constant for capillary flow u, velocity of blood flow in shunt vessel P,. initial arterial hydrogen tension Pf,final arterial hydrogen tension T~ and TC, clearance time constants for arteries

Value 2.0 x cm3 H2/cmSblood/ Torr 2.0 x IW5 cm3 H21cmqblood/ Torr 3.5 x 10-5 cm2/sec 3.5 x cmz/sec 10 pm 440 pm 1000 p m 0.00167 sec-' 2800 pm/sec 71 Torr 0 8 sec

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a number of these parameters, such as Cf, v, L, RT, Rv, and r p , over a range of values. We found that the hydrogen tensions at various points in the tissue cylinder vary with time according to a biexponential function near the arterial end close to the large vessel, and according to a monoexponential function near the venous end or far from the large vessel. Figure 6 shows a typical clearance curve from the model at the arterial end of the large vessel for various radial distances away from the vessel. There is a rapidly falling tension near the vessel wall, with slower decay further from the vessel. When these curves are fitted to a biexponential function, P

=

PO + CSexp(-Kst)

+ C F exp(-KFt)

(37) the slow rate constants KS are in the range 0.12-0.58 min-', while the fast rate constants KF vary from around twice the slow rate constant (as low as 0.34 min-') to as high as 15 min-'. These values compare well with experimental measurements of hydrogen clearance rate constants.

120.

100.

80.

60.

40.

20.

0.0

Time, sec FIG. 6. Hydrogen clearance curves predicted by model at the arterial end of the large vessel, for several radial distances from the vessel center (wall of vessel = 10-pm radius corresponds to fastest clearance). Curves are shown for wall of vessel (-), 50 p m (---), 90 pm (...), 130 pm (-.-), 170 p m (- -), and 210 pm (-).

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Figure 7 shows the variation of the fast rate constants KF with distance from the large shunt vessel, for various axial locations (distances from the arterial end of the vessel). It may be seen that the fast rate constant is greatest at the wall of the vessel at the arterial end and decreases with radial distance from the vessel, as well as with axial distance (moving toward the venous end). T h e largest value (7.5 min-') corresponds to the clearance rate of hydrogen in arterial blood. Figure 8 shows that the contribution or weight of the fast component, expressed as a percentage [ 100% * CF/(Cs + CF)], also generally decreases with both radial and axial distance, with an abrupt transition to zero where the biexponential clearance function changes to monoexponential. Figure 9 shows the distribution of fast rate constants sampled at various positions in the Krogh cylinder. This may be compared with Figure 10, a histogram of experimental clearance rate constants expressed as flows. (The flow F = AKF where A is the partition coefficient of hydrogen in tissue, A = 100 ml H2/100 gm tissue).

I00

200

300

400

Radial Coordinate R. urn

FIG. 7. Dependence of calculated fast rate constants on radial distance from the vessel center for several values of axial distance (arterialend = 0). Curves ate shown for wall of vessel (-), 100 p m (---), 200 p m (. . .), 300 prn (-.-), 400 p m (- -), and 500 p m (...) .

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BLOOD FLOW A N D OXYGEN DELIVEKY IN NEUROPATHY

FIG.8. Dependence of fast component weight on radial distance from the vessel center for several values of axial distance (arterial end = 0). Curves are shown for wall of vessel (-), 100 p m (---), 200 p m (. . .), 300 p m (-.-), 400 pm (- -), and 500 p m (...) .

I

3

4

5

6

7

Fast Rate Constant, min-'

FIG. 9. Histogram of fast rate constants resulting from model of hydrogen clearance with normal parameters.

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$ E

n

3

z

400

200

0

100

300

800

600

500

700

900

1,OOO 1,200 1,400 21,500 1,100 1,300 1,500

Rate constant, rnl/min/lOOg FIG. 10. Histogram of all rate constants derived from nerve blood flow measurements and arterial H2 clearance curves. (From Day et al., 1989.)

VI. Diabetic Neuropothy

A. EVIDENCE OF ENDONEURIAL ISCHEMIAI N DIABETIC NEUROPATHY That there is microvascular pathology in human diabetic neuropathy (HDN) is incontrovertible. There is strong evidence that the fiber degeneration is, at least in part, ischemic. There are four major lines of evidence for peripheral nerve ischemia in HDN. First, there is the close statistical association of diabetic retinopathy, neuropathy, and nephropathy (Fagerberg, 1959; Pirart, 1978). Since there is good evidence of a microvascular basis of retinopathy and neophropathy, there is epidemiologic support for a microvascular mechanism of neuropathy (Fagerberg, 1959). Second, in addition to well-documented evidence of accelerated macrovascular atherogenesis in diabetes, there is now clear-cut evidence of a similar microvascular process (Thomas and Eliasson, 1984). There is endothelial cell hypertrophy and hyperplasia, intimal and smooth muscle cell proliferation, and capillary thrombosis (Timperley et al., 1976) and closure (Dyck et al., 1985). The percentage of closed capillaries was found to be directly related to the severity of nerve pathology

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in both insulin-dependent and non-insulin-dependent diabetic neuropathy (Dyck et al., 1985). Third, there is basement membrane thickening and redundancy (Johnson et al., 1986). Fourth, there is a multifocal loss of myelinated nerve fibers, a hallmark of nerve ischemia (Dyck et al., 1986; Johnson et al., 1986). This multifocal loss of fibers is well seen in a reconstruction of the distribution of fiber loss at different levels of peripheral nerve in HDN with “diffuse” distribution of neuropathy. The changes are well seen in proximal segments. In more distal sites the cumulative fiber loss may lead ultimately to severe and diffuse loss of myelinated nerve fibers.

B. THEHYPOXIC HYPOTHESIS The observations on HDN and our observation on chronic progressive experimental diabetic neuropathy (CPEDN) led us to suggest that much of diabetic neuropathy might be generated by endoneurial hypoxia. At that time, the mechanism of diabetic neuropathy was suggested as being due to myoinositol deficiency (Greene, 1983) resulting in a metabolic disorder of the axon o r Schwann cell. Although there clearly appeared to be an acute metabolic effect of diabetes on peripheral nerve, resulting in conduction slowing of 10-15%, normalization of nerve free myoinositol ameliorated the acute metabolic effects but did not result in improvement of CPEDN or progressive HDN. Furthermore, nerve myosinositol was found to be normal in HDN and CPEDN (Dyck et al., 1980, 1988; Cameron et al., 1986). T h e hypoxic hypothesis is based on the premise that fiber degeneration and the pathophysiology of diabetic symptoms are due in large part to microvascular ischemia and hypoxia. According to this hypothesis, CPEDN is a metabolic disorder in which the brunt of the insult is borne by the microvessels of nerve at an early stage of the neuropathy, and fiber degeneration is due to endoneurial hypoxia. We hypothesized that the diabetic state results in hemorheologic and capillary abnormalities that lead to a reduction in NBF and oxygen delivery leading to multifocal fiber degeneration as a major mechanism of neuropathy (Low, 1987; Tuck et al., 1984). While there is unequivocal histopathological evidence of ischemic fiber degeneration in HDN, studies do not indicate whether these changes indicate a central pathogenetic role of microvascular ischemia or whether the changes are secondary and represent a late stage. T o approach this important issue we studied chronic experimental Streptozotocin (STZ) diabetic neuropathy, seeking evidence of endoneurial hypoxia and ischemia at a stage before florid fiber degeneration

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resulted. It was necessary to demonstrate that endoneurial hypoxia preceded fiber degeneration.

C. EVIDENCE FOR

THE

HYPOXIC HYPOTHESIS

T h e following is a summary of evidence in support of the hypoxic hypothesis.

1. Endoneurial hypoxia is present in CPEDN and in HDN. We found that sciatic nerve creatine phosphate was significantly reduced and lactate significantly increased in STZ diabetic rats at both the 1- and 4-month timepoints (Low et al., 1985b). Creatine phosphate is a storage form of adenosine triphosphate and alteration in creatine phosphate and lactate are the most sensitive metabolite responses to mild hypoxia (Low et al., 1985b). These biochemical changes provide indirect evidence for endoneurial hypoxia and in and of themselves represent relatively weak evidence. Direct evidence was found when we measured NBF in CPEDN (4 months) using microelectrode hydrogen polarography. NBF was reduced by at least one-third (Tuck et al., 1984). The reduction in NBF was likely due to microvascular changes and/or hemorheologic changes since there was a marked increase in nerve vascular resistance to 170% of normal (Low, 1987; Tuck et al., 1984). However, the reduced NBF could theoretically have been due to reduced oxygen requirements secondary to deranged metabolism of peripheral nerve (Greene, 1983) or due to fiber loss. To resolve this issue, we systematically measured endoneurial oxygen tensions using 02-sensitive microelectrodes. Oxygen histograms were obtained in the sciatic nerve of these rats. Endoneurial 0 2 (PNOp) reduction should occur if reduced NBF were primary, whereas it should be increased if NBF reduction were secondary to reduced tissue demands. After 4 months of EDN, PN02 was significantly reduced; 60% of measurements were <25 Torr, compared with only 19% in controls (Tuck et al., 1984). T h e critical oxygen tension in mammalian nerve is about 25 Torr (Low et al., 1985a), so that after 4 months of diabetes, the majority of nerve fibers were in a hypoxic state. Furthermore, we demonstrated that hypoxia per se will reproduce the lipid biosynthetic (Yao and Low, 1986) and electrophysiological abnormalities (Low et al., 1985b). Subsequently endoneurial oxygen tension was directly measured by oxygen microelectrodes, in human sural nerve of patients with diabetic neuropathy, and endoneurial hypoxia was found to be present (Newrick et al., 1986).

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2. Peripheral nerve is susceptible to hypoxia. An important question is the susceptibility of peripheral nerve to chronic hypoxia. We asked if nerve was susceptible to hypoxia if we increased intercapillary distance (ICD). ICD increase was achieved by making nerve edematous (experimental galactose neuropathy). T h e edema has a particular topography, being maximal in the subperineurial area. P N 0 2 and NBF reductions exactly ..paralleled the distribution of edema and of ICD increase, confirming that nerve was indeed susceptible to ICD increase (Low et al., 1985a; McManis et al., 1986). The effects of chronic hypoxia on normal rat peripheral nerve will reproduce the conduction slowing and resistance to ischemic conduction failure (RICF) (Low et al., 198613) as well as the pattern of [I4C]acetate incorporation alterations (Yao and Low, 1986). This susceptibility is not surprising. Previous clinical descriptions of hypoxic neuropathy are available. Hypoxia secondary to chronic obstructive pulmonary disease (COPD) is associated with neuropathy (Appenzeller et al., 1968; Faden et al., 1981), and this observation has been confirmed by a prospective study of 43 patients with COPD (Paramelle et al., 1986). These workers found electrophysiological evidence of significant neuropathy in 15 patients and of mild neuropathy in 17 patients. Nerve and muscle biopsies were performed in 8 cases. Findings were those of neurogenic atrophy in muscle, mixed axonal and demyelinating changes in nerve, and basal membrane thickening in both muscle and nerve capillaries. Although early in uitro studies of isolated mitochondria suggested marked insusceptibility to hypoxia, all subsequent in uiuo studies have demonstrated consistent hypoxic injury to tissue. For instance, at P A 0 2 of 35 Torr, synthesis of catecholamines and indoleamines is impaired (Davis and Carlsson, 1973). At a P ~ 0 2of 42-57 Torr, acetylcholine synthesis decreases by 40-50% (Gibson and Duffy, 1981). Glycolysis is stimulated by a P A 0 2 of 50 Torr (Norberg and Siesjo, 1975). With severe hypoxia, major changes in cyclic nucleotides (Benzi and Villa, 1976; Folbergrova et ul., 1981) and phospholipids of nerve cell membrane, with massive release of free fatty acids, occur (Gardiner et al., 1981). Nukada et al. (1986) have found that endoneurial hypoxia was associated with axonal pathology, suggesting that chronic mild hypoxia will have morphological effects. 3. Oxygen treatment will prevent and reverse certain electrophysiological and biochemical abnormalities of EDN. When rats with EDN were reared in an 02-enriched environment (40% 0 2 ) , nerve conduction slowing and RICF were partly prevented. T h e levels of nerve free sugars (glucose, fructose, sorbitol) were markedly increased in EDN. Oxygen supplementation resulted in no change in plasma glucose but

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the endoneurial sugars returned toward normal (by 60, 30 and 34% respectively). [I4C] Acetate incorporation in EDN was 57% lower than controls. After 0 2 supplementation, the changes were no longer significantly different from controls (Yao and Low, 1986). By combined high-performance thin layer chromatography and fluorography, we found that proportionately less [ 14C]acetate was incorporated into cholesterol and more into free fatty acids of diabetic nerves. Oxygen supplementation largely prevented these abnormalities (Yao and Low, 1986). T h e increase in P ~ 0 with 2 oxygen supplementation is small, likely too small to benefit established CPEDN. It is possible, however, to greatly increase PN02 by hyperbaric oxygenation (HBO). We found that treatment with HBO (100% 0 2 , 2.5 atm 2 hriday for 4 weeks) will result in nerve hypermetabolism and will normalize nerve action potential and RICF in well-established EDN (Low d al., 1988).

D. MECHANISMS OF MICROVASCULAR ISCHEMIA 1 . Macrovascular Disease

Atherosclerosis is more prevalent and more advanced for age in diabetics than nondiabetics (Thomas and Eliasson, 1984) and will result in microvascular ischemia.

2. Capillav Caliber Reduction or Closure Nerve ischemia ensues if capillary radius is reduced or if there is capillary closure, resulting in an increase in ICD. T h e evidence for microvascular ischemia in diabetic neuropathy has been described in Section V1,A. Although microvascular pathology has been well documented, the focus does not appear to have been sufficiently quantitative to address the crucial question of oxygen delivery. For instance, in the detailed morphometric study by Yasuda and Dyck (1987), increased variance of capillary lumen area and an increased percentage of closed capillaries were noted, but the data do not lend themselves to a quantitative analysis of oxygen delivery. 3 . Hemorrheologic Mechanism

In diabetes, the combination of increased whole blood or plasma viscosity, increased aggregability, and reduced red cell deformability results in a reduction of blood flow and stagnation hypoxia. The viscosity of blood is increased in both HDN and EDN (Skovborg et al., 1966; Isogai et al., 1976; Hoare et al., 1976; Barnes et al., 1977) and there is

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reduced red cell deformability (McMillan et al., 1983). T h e increased aggregability (McMillan, 1984) coupled with the reduced deformability leads to increased thixotropy of diabetic blood (McMillan and Utterback, 198 l), a phenomenon of increased resistance to acceleration of blood flow (McMillan, 1983) that results in increased shear stress on the microvascular wall leading to increased permeability and stimulation of synthesis of connective tissue (McMillan, 1984). T h e increased blood viscosity preceeds clinically detectable retinopathy, a site of prominent angiopathy (Lowe et al., 1980). Blood hematocrit is the major determinant of whole blood viscosity, but erythrocyte aggregability and deformability are also important contributors to blood viscosity (Dintenfass, 1979; Palinski et al., 1983). Plasma or serum viscosity is increased in diabetics (Cogan et al., 1961; McMillan, 1974, 1984; Isogai et al., 1976; Dintenfass, 1979; Lowe et al., 1980; Oughton et al., 1981). Plasma fibrinogen is increased (Skovborg et al., 1966; Hoare et al., 1976; Barnes et al., 1977; Ditzel, 1968; Mosora et al., 1972) as is a2-globulin, but albumin is reduced (Skovborg et al., 1966). 4. Vasoregulatory Abnormalities Prostacylin is the major vasodilator and inhibitor of platelet aggregation and thromboxane A2 has the opposite effects. T h e ratio of prostacyclin to thromboxane A2 is considered to be important in the maintenance of vascular tone (Moncada and Vane, 1979). Lipid hydroperoxides increase cyclooxygenase activity but in hibit prostacyclin synthetase activity (Moncada et al., 1976) such that the prostacyclinthromboxane ratio is reduced, favoring vasoconstriction and platelet aggregation in diabetes, and further aggravating microvascular ischemia-hypoxia. An increase in platelet thromboxane synthesis has been consistently found in human and experimental diabetes (Ward et al., 1989) and plasma thromboxane B2 (TxB2) synthesis is a linear function of plasma glucose. Prostacyclin is produced almost entirely in microvessels. We measured sciatic nerve 6-keto prostaglandin F1, (GKPGFl,), the stable metabolite of prostacyclin. We found that 6KPGF1, was normal at 1 month but reduced by 43%by 4 months (Ward et al., 1989). This reduction in prostacyclin, in conjunction with the known increase in platelet TxB2, would result in vasoconstriction and susceptibility to platelet aggregation and adhesion (Ward et al., 1989) and may result in further ischemia, hypoxia, and capillary damage. The delay in reduction of prostacyclin is in keeping with its role in CPEDN. Norepinephrine (NE) is reduced in peripheral nerve of chronically diabetic rats (Dhital et al., 1986; Ward et al., 1989). Whether this reduction results in denervation supersensitivity with excessive vasoconstriction is not known for nerve.

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5 . OFR-Mediated Mechanism OFRs may potentially cause microvascular ischemia by several mechanisms. These include the inhibition of prostacyclin synthetase (see Section VI,D,4), disruption of the blood-nerve barrier (BNB), and a direct effect on nerve (see Section Vl1,E). 6. Advanced Glycosylation End Products Advanced glycosylation endproducts may be an important mechanism mediating the effect of hyperglycemia on microvessels. This topic is further discussed in Section V1.F. 7. Impairment of Oxygen Release Oxygen release is affected by hemoglobin AIC, 2,3-diphosphoglycerate, hematocrit, pH, and perhaps by insulin administration itself (see Section III,B,6). 8 . Alherogenic Effects of Insulin There is strong epidemiologic evidence that insulin is atherogenic to nondiabetics. Three prospective population-based studies have independently shown that high plasma insulin, fasting or after oral glucose load, is associated with increased risk of major coronary artery disease in nondiabetics [Helsinki Policeman Study (Pyorala 1979); Busselton Study (Welborn and Wearne, 1979); Paris prospective study (Ducimetiere et al., 1980)l. Multivariate analysis showed that the insulin risk factor was independent of other coronary artery disease risk factors including plasma cholesterol, triglyceride, BP, and obesity. There have been no prospective studies on the influence of hyperinsulinemia as an independent risk factor in diabetics. However, there is substantial clinical and cross-sectional evidence of an association. T h e majority of patients with non-insulin-dependent diabetes mellitus (NIDDM) have increased insulin and glucose, and patients with insulin-dependent diabetes mellitus (IDDM) receiving exogenous insulin have increased plasma free insulin in the peripheral circulation between meals and at night (Reaven et al., 1972). Part of the explanation of hyperinsulinemia is the unphysiological route of insulin administration. Normally, insulin is secreted by the pancreas into the portal system and the liver degrades about 50% of the insulin in its first passage. Thus in the normal person insulin concentrations in the portal vein are much higher than in the systemic circulation (Blackard and Nelson, 1970). Since insulin is usually administered subcutaneously the reverse occurs in diabetics receiving insulin. Insulin antibodies form with long-term

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insulin administration, further increasing insulin requirements (Rasmussen et al., 1975). High fasting or postglucose insulin levels have a predictive value in the increased incidence of coronary artery disease and cardiac mortality (Stout, 1981). Kashyap et a1. (1970) and Santen et al. (1972) first observed higher plasma insulin in diabetics with atherosclerotic vascular disease than in those without. Diabetics who are obese or who have received treatment with insulin have elevated insulin levels nd have an increased incidence of coronary artery disease. Stand1 and Janka (1985) in a study of patients with NIDDM found that fasting C-peptide level was significantly higher in diabetics with atherosclerotic vascular disease (coronary artery disease, carotid disease, or peripheral vascular disease) than in those without atherosclerotic vascular disease. They also found that patients with NIDDM with atherosclerotic vascular disease had higher insulin requirements than those without atherosclerotic vascular disease. Hillson et al. (1984) studied newly diagnosed NIDDM patients and found that patients with electrocardiographic abnormalities had a significantly higher fasting and postglucose plasma insulin than patients without abnormalities. They also found a greater incidence of the appearance of new electrocardiographic abnormalities in patients with increased insulin. At the cellular and molecular level, insulin receptors are found in endothelial cells of both large and small vessels including pulmonary artery (Bar, 1982; King et al., 1985), human umbilical vein and artery (Bar, 1982; Bar et al., 1978), bovine fat capillary (Bar and Boes, 1985), and brain capillary (HaskeI1et al., 1985). This topic has been reviewed by Jacobs and Cuatrecasas (1983). Insulin and insulinlike growth factors have mitogenic effects on blood vessel wall by binding to receptors, and there are major differences between large vessels and microvessels (Vinters and Berliner, 1987). Insulinlike growth factors including IGF-I, IGF-11, somatomedins A and C, and multiplication-stimulating activity (MSA) are 7-9-kDa proteins that share several chemical, biological, and receptor binding properties (Bar, 1982). Microvascular endothelium is more susceptible to many of the effects of insulin than large vessels (Vinters and Berliner, 1987). For instance, insulin-induced stimulation of DNA synthesis or [ 14C]glucoseincorporation into glycogen occurs in retinal capillary, but not in aortic endothelium of human umbilical vein endothelium (King et al., 1983; Vinters and Berliner, 1987). Mouse brain microvascular endothelium showed greater uptake of aminoisobutyric acid than bovine aortic endothelium at the same insulin level (Berliner et al., 1983). A similar selectivity has been found with other growth factors. For

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instance, retinal endothelium had a three-fold increase in [‘Hlthymidine incorporation into DNA under the influence of IGF-I and MSA, but aortic endothelium was unaffected (King et al., 1985). For retinal endothelium, the degree of mitogenicity, in decreasing order, was insulin > IGF-I > MSA. Dose-response curves for the effect in all cell types examined showed a peak stimulation of DNA synthesis at nonphysiologic concentrations, usually 103- 1O4 ng/ml (Vinters and Berliner, 1987). However, mitogenic effects of insulin are seen in concentrations as low as 10-100 ng/ml (Vinters and Berliner, 1987). Relating these findings to levels in vivo, retinal capillary proliferation would be predicted (and is observed in diabetic retinopathy) with high circulating insulin levels, such as are seen during aggressive insulin therapy (Vinters and Berliner, 1987). Plasma levels of <20ng/ml are seen in patients treated with an artificial endocrine pancreas, and even higher levels are seen in patients with insulin resistance. Furthermore, the smooth muscle response to insulin and IGFs may be additive at near-physiologic ranges (King et al., 1985). There is some evidence that insulin levels and sensitivity may be affected by exercise. Improvement in glucose tolerance was described after 1 week of exercise in patients with mild NIDDM and this improvement was associated with a reduction in insulin levels, indicating increased insulin sensitivity (Rogers et al., 1988).

E. SUGGESTED PATHOGENESIS OF DIABETIC NEUROPATHY The pathogenesis of diabetic neuropathy remains incompletely understood. Central to the pathogenesis of diabetic neuropathy is chronic hyperglycemia. In the past decade, several hypotheses with either prophylactic or interventional treatment implications have been developed, and testing of these hypotheses has been a cogent stimulus to much of the data generated. T h e myoinositol (MI) hypothesis states that a reduction in nerve free MI results in neuropathy, perhaps via a reduction in Na+,K+-ATPase (Greene and Lattimer, 1987). The reduced nerve free inositol is thought to be secondary to hyperglycemic activation of the polyol pathway. Another effect of hyperglycemia is a reduction in sodium-dependent active transport of MI into nerve. T h e importance of this hypothesis relates to the treatment implications. Nerve free MI is reduced and can be normalized by control of hyperglycemia, by aldose reductase inhibition, and by MI supplementation in acute short-duration experimental diabetic neuropathy. It has been much more difficult to postulate a role of MI deficiency

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in chronic progressive diabetic neuropathy, in which M I returns to normal in spite of progression of the neuropathy (Cameron et al., 1986), and in chronic human diabetic neuropathy, in which MI has been found to be normal in biopsied sural nerve (Dyck et al., 1980, 1988). Hyperglycemia has been suggested to produce neuropathy by other mechanisms, such as the generation of advanced glycosylation end products, which leads to the trapping of low-density lipoproteins and macrophage activation, resulting in microvascular atherogenesis (Brownlee et al., 1988). Another postulated mechanism is the effect of hyperglycemia on slow axonal transport. Hyperglycemia causes an impairment in slow transport of structural proteins, resulting in proximal axonal enlargement and distal attenuation (Medori et al., 1985). The mechanism of the transport abnormality is not known. Possibilities include protein glycosylation or phosphorylation. Hypoxia does not appear to impair slow transport. The suggested pathogenetic scheme (Fig. 1 1 ) is presented to incorporate recent advances and to provide a framework for further conceptual development and research. It is biased towards a microvascular basis. T h e suggested scheme has been previously described (Low, 1987). Chronic hyperglycemia in experimental diabetic neuropathy results in a reduction in nerve blood flow and endoneurial hypoxia. Two components of the scheme are of crucial importance. T h e first is the mechanism by which hyperglycemia will generate microvascular changes. Chronic hyperglycemia has been suggested to cause macro- and microvascular atherogenesis by the generation of advanced glycosylation end products (AGE) (Brownlee et al., 1988). T h e latter, by increasing

I

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+

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FIG. 1 1. Suggested pathogenesis of diabetic neuropathy.

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macrophage recognition and uptake, stimulates macrophage-derived growth factor. This is one mechanism that has been suggested to result in smooth muscle proliferation and atherogenesis. There is also an AGE-induced increase in low-density lipoproteins and other growth factors in vessel wall. There is considerable experimental support for this hypothesis (Brownlee et al., 1988). These findings also have important treatment implications. Administration of aminoguanidine hydrochloride results in the formation of unreactive early glycosylation products rather than AGE. These workers have reported that rats treated with aminoguanidine for 10 months did not develop the renal glomerular basement membrane thickening seen in untreated diabetic animals (Brownlee et al., 1988). A second factor of crucial importance is the mechanism in which hypoxia, once established, activates a vicious cycle of further capillary damage and escalating hypoxia (Fig. 12). Endoneurial hypoxia is secondary to NBF reduction and increased endoneurial vascular resistance (Low, 1987; Tuck et al., 1984). Endoneurial ischemia-hypoxia results in the breakdown of ATP to hypoxanthine (Low et al., 1985b), the generation of reducing equivalents including NADPH (Low et al., 1985b), and the conversion of xanthine dehydrogenase to xanthine oxidase, its active form (McCord, 1985). T h e availability of substrate, enzyme, and cofactor would create an an vivo free radical generating system (McCord, 1985) producing the superoxide radical. Intermittent ischemia may be more deleterious, since reperfusion results in acceler-

FIG. 12. Suggested ischemic-hypoxic cycle in the pathogenesis of diabetic neuropathy. For details, see text.

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ated free radical formation by introducing oxygen into a system that has been primed to produce free radicals. Superoxide radical is converted to hydrogen peroxide by superoxide dismutase, which is reduced in some diabetic tissues including diabetic peripheral nerve (Ward et al., 1989). T h e superoxide generated may reduce metal chelates by the HaberWeiss reaction, generating hydroxyl radicals, which damage endothelial cells. T h e blood-nerve barrier has been reported to be defective in both human (Ohi et al., 1985) and experimental diabetics (Rechthand et al., 1987). OFRs react with several compounds including lipids to generate lipid hydroperoxides. Ischemia will activate phospholipases (especially A2) to break down membrane phospholipids and activate the arachidonic acid cascade generating leukotrienes (further damaging endothelial cells) and prostaglandins. Lipid hydroperoxides increase cyclooxygenase activity but inhibit prostacyclin synthetase activity (Moncada et al., 1976) such that the prostacyclin-thromboxane ratio is reduced, favoring vasoconstriction and platelet aggregation in diabetes and further aggravating microvascular ischemia-hypoxia. An increase in platelet thromboxane synthesis has been consistently found in human and experimental diabetics (Gerrard et al., 1980; Halushka et al., 1981; Karpen et al., 1982; Ziboh et al., 1979), and plasma TxB2 synthesis is linearly related to plasma glucose concentration. T h e composite suggested pathogenesis of chronic progressive diabetic neuropathy (CPDN), focusing on the role of hyperglycemia in atherogenesis and the ischemic-hypoxic cycle, is shown in Fig. 13. Hyperglycemia results in metabolic effects on nerve fibers as well as microvessels. T h e metabolic neuropathy that develops within a week or two of hyperglycemia is related, at least in part, to nerve free M I reduction and is prevented or normalized by euglycemia or by the administration of MI or aldose reductase inhibitors. T h e transient improvement of nerve conduction velocity of 2-3 m/sec following institution of near-euglycemia may be the human counterpart of this phenomenon. T h e relationship of this metabolic “neuropathy” to CPDN is uncertain and may be less important than was previously thought (Dyck et al., 1988). T h e major metabolic effect of hyperglycemia in CPEDN is suggested to be on microvessels acting via AGE and rheologic mechanisms to produce microvascular pathology, reduced NBF, and endoneurial hypoxia. A vicious cycle supervenes in which eicosanoids (especially with reduction in the prostaglandin-thromboxane A2 ratio), leucotrienes, AGE, and OFR, as well as continued rheologic mechanisms, are important. Insulin per se may also have an atherogenic role. T h e role of M I or sorbitol is uncertain. The progressive microvascular and macrovas-

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Structural L

MI-sorbitol derangements

e m

Metabolic neuropathy

rbc deformability neuropathy Eicosanoidsl leukotrienes Oxygen-free radical

FIG. 13. Composite pathogenetic scheme of the pathogenesis of chronic progressive diabetic neuropathy.

cular pathology results in the nerve fiber pathology of multifocal fiber loss. The hypoxic-ischemia mechanism is likely to be of great importance in the pathogenesis of CPEDN and CPDN, but other factors, including genetic susceptibility and immune mechanisms, are also likely to be important in so heterogenous a disorder as HDN.

F. RESULTSOF MATHEMATICAL MODELING The hypothesis that reduction in endoneurial oxygen tension in diabetic neuropathy may be due to reduced nerve blood flow is in agreement with the results of the mathematical model of nerve oxygen supply. Measurements of oxygen tensions in sciatic nerves of diabetic rats (Tuck et al., 1984; Low et al., 1984) show a mean oxygen tension of 23 Torr, which is 8 Torr less than in normal control animals without neuropathy. The mean blood flow in diabetic rat sciatic nerve was 0.087 cm3 blood/cm3 tissue/min, only 67% of the value (0.130) in normal rats (Low et al., 1984). Using a blood flow velocity of 67% of normal in our model, we would predict a mean tissue oxygen tension of 26.2 Torr, which is 6 Torr less than that with normal flow. Measured oxygen tensions ranged from under 10 to around 45 Torr in diabetic animals, while values predicted by our model at 67% flow range from 16.8 Torr

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at the lethal corner to 87.2 Torr at the arterial end adjacent to the capillary. Figure 14 compares histograms of experimentally measured oxygen tensions with those generated by the model. A 33% reduction in flow can probably explain most of the reduced endoneurial oxygen tension measured in experimental diabetic neuropathy. Factors other than blood velocity may also contribute to reduced oxygen tensions, however. For example, occlusion of some capillaries would increase effective intercapillary distance, and alterations in the oxygen dissociation curve could result from the changes in erythrocyte 2,3-DPG and HbAlC concentrations in diabetes.

VII. Ischemic Neuropathy

A reduction in nutrient blood supply has been recognized as a cause of peripheral nerve disease since 1863, when Patry described three young patients with typhoid fever who developed gangrene in one limb. All three were severely ill and two of the victims died. Each patient complained of the abrupt onset of intense pain in the involved limb

+.

C a,

FIG. 14. Histogram of oxygen tensions calculated from 2500 simulations of model with velocity 67% of normal (dashed line), compared with experimental histogram in 308 diabetic rats (solid line).

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which was followed by the development of dry gangrene and limb anesthesia. In the two autopsied cases, Patry found arterial and venous thromboses in the major vessels of the affected leg and felt that these had caused nerve infarction, which accounted for the patients’ pain and sensory symptoms. Since that time, many publications have been written on the relationship between ischemia and nerve dysfunction. It has been recognized that nerve damage can occur with extensive microvascular disease (e.g., polyarteritis nodosa and, in all likelihood, diabetes), as well as with the complete arrest of circulation in a limb that produces gangrene. Most of the early descriptions of the connection between peripheral neuropathy and vascular disease discussed the results of a severe reduction in blood flow accompanying arterial thrombosis and embolism, but Kernohan and Woltman ( 1938) indicated that the neuropathy seen in polyarteritis nodosa is due to ischemia. Subsequently, Fetterman and Spitler (1940) ascribed pathological changes in nerves to other disorders affecting circulation in small blood vessels, such as syphilis and p l y cythemia. When a disease process causes a complete loss of blood flow in a limb, the resulting gangrene is associated with infarction of the entire nerve trunk of involved nerves. Mild to moderate degrees of ischemia do not produce any pathological alteration in peripheral nerves. Between these two extremes there may occur a peculiar but very characteristic pathological lesion of nerves that are subjected to severe ischemia with some preservation of blood flow into the affected limb. At predictable sites along the course of these nerves, focal areas of infarction appear that involve only the centers of large-diameter nerve fascicles, particularly those in the center of multifascicular nerves, or wedge-shaped infarcts with the bases of the wedges towards the middle of the nerve (Dyck et al., 1972). Small-diameter fascicles are relatively or completely spared. Research efforts in ischemic neuropathy have been directed towards identifying the pathophysiological substrate for this pattern of nerve damage. Theories that have been postulated include (1) relative sparing of blood flow to the outer portion of nerves, possibly due to superior anastomotic flow to this region under ischemic conditions (see, e.g., Sladky et al., 1985); ( 2 ) a greater safety margin against ischemia in the peripheral region because of greater capillary density (Nukada et al., 1985); and (3) diffusion of substances into or out of the subperineurial region during ischemia (Bentley and Schlapp, 1943; Blunt, 1960; McManis and Low, 1988). We believe that the weight of evidence favors the last hypothesis.

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A. EARLY OBSERVATIONS In 1943 Bentley and Schlapp published their observations on the blood supply of nerves. At that time, it was known that the excitability of nerves is dependent on an intact blood supply and that limb ischemia induced by a tourniquet on a limb causes anesthesia, beginning distally and spreading proximally. In the first experiments of their kind, Bentley and Schlapp examined the importance of blood flow to nerves in vivo. They studied nerve conduction in the sciatic nerves of cats and found in preliminary testing that several of the regional nutrient arteries could be cut without affecting the ability of the nerve to conduct electrical impulses. If the nerve was made completely ischemic by severing all the segmental arteries and cutting the nerve above and below the length of nerve being tested, however, conduction failed. I n the distal parts of the limb, conduction failed in 30 min, but the time to failure of conduction in nerve segments lying in well-vascularized muscle in the thigh was 2 hr. The authors postulated that an environmental effect, possibly diffusion of substances from muscle into the ischemic nerve, was the explanation for this phenomenon. Additional evidence for the diffusion hypothesis was provided in three further experiments. First, the authors showed that conduction failed in the thigh much earlier when the surrounding muscles were made ischemic. Second, conduction in distal nerve segments was maintained when these parts of the nerve were transposed into the thigh muscles adjacent to the proximal nerve, which they had shown to be more resistant to ischemia. Finally, wrapping the nerve in a sheet of rubber caused a substantial reduction in the time to conduction failure in the thigh but not in the distal leg. These findings were interpreted as indicating that the muscle bulk in the thigh was acting as a reservoir of oxygen that could diffuse into the ischemic nerve in the thigh, whereas the distal nerve lacked such a reservoir. These authors showed that the blood flow requirements of nerve are low. They demonstrated that a tourniquet inflated to just above arterial pressure would allow a trickle of blood to flow in the artery and that this was enough to maintain nerve conduction for considerably longer than when all blood flow was stopped by inflating the cuff to a much greater pressure. Blunt (1960) extended these observations and confirmed the importance of diffusion in preserving conduction in ischemic nerves. He showed that ligating all of the regional nutrient vessels of the sciatic nerve in the thigh of the rabbit caused no functional or pathological

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change. Similarly, wrapping the nerve in a film of polyethylene had no effect when the vascular supply was left intact. In contrast, the combination of these two procedures caused clinical deficits consistent with a sciatic neuropathy and degenerative changes on microscopic examination in every animal. These and other early studies established that nerve is resistant to ischemia because its NBF requirements are low. They also provide compelling evidence for the important role played by diffusion in (1) maintaining conduction in ischemic nerves and (2) preventing ischemic degenerative changes. It is important to note that in none of Blunt’s rabbits was there panfascicular ischemic necrosis. Some nerves had only scattered fiber degeneration and the maximal extent of damage was the loss of about 50% of the myelinated axons. Blunt did not describe the distribution of the fiber loss, so it is unclear whether there was a centrifascicular pattern to the ischemic degeneration. Since oxygen can diffuse only partway into large nerve fascicles (McManis and Low, 1988), it is possible that these experiments and those of Bentley and Schlapp were the first models of the sparing of subperineurial fibers in nerve ischemia.

B. EXPERIMENTAL MODELSOF ISCHEMIC NEUROPATHY Recent investigations into nerve ischemia have concentrated on the distribution of ischemic damage and on the mechanisms for the centrifascicular pattern. The following models for examining nerve ischemia have been established. T h e discussion includes comments on the major discoveries made with each technique. 1. Arterial Ligation

The most common method of reducing nerve blood flow is the ligation of arteries. Some experiments have involved the ligation of one or more of the major arteries supplying the limb while other researchers have tied off all the small nutrient arteries to the segment of nerve being studied, with or without interruption of the longitudinal plexus of vessels on and within the nerve trunk. Early in the course of research into nerve ischemia, it was determined that the ligation of the major limb artery alone does not cause any pathological change in nerve. Similarly, cutting the regional arteries does not by itself produce ischemia of sufficient severity to cause conduction failure or degeneration. Combining these two procedures, placing a plastic o r rubber film around the nerve, or ligating multiple

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major limb arteries will cause degrees of nerve ischemia that vary in severity among species. T h e first effective model which did not require mobilization of the nerve from its bed was developed by Korthals and Wisniewski (1975). These researchers tied the abdominal aorta and the femoral artery supplying the limb being studied in cats. The four cats in which these two vessels were tied simultaneously all developed severe lower limb paralysis consistent with a distal sciatic nerve palsy. All had total necrosis of the proximal parts of the peroneal and tibial nerves up to the sciatic bifurcation as early as the third postoperative day. At the edges of the zone of total necrosis there were regions with centrifascicular infarction and preserved subperineurial axons, although by the tenth postoperative day this appearance in the distal adjacent segment was obscured by Wallerian degeneration. If the aorta or the femoral artery was ligated in isolation there was degeneration of only rare axons in the peroneal and tibial nerves, and there was a similar lack of pathology if the ligation of the two arteries was separated by 2 months. Tying off the femoral artery 3 days after aortic ligation caused extensive necrosis. In rabbits, aortic ligation was universally fatal, and simultaneous ligation of the common iliac and femoral arteries caused much more extensive damage than in the cat model described above. In a subsequent study, these authors (Korthals et al., 1978) used the same experimental model to show that organelles accumulate in axons at the borders of the nerve infarct, probably due initially to failure of fast axonal transport and later to a mechanical block of axoplasmic transport. They also confirmed that larger nerve fascicles are more vulnerable to ischemia than smaller ones and that the center of fascicles is more vulnerable than the periphery. These findings are consistent with the observations of Dyck et al. (1972) in human vasculitic neuropathy and our later demonstration of the ability of oxygen to diffuse partway into ischemic nerves (McManis and Low, 1988). A similar double arterial ligation model has been developed for the rabbit by Hess et al. (1979) using a variation of Korthals’s technique. Aortic ligation in a single rabbit did not prove fatal, contrary to the experience of Korthals and Wisniewski (1975). Hess et al. variously tied off the common, external, and internal iliac arteries in combination and found that ischemic changes were most reliably produced by ligating the internal iliac together with either the external or the common iliac arteries. Like previous investigators, they found that occluding the femoral artery alone did not Cause fiber degeneration. The ligation of two proximal arteries caused less severe and less focal ischemia than in Korthals’s cats, but the evidence pointed to the sciatic bifurcation region

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as the most affected site, as all other studies have indicated. In addition, Hess et al. found that the nerve fiber degeneration was maximal in the centrifascicular region, and in at least one rabbit there was a large fascicle containing a centrifascicular infarct with two adjacent smaller fascicles being completely unaffected. This model of ischemic neuropathy causes less severe and less consistent ischemic lesions than the cat model, but it is still a useful one in that it may be advantageous for certain experiments to have a model that does not produce total necrosis of axons. In a physiological study of the effects of nerve ischemia, Sladky et al. (1985) measured NBF in rats after ligating the femoral artery alone. They were interested in the relative distribution of ischemia in the peripheral nerves and were not concerned that this degree of ischemia was unlikely to cause ischemic fiber damage. Using iodoantipyrine autoradiography, they found that acute occlusion of the femoral artery reduced NBF maximally at the sciatic nerve bifurcation. Their recordings suggested that blood flow was lowest in the center of the proximal tibia1 nerve. This observation was felt to explain the centrifascicular infarct pattern. They postulated that these sites were most affected because they lie in a watershed zone between regional arterial territories. We provided further evidence to support this hypothesis (McManis and Low, 1988) by showing that ligation of regional arteries proximal o r distal to the sciatic bifurcation (hypogastric trunk and femoral artery, respectively) caused a substantial reduction in NBF at the bifurcation in both cases. In contrast, the blood flow in distal parts of the nerve was unaffected or minimally reduced by hypogastric trunk ligation and femoral artery ligation had little impact on proximal NBF. We were unable to confirm that there was a differential reduction in NBF in the subperineurial and centrifascicular regions in our experiments. Using high-resolution hydrogen polarography, we identified well-defined arterial territories for each of the major vessels and found that tying these arteries produced large reductions in NBF. However, there was never any significant difference in NBF between the central and subperineurial regions. We speculated that the differences identified by Sladky et ul. were artifactual and the result of including the epineurium in the “outer annulus” oftheir nerves. It is known that the blood flow in the epineurium is approximately double that of the endoneurium (see, e.g., Rundqvist et al., 1985) because of the many large vessels that form the longitudinal plexus. It is reasonable to assume that flow in these superficial anastomotic vessels will be as high as possible when one nerve segment is ischemic. This would create a relatively

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higher blood flow in the outer than the inner annulus and give a false impression of relative ischemia in the center of the nerve on the autoradiograph.

2. Hypotension Another method of reducing NBF is to lower mean arterial blood pressure. Peripheral nerve autoregulates poorly and there is a direct relationship between blood pressure and nerve blood flow (Low and Tuck, 1984; Sundqvist et al., 1985; McManis and Low, 1988). Hypotension can be induced by progressive exsanguination or by connecting the vascular compartment of the animal to a gravity-controlled reservoir. The advantage of these methods is that they do not require the use of pharmacological agents, which affect vascular tone directly and produce spurious changes in NBF. Hypotension is a useful means of inducing nerve ischemia because the degree of reduction in perfusion pressure is the same in all arteries of comparable caliber. This permits the comparison of NBF in different regions without having to consider the effects of anastomoses between different arterial territories. We employed this method (McManis and Low, 1988) to show that the lack of a difference in NBF between the central and subperineurial regions was not the result of anastomotic flow. It could otherwise have been postulated that blood flow in nonligated arteries was preferentially supplying the subperineurial region to equalize NBF reductions in the two regions. Another advantage of the hypotension model is that it stimulates microvascular disease more closely than does the ligation of major arteries.

3. Microembolization Microemboli have been used to measure NBF, to study the effects of vascular occlusion in various organs, and to study arteriovenous shunts. Nukada has developed a unique model of ischemic neuropathy that employs microemboli to occlude microvascular channels (Nukada and Dyck, 1984; Nukada et al., 1985). Microspheres with a diameter of 15 pm were chosen since spheres of this size lodged in capillaries rather than larger proximal vessels and did not pass through the microcirculation. It was found that the injection of lo6 microspheres did not cause significant fiber pathology whereas 30 X lofimicrospheres produced severe ischemic damage but plugged arteries as well as capillaries. Since a model of microvascular disease was being sought, a final dose of 6 x lo6 spheres was chosen. T h e injection of this dose resulted in clinical and pathological changes in the distal sciatic nerve with vascular occlusion

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restricted to capillaries. Pathological examination revealed centrifascicular fiber degeneration of affected nerves without transneural infarction or ischemic changes in the soft tissues of the leg o r foot. T o perform this embolization technique, Nukada cannulated the major arteries supplying the sciatic nerve of rats with small-diameter (PE 10) plastic tubing. The most effective dose of 5.6 x lo6 microspheres was injected into the external iliac, internal iliac, and superior gluteal arteries in divided doses of 2.6 X lo6, 2.1 X lofi, and 9 x lo5 microspheres, respectively. The arteries were clamped proximal to the injection site during the embolization procedure. After injection the arteriotomies were repaired with ophthalmic suture material and the rats were allowed to recover from anesthesia. Pathological studies were performed at intervals from 6 hr to 6 weeks with the greatest number being done at 7 days. This model provides the closest approximation of microvascular ischemic neuropathy such as is seen in human vasculitis. It reliably causes centrifascicular infarction in the rat without necrosis of skin. Nukada and Dyck did not examine muscle histologically but it seems unlikely that this technique would produce significant muscle damage. Using the microembolization technique, these authors (Nukada and Dyck, 1984; Nukada et al., 1985) found that capillaries are more plentiful in the subperineurial region of some parts of the nerve and postulated that this may account for the sparing of subperineurial axons in nerve ischemia. They compared capillary density and minimal intercapillary distances in the inner and outer “contour areas” of nerve fascicles and found that there was a significant difference in capillary density between outer and inner areas of the sciatic and proximal tibial nerves, with the density being roughly twice as great in the outer contour area. Such a difference could not be found in the peroneal or distal tibial nerves. Statistically significant differences in minimum intercapillary distances were found only in the sciatic nerves. The authors argued that the greater capillary densities in the subperineurial regions would maintain adequate oxygenation during ischemia because of the shorter diffusion distances between capillaries, whereas the center of the fascicle would be relatively hypoxic. They further suggested that some of these capillaries could be in excess of the needs of the nerve when NBF is normal and that reserve vessels could open up under ischemic stress. There are several lines of evidence that suggest that these differences in capillary density cannot account for ischemic subperineurial sparing. First, there is no difference in resting NBF in the two regions, as would be expected if the capillary differences were physiologically significant (Sladky et al., 1985; McManis and Low, 1988). Second, the

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parts of the nerve with significant differences in capillary density do not correspond precisely to the segments of nerve that develop centrifascicular infarcts. Finally (and most relevantly), there is no sparing of NBF or oxygen tension in the subperineurial region during ischemia (McManis and Low, 1988). 4. Arachidonic Acid Injection Parry and Brown (1981) demonstrated that the injection of arachidonic acid into the femoral artery of rats caused focal infarction in the proximal tibia1 nerve similar to that produced by arterial ligation in the cat or rabbit. This is the result of the combination of femoral artery ligation and microvascular occlusion from platelet aggregation and vasospasm. Parry and Linn (1986) subsequently showed that low doses of arachidonic acid will cause transient conduction block at the same site without fiber degeneration or persistent clinical deficit.

C. PATHOLOGY OF NERVE ISCHEMIA Prior to the investigations of Blunt in 1960, the evidence for pathological changes induced by ischemia was scant. Virtually every study had found minimal or no fiber degeneration after arterial litigation with the single exception of a study by Okada in 1905 (cited in Blunt, 1960). The reason these experiments were unsuccessful in creating ischemic lesions is that they involved single artery ligation. This is not sufficiently severe an insult to cause degenerative changes because of the low metabolic demand and the wide safety margin of perfusion in peripheral nerve. Blunt found that the combination of the ligation of regional nutrient arteries and ensheathment of the sciatic nerve with a polyethylene film caused fiber degeneration in all cases. The extent of the abnormalities varied from the loss of a few scattered fibers to a 50% reduction in myelinated fiber numbers. He felt that the changes differed from crush-induced Wallerian degeneration in that the fiber loss was scattered rather than uniform. Fragmentation of axons, fusiform swellings, and vacuolation, indistinguishable from crush-induced changes, were seen on teased fiber preparations. Subsequent studies (Korthals and Wisniewski, 1975; Korthals et al., 1978; Hess et aE., 1979; Nukada and Dyck, 1984) have characterized the pathology of ischemic neuropathy. In rabbits the changes induced by arterial ligation were largely confined to small intramuscular nerves, but there were reductions in myelinated fiber numbers distal to the sciatic

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bifurcation, with paranodal demylination between normal and degenerating parts of axons. In contrast to the subperineurial lesions caused by nerve compression, transverse sections revealed centrifascicular degeneration in large fibers. Small-diameter fibers were generally spared. I n the cat model of Korthals and Wisniewski, there was more severe ischemic damage with total necrosis of the entire nerve at typical sites. Most myelin sheaths were disrupted at these sites and dark axons were present by the third day. The changes differed from Wallerian degeneration in that there was necrosis of Schwann cells and blood vessel walls as well as other endoneurial cells. Outside the zone of necrosis, there were adjacent centrifascicular lesions with swollen or collapsed nerve fibers and retraction of myelin at the nodes of Ranvier. Splits in the myelin sheath were shown by electron microscopy to be vesicular disruption of myelin lamellae. By 2 1 days, there had been extensive phagocytosis, which began after 5 days, together with thickening of the perineurium and an increase in endoneurial collagen. Axonal degeneration indistinguishable from Wallerian changes was seen distal to the infarcted region. I n a subsequent study, Korthals et al. (1978) showed that organelles accumulate at the proximal and distal ends of the nerve infarct. These organelles consisted primarily of mitochondria, dense bodies, and vesicles. Other structures seen included axoplasmic reticulum and membranous bodies. Centrifascicular degeneration in the rat is marked by axons that are dark, attenuated, vacuolated, and demyelinated (Nukada and Dyck, 1984). Other pathological changes include fiber separation, flattening of myelin profiles, and axonal cytolysis. The most subtle change is the presence of dark axons. Teased fibers revealed that more than half the axons have paranodal demyelination.

D. NERVECONDUCTION Many studies have documented that conduction fails in about 30 min in peripheral nerve that is totally ischemic. Nerves that are subjected to chronic endoneurial hypoxia are resistant to ischemia and will continue to conduct for longer periods (Low et al., 1986b). This phenomenon is thought to be the result of downregulation of endoneurial metabolism and a change from aerobic to anaerobic metabolism. The latter change is promoted by the lack of oxygen and the abundant supply of substrates in disorders such as diabetes and galactosemia. In normal nerve, conduction is rapidly reestablished with restoration

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of blood flow if the period of ischemia does not exceed 60-90 min. Recovery begins in 30 sec after blood flow returns and is complete within 5 min (Bentley and Schlapp, 1943). Schmelzer et al. (1989) have shown that 3 hr of ischemia induced by aortic occlusion caused conduction block in the region of the sciatic bifurcation to persist for at least 7 days. This was associated with incomplete restoration of flow, even after 2 hr of reperfusion. T h e site of conduction block was at the region known to be the most vulnerable to ischemia because it lies in a watershed zone between regional arterial territories. Prolonged ischemia also caused impaired function of the blood-nerve barrier. This damage to the blood-nerve barrier was aggravated during reperfusion, suggesting that peripheral nerve is vulnerable to reperfusion injury, which may be the result of the generation of oxygen free radicals.

E. PHYSIOLOGY OF CENTRIFASCICULAR INFARCTION It is clear that many models of peripheral nerve ischemia cause a reduction in NBF that is sufficiently severe to produce focal ischemic damage. In all of these models, the ischemic lesions occur at a highly characteristic site at the bifurcation of the sciatic nerve into the peroneal and tibia1 branches. A unique feature of these lesions and those that occur in many human ischemic neuropathies is the sparing of subperineurial axons around a core of fiber degeneration. As previously discussed, the mechanism for this pattern remains in dispute. Early studies in ischemic neuropathy did not address this question because the pattern was not recognized prior to 1972. Nevertheless, the work of Bentley and Schlapp (1943) and of Blunt (1960) provided very clear evidence for diffusion of substances, presumed to be oxygen, into the ischemic nerve. The findings and their significance have largely been ignored by researchers seeking the explanation for the subperineurial fiber-sparing-centrifascicular infarct pattern. This has opened the way to speculation on differential blood flow reductions in the two regions during ischemia from either better anastomotic flow to the subperineurial region or the protective effect of greater capillary density in the subperineurium. In spite of the findings of Sladky et al., (1985) we believe that there are no differences in the degree of NBF reduction in the central and subperineurial regions. Our test of this theory, using a technique for measuring NBF with high resolution and a high degree of reliability and reproducibility and several different models of nerve ischemia, provided no evidence for differential NBF reduction, making this hypothesis untenable. To examine this question further, we measured oxygen tensions in

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the central and subperineurial regions simultaneously, using microelectrodes and polarographic techniques (McManis and Low, 1988). These measurements were made at rest and in ischemic nerve under two sets of conditions; first in an anoxic environment, and second in an oxygenenriched environment. We found that oxygen tended to diffuse out of the nerve down a concentration gradient into the anoxic medium, rendering the subperineurial region less well oxygenated than the center of the nerve during ischemia. This finding makes it unlikely that there is better oxygenation of the subperineurium of ischemic nerve from the greater capillary density in this region. Noting the observations of Bentley and Schlapp (1943) and of Blunt (1960), we added oxygen to the pool of oil surrounding the nerve and found that oxygen is able to diffuse into nerve, particularly ischemic nerve, but that the diffusion was restricted to the subperineurial area. These findings fit very well with the observed effects of an insulating film between relatively well-perfused surrounding tissues and the ischemic nerve. Without the film, oxygen diffusion permits the ischemic nerve to continue to conduct for longer than when diffusion is blocked by a barrier impermeable to oxygen. It appears that oxygen diffusion can maintain the viability of the subperineurium better than the center of the nerve when the fascicles are larger than a certain diameter. Oxygen diffusion also accounts for the sparing of small-diameter fascicles, since the short diffusion distance permits oxygen to diffuse throughout the fascicle and prevent fiber loss. In multifascicular nerves the outer segments of some fascicles will receive sufficient oxygen by diffusion to minimize ischemic degeneration. This may explain the wedge-shaped infarcts seen in some fascicles. The metabolic demands and oxygen requirements of nerve are low (Low et al., 1985b). No functional or structural changes occur unless the reduction in perfusion is severe. Nerve fibers must be almost completely anoxic before undergoing degenerative changes. Thus, it is entirely feasible that the diffusion of a small amount of oxygen from surrounding tissues could support the axons in the subperineurium while central axons degenerate. Potential sources of oxygen include any adjacent well-perfused tissue when the ischemia is restricted to nerve. When the entire limb is ischemic, myoglobin in surrounding muscle may act as a reservoir of oxygen, particularly if muscle oxygen needs are low. In addition, fat surrounding the nerve could act as a potential oxygen source. A large amount of oxygen could be stored in this tissue because oxygen is six times as soluble in fat tissue as it is in nerve. We conclude from these studies that nerve ischemia causes fiber degeneration at characteristic sites along the nerve because these regions

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lie in watershed areas between the territories of major nutrient arteries, and that the centrifascicular fiber degeneration-subperineurial sparing pattern is the result of diffusion of oxygen from surrounding viable tissues partway into large nerve fascicles. MECHANISMS OF NERVE ISCHEMIA F. MOLECULAR

Essentially no information is available on the molecular mechanisms of nerve ischemia. I n other systems, especially intestine and heart, the ischemic insult is mediated, in large part, by OFR effects and perhaps by intracellular calcium influx during the period of ischemia and to a greater extent during reperfusion. There is also a complex interplay of ischemia, eicosanoids, and norepinephrine. Hypoxia-ischemia results in an increase in tissue reducing equivalents (Neely and Feuvray, 1981; Low et al., 1985b) and an increase in tissue xanthine (Jennings and Reimer, 1982). Ischemia or hypoxia has been suggested to activate a calcium-dependent protease (possibly calpain) that converts cytosolic xanthine dehydrogenase to xanthine oxidase (Batelli et al., 1972). This important reaction occurs in capillary endothelium (Jarasch et al., 1981). The above three conditions create an oxygen free radical-generating system. Furthermore, reperfusion accelerates OFR formation by introducing 0 2 into a system primed and generating OFR (McCord, 1985). Ischemia results in the release of norepinephrine from sympathetic nerve terminals and the release of OFRs (Bovaris and Chance, 1973). Catecholamine release results in the increased synthesis and release of prostaglandin 1 2 (PGI2) metabolites (Gilmore et al., 1968; Greenberg, 1978) by an a-receptor-mediated and calcium-calmodulin-dependent mechanism. Calmodulin is known to activate phospholipase A2 (Wong and Chung, 1979), resulting in a breakdown of membrane phospholipids and activation of the arachidonic acid cascade (Burton et al., 1986) generating leukotrienes (further damaging endothelial cells) and prostaglandins. Prostacylin, localized in the vascular endothelial cell (MacIntyre et al., 1978), is the major vasodilator and inhibitor of platelet aggregation described, acting by stimulating platelet adenylate cyclase (Gorman et al., 1977). PGIp synthetase is rich in vascular endothelial cells (Moncada et al., 1977; Weksler et al., 1977) and is inhibited by lipid endoperoxides (Moncada, 1982). The ratio of prostacyclin to thromboxane A2 (TxA2) is considered to be important in the maintenance of vascular tone (Moncada, 1982). T h e effects of ischemia in several tissues are amplified during reperfusion, a phenomenon referred to as reperfusion injury or re-

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duced reflow (McCord, 1985; Demopoulos et al., 1980; Hearse et al., 1986; Granger et al., 1986). We have found that such a phenomenon exists in nerve but only if the ischemia is near total and lasts at least 60 min (Day et al., 1989; Schmelzer et al., 1989). As described earlier, a major mechanism of reperfusion injury is OFR-mediated. There is a massive increase in cellular calcium (Cheung et al., 1986), leading to the suggestion that a key pathogenetic mechanism of ischemia is cytosolic calcium-mediated activation of phospholipases and the production of free fatty acids and lysophospholipids. However, a rise in cytosolic free calcium as an early event has been consistently found to be absent (Cheung et al., 1986). However, calcium may potentiate OFR toxicity (Malis and Bonventre, 1986) and the beneficial effects of calcium channel blockers may be mediated by blocking this potentiating effect and by reducing catecholamine-induced vasoconstriction (Cheung et al., 1986). There is some indirect evidence that similar ischemic mechanisms are operative in peripheral nerve. Calcium ionophores will cause vesicular disruption of myelin (Schlaepfer, 1977; Smith et al., 1985) and axonal degeneration (Schlaepfer, 1974), and nerve reconnection in a calciumfree medium resulted in improved functional recovery (de Medinaceli et al., 1983). The vasoconstriction and microvascular ischemia-hypoxia in disorders such as diabetes has been in part ascribed to perturbations of prostaglandins and OFR generation. Lipid hydroperoxides are increased and inhibit prostacyclin synthetase activity, resulting in a reduced prostacyclin-thromboxane ratio, vasoconstriction, and platelet aggregation (Ziboh et al., 1979). Three abnormalities in PGI2 have been described in diabetes. Apart from a reduced synthesis of PGIp, serum from patients with diabetes o r animals with STZ-induced diabetes contains less PGIZ releasestimulating factor or may have more of an inhibitory factor (Lubawy and Velentovic, 1982). The presence of a stimulating factor appears to be well established in normal serum (Seid et al., 1983; Snopko et al., 1987). In addition, platelets of diabetic patients have reduced responsiveness to PGIp (Betteridge et al., 1982), and dipyridamole will restore responsiveness (Karpen et al., 1982). Phospholipase A2 activity is increased in diabetes (Takada et al., 1981). We propose the hypothesis that the molecular mechanisms of nerve ischemia are similar to those in tissues like heart, gut, and brain but that nerve differs in its threshold to ischemic and reperfusion damage. Ischemia would result in phospholipase activation and phospholipid breakdown, liberating arachidonic acid and activating a cascade to produce the prostaglandins and leucotrienes. OFR would be generated

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during ischemia and especially during reperfusion by mechanisms described earlier. T h e formation of lipid hydroperoxides would inhibit prostacyclin synthetase. The increased biosynthetic rate of TxA2, coupled with the ischemia-related inhibition of prostacyclin production by endothelial cells, would result in vasoconstriction aggravating the ischemic insult. Mechanisms of blood-nerve barrier damage would include OFR, the leucotrienes (Moore, 1985), and arachidonic acid (Chan et ad., 1983). There are special problems in the study of peripheral nerve ischemia. As described in Section VII,A, reasonable models of nerve ischemia have only recently been developed. Another problem relates to the choice of indices of OFR effect on peripheral nerve. Malondialdehyde estimation has the advantages of demonstrating lipid peroxidation but has the disadvantage of limited specificity and being metabolized (Freeman and Crapo, 1982). Part of the problem is overcome by the use of a high-pressure liquid chromatographic method, thereby increasing sensitivity and specificity (Yu et al., 1986). Reduction of extractable membrane cholesterol and arachidonic acid and alterations in fatty acid profile are more sensitive but less direct indices. We have chosen a combination of indices, combining nerve malondialdehye assays, conjugated dienes and lipid hydroperoxide measurements, and determination of the BNB integrity. Finally, methods need to be refined to handle amounts of tissue measured in milligrams. Using our model, we are able to produce ischemic conduction failure consistently within 30 min of ischemia, with prompt recovery of impulse transmission following 1 hr of ischemia, but persistent conduction failure and conduction block followed 3 hr of ischemia. Reperfusion failure was evident in peripheral nerve after 1 and, especially, 3 hr of ischemia. NBF was not completely restored with reperfusion and there was a suggestion of progressive reduction in NBF with increasing duration of reperfusion. We used the BNB as a physiological index of the OFR effect, since OFRs are known to increase microvascular permeability (del Maestro et al., 1981 ; Korthuis et al., 1985). T h e permeability-surface-area product (PA) may be increased as a result of an increase in permeability or an increase in the surface area of endoneurial capillaries. In our study (Schmelzer et al., 1989), NBF remained persistently reduced with reperfusion. Therefore, the progressive increase in PA must be due to an increase in permeability. Our results suggest that reperfusion injury may exist in nerve if the ischemia is near total and of long duration, since it occurs following 3 hr but not 1 hr of occlusion. Based on the progressive increase in PA with

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reperfusion and the preliminary results of an increase in conjugated dienes and malondiddehyde with reperfusion (N. Pdrinandi, unpublished observations), we propose the hypothesis that nerve ischemia may be due to the generation of OFRs that damage the endothelial barrier and, subsequently, endoneurial contents. The mechanisms are likely to be similar to those involved in heart, gut, and brain but modified in terms of the threshold of each tissue to ischemic and reperfusion damage. The elucidation of the mechanisms of reperfusion injury and strategies to ameliorate this damage are particularly relevant in nerve, in which the ischemic threshold is much higher and the development of ischemic and reperfusion injury occurs over many hours, thus permitting ample time to institute treatment, should it become available.

G. RESULTSOF MATHEMATICAL MODELING

Mathematical modeling shows that the major effects of changes in arterial oxygen tension on the endoneurial oxygen tension are at the arterial end of the system. For much of the length of the capillary away from the arterial end, the difference in oxygen tensions resulting from entrance oxygen tensions ranging from 67.5 to 235 Torr is under 10 Torr. With a 50% reduction in arterial oxygen tension (to 45 Torr), which corresponds to breathing about 10% inspired oxygen concentration, the model predicts a mean tissue oxygen tension of 20.1 Torr, which is 12 Torr less than that with normal arterial oxygen tension. Oxygen tensions range from 11.9 Torr at the lethal corner to 42.7Torr at the arterial end adjacent to the capillary. The major effects of changes in blood flow on the endoneurial oxygen tension, however, are at the venous end of the system. With a 50% reduction in blood flow velocity in capillaries, the model predicts a mean tissue oxygen tension of 22.1 Torr, which is 10 Torr less than that with normal blood flow. Oxygen tensions range from 6.3 Torr at the lethal corner to 86.9 Torr at the arterial end adjacent to the capillary.

VIII. Edematous Neuropathy

In some peripheral neuropathies there is an accumulation of free water or other substances within the nerves. These accumulated substances may play an important role in the genesis and progression of nerve dysfunction by altering the nerve microenvironment. It is likely

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that the edema itself is the direct cause of the endoneurial ischemia and hypoxia that have been identified in experimental models of edematous neuropathy. Nerve edema may also be involved in the pathogenesis of diabetic neuropathy since sorbitol, an osmotically active sugar alcohol, accumulates within peripheral nerves in diabetics.

A. PATHOPHYSIOLOGY Nerve edema refers to a state of excess hydration. It is a common sequela of physical injury to the nerve but also occurs in response to the presence of agents that act to increase the osmotic gradient across the nerve membrane and in response to intoxication by substances that are directly neurotoxic. T h e increase in endoneurial fluid may be widespread throughout the endoneurium or it may be restricted to certain structures. Vasogenic edema, which is the type of edema seen as a result of thermal injury or other local trauma, causes a diffuse increase in fluid, whereas cytotoxic edema due to poisons such as hexachlorophene or triethyltin tends to be restricted to the myelin sheath, producing intram yelinic edema. Peripheral nerve is invested by a relatively inelastic perineurium that has only limited abilities to expand. Small increases in endoneurial volume can be accommodated but any increase beyond these limits will produce an elevation in endoneurial fluid pressure. This pressure elevation appears to be implicated in the pathogenesis of the neuropathy associated with nerve edema, particularly in the edematous neuropathies seen with osmotically active agents that are not toxic to nerve fibers, such as galactose (Sharma et al., 1976). In these conditions, the neural dysfunction and axonal abnormalities are likely to be the result of chronic endoneurial ischemia and hypoxia. 1 . Blood-Nerwe Bam’er Vasogenic edema occurs most commonly as the result of a breakdown in the function of the blood-nerve barrier. T h e barrier is formed by the perineurium and by the capillary endothelium within nerves. As in the brain, the capillaries have tight junctions between endothelial cells that make the vessels relatively impermeable. The passage of water, proteins, solutes, and electrolytes is largely dependent on active and facilitated transport mechanisms. Physical injury of the vessels will impair the ability of the blood-nerve barrier to control net water movement and result in edema. Similarly, substances that affect endothelial cell function such as hyperosmolar agents will produce a

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breakdown in the barrier. This property is employed clinically in the management of tissue edema; opening the barrier with hyperosmolar agents can cause an increase in the net movement of water out of the swollen tissues. In contrast, nonedematous tissues will have an increase in fluids and solutes when the barrier is opened. The mechanism by which the damage is produced is uncertain. It may be the result of mechanical distortion or shrinkage of endothelial cells or due to vascular dilatation) Powell et al., 1980). Substances that cannot cross the blood-nerve barrier by simple diffusion can move through capillaries by way of specialized systems, which tend to be selective. These include active, facilitated, and vesicular transport system. Small and medium-sized molecules used in energy production, such as glucose and amino acids, as well as the by-products of anaerobic glycolysis, are transferred by facilitated transport (Powell el al., 1980). Electrolytes, especially sodium and potassium, depend on active transport, which is an energy-requiring process. Active transport in the form of the sodium-potassium pump is one of the major energy-dependent metabolic activities in peripheral nerve. Transfer of substances across membranes by way of pinocytotic vesicles is a method that is not very active in neural tissues unless there is increased membrane permeability (Klatzo, 1977). Vesicular and facilitated transport are, in general, bidirectional and move substances down concentration gradients (Klatzo, 1977). Although water is able to pass freely through the blood-nerve barrier in either direction, its movement through the barrier and the nerve water content are effectively controlled in normal nerve by concentration gradients. Thus, nerve edema often is not the result of a change in barrier permeability to water but rather is due to alterations in transendothelial movement of substances that affect tissue osmotic pressure. Net water movement is determined by the difference between combined osmotic and hydrostatic pressures in the vascular and tissue compartments. The accumulation of certain substances within the endoneurium will increase tissue osmotic pressure and cause an increase in tissue water content (i.e., edema).

2. Cell Injury Some neurotoxins produce intraneural swelling by causing edema of the myelin sheath. Both hexachlorophene and triethyltin cause intramyelinic edema in the central nervous system. In the brain triethyltin produces dramatic swelling of myelin without any accumulation of fluid in the extracellular space or any reduction in the function of the

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blood-brain barrier. This may be the result of an increase in permeability of myelin membranes (Torack et al., 1960). T h e changes seen in the peripheral nerves are similar though less dramatic. On the other hand, hexachlorophene intoxication will result in severe abnormalities of peripheral myelin in addition to its effects on the brain. This intramyelinic edema occurs in the presence of an intact blood-nerve barrier and structurally normal myelin-axonal junctions at the nodes of Ranvier (Powell et al., 1979). The edema is restricted to myelin sheaths during continued exposure but severe interstitial edema may develop upon withdrawal of the toxin (Powell et al., 1980). Pathological changes are not confined to the myelin, however, since axonal degeneration is a common sequel to exposure to both hexachlorophene and triethyltin (Graham and Gonatas, 1973). Powell et al. (1980) speculate that this could be the result of the elevated endoneurial pressure that accompanies nerve edema. Arguing against this is the fact that the pressure elevations seen in edematous neuropathies are uniformly modest, being less than 10 Torr in all cases. This is much less than the pressure required to produce axonal damage with external pressure, and it seems unlikely that axonal degeneration could occur with intraneural pressures of 5 or 6 Torr, although this possibility cannot be completely discounted. Intraneural edema causes an increase in intercapillary distance in both experimental lead and galactose intoxication (Low and Dyck, 1977; Low et al., 1982). This separation of capillaries is associated with a reduction in tissue perfusion, particularly in the most edematous regions (McManis et al., 1986). Tissue perfusion is affected more by increases in intercapillary distance than by changes in any other parameter (McManis et al., 1986; Lagerlund and Low, 1987). Chronic endoneurial ischemia and hypoxia are associated with low-grade fiber pathology (Low et al., 1982). This undoubtedly plays a role in the genesis of the axonal damage in edematous neuropathies but cannot account for the relatively greater fiber pathology in lead neuropathy as compared with galactose intoxication. A third possible explanation for these axonal changes is that lead is directly toxic to axons. Although lead causes predominantly myelinic abnormalities in rats, human lead neuropathy is marked by axonal destruction rather than demyelination. It is probable that a combination of these factors is implicated in the fiber damage seen in experimental lead neuropathy. Experimental lead neuropathy produces capillary abnormalities within nerve that decrease the effectiveness of the blood-nerve barrier. Lead is selectively taken up by capillary endothelial cells, resulting in an increase in vascular permeability and vasogenic edema (Low and Dyck,

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1977). Lead is also taken up by Schwann cells, with resulting demyelination (Low and Dyck, 1977), but the diffuse intramyelinic edema produced by hexachlorophene does not occur. 3. Osmotic Edema

As discussed above, the accumulation of substances that increase tissue osmotic pressure can produce edema without a breakdown in the blood-nerve barrier. Water moves passively into the endoneurium down a concentration gradient, although this process may be aided by endothelial cell disruption. In human diabetics, endoneurial edema is at least partly due to a buildup of sorbitol within nerves. Whether this edema is causally related to the neuropathy of diabetes has been studied with the experimental model of galactose intoxication in rats. T h e chronic ingestion of 40% galactose produces neuropathy in rats marked by endoneurial galactitol storage. Both sorbitol and galactitol are sugar alcohols that are manufactured by the action of aldose reductase on glucose and galactose, respectively. Galactitol and galactose are not directly toxic to nerve, so their effects are mediated by other mechanisms (Sharma et al., 1976). Nerve edema is the result of the altered tissue osmotic pressure; there is no disruption of the barrier function provided by the perineurium and the endothelium of neural blood vessels (Malmgren et al., 1979). Thus, this experimental model provides a useful means of testing the direct effects of sugar alcohol accumulation. Low et al. (1982) showed that the pathological abnormalities in galactose neuropathy are mild and much less severe than the changes produced by diabetes, suggesting that the storage of sorbitol is unlikely to be an important mechanism in diabetic neuropathy. Additional evidence supporting this conclusion comes from studies of the effects of aldose reductase inhibitors in human and experimental neuropathies, which have shown only a small improvement in nerve function.

4. Nerve Crush Peripheral nerves that have been damaged mechanically exhibit edema at the site of injury, probably due to vascular disruption and perineurial damage, which diminish the effectiveness of the bloodnerve barrier. T h e edema may not be restricted to the site of injury when Wallerian degeneration occurs subsequently. T h e observed increases in endoneurial fluid and intraneural pressure may be related to a fivefold increase in mast cells and elevated serotonin levels in the distal nerve segment. Endoneurial fluid pressure, measured at a site distant (2 cm) from the injury, rises within 90 min of nerve crush and continues

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to rise during the first week afterwards, followed by a return to normal by 3 weeks (Powell et al., 1979). 5. Twitcher Mouse

Another experimental model of nerve edema is the Twitcher mouse. These mice have an autosomal recessive illness characterized by muscular weakness and tremor that leads to a premature death. Deficiency of the enzyme galactosylceramidase causes central and peripheral demyelination together with severe endoneurial edema and increased endoneurial pressure (Powell et al., 1983). Vascular permeability is increased in these mice with ensuing endoneurial (particularly subperineurial edema).

B. ENDONEURIAL PRESSURE Peripheral nerve is enveloped in a closely apposed cell layer, the perineurium. This structure is able to expand in an elastic fashion in response to increases in endoneurial fluid, within certain limits. When these limits are reached, the pressure within the nerve will rise. This sequence of events has been examined in depth because it is believed that the edema and the pressure rises are implicated in the pathogenesis of some neuropathies. All of the models of edematous neuropathy discussed above are associated with elevated endoneurial pressure at some point. Measurements of increased endoneurial pressure were first made by Low et al. in 1977 using polyethylene matrix capsules embedded in nerves of rats with lead neuropathy. Since that time it has been shown that endoneurial edema causes a rise in pressure in many different neuropathies. It has been postulated that the increased pressures are the cause of pathological changes, either by direct compression of axons, by compression of vessels within nerve, or by vessels traversing the perineurium. In assessing the potential pathophysiological mechanisms, it is critical to be aware of the time course of pressure changes within edematous nerves. Low (1981) summarized the events related to progressively increasing endoneurial fluid. Initial small volume increments do not cause pressure changes because there is normally some laxity in the perineurium, and reorientation of loose elastin and collagen fibers may occur. Further increases in volume in the acute model cause a reversible elevation of pressure. This is the elastic part of the volume-pressure

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curve and is presumably modulated by elastin. Pressure increases beyond the elastic limits of the tissue in the acute model result in steep rises in pressure, but in the chronic model this does not occur because of relaxation of the perineurium. Acute increases in endoneurial pressure such as might occur with hemorrhage into nerve or with a thermal injury can cause the pressure to rise as high as 20 mm Hg. This pressure is sufficient to collapse capillaries within nerve and produce ischemia. In chronic edematous neuropathies the endoneurial pressure never exceeds 10 mm Hg and is almost always less than 7 mm Hg. These pressure are clearly below capillary pressures. In serial studies of chronic endoneurial edema using either galactose (Low et al., 1980) o r lead (Powell et al., 1980), it has been demonstrated that there is only a small rise in pressure and that the pressure returns towards normal after 16-26 weeks. This may be due to stress relaxation of the perineurium and to “creep” of the perineurium, with the same stresses producing greater deformities the longer the pressure increases have been present. This suggests that the tissues have become plastic rather than elastic in their response to pressure increases. Neuropathy progresses despite this reduction in pressure with long-standing edema. This fact and the modest increases in pressure that occur make it unlikely that compression of intraneural vessels is the cause of neuropathy.

C. BLOODFLOWIN NERVEEDEMA Nerve edema is associated with chronic endoneurial hypoxia (Low et al., 1985a). This appears to be due to increased intercapillary distances in nerve edema. Experimental galactose neuropathy was chosen as the model to study this relationship because galactose and galactitol are not direct neurotoxins. It was shown that endoneurial oxygen tensions are significantly lower in nerve with galactose neuropathy than in control nerves, with many oxygen tensions falling below the critical level. Oxygen was particularly deficient in the subperineurial region, where intercapillary distances are greatest in edematous neuropathies. Myers and Powell (1984) measured nerve blood flow in galactose neuropathy using iodoantipyrine autoradiography and found that NBF was reduced. The degree of the reduction was correlated with the severity of edema as quantitated by measuring nerve water content. They postulated that ischemia and increased endoneurial pressure were responsible for the pathological changes. As discussed above, it is unlikely that pressure plays any part in the genesis of these changes. The role of ischemia has also been studied in our laboratory. We, too, found that

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there is a reduction in NBF in galactose neuropathy. Using a double microelectrode technique and hydrogen polarography to improve resolution of NBF recordings, we were able to show that the greatest reductions in NBF were in the subperineurial regions (McManis et al., 1986). This finding correlated well with the earlier demonstration of a similar distribution of hypoxia and provides compelling evidence that the fiber pathology of isolated endoneurial edema is due to hypoxia resulting from chronic ischemia which, in turn, is produced by increases in intercapillary distance. Nerve blood flow is also reduced in other edematous neuropathies. Myers et al. (1982) have demonstrated markedly reduced NBF in hexachlorophene neuropathy. In subsequent studies, Myers et al. (1986) have postulated that the decrease in NBF could be due to a pinching of small blood vessels as they traverse the stretched perineurium. They developed a biomechanical computer model that suggests that transperineurial vessels might undergo elliptical stretching in the circumferential plane with associated flattening of the vessel and loss of cross-sectional area. T h e model indicated that there could be a 25% reduction in the lumen of the vessels crossing the perineurium of edematous nerves. Morphological evidence to support this hypothesis is deficient since the authors have identified only a single vessel distorted in the manner they describe, but it is possible that this mechanism could aggravate the ischemia resulting from edema and increased intercapillary distances.

D. RESULTSOF MATHEMATICAL MODELING T h e results of mathematical modeling support the hypothesis that the reduced endoneurial oxygen tension in experimental galactose neuropathy is due to increased intercapillary distance caused by nerve edema. Measurements of oxygen tension in the sciatic nerves of rats with experimental galactose neuropathy (Low et al., 1985a) show a mean oxygen tension of 16 Torr, or 12 Torr less than the mean in normal controls. The number of capillaries per unit area of nerve was 30.6/mm2 in cases of galactose neuropathy, compared with 45.6/mm2 in normal animals. This corresponds to a mean area per vessel of 32,700 pm2 in cases of galactose neuropathy, which is 50% greater than that in normal nerves (21,900 pm2). In this case, the tissue cylinder radius would be 22% greater than normal. Using a tissue cylinder radius of 24% above normal (78 p m instead of 63 p m ) in our model, we predict a mean oxygen tension of 18.9 Torr, o r 13 Torr less than that with a normal radius. Measured oxygen tensions ranged from under 4 to about 44 Torr in galactose neuropathy, while values predicted by the model at

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45.-

0 *

0 k

40.-

35.-

1

-0.5 1 3 . 5 27.5 4 1 . 5 55.5 69.5 Partial

Pressure, Torr

FIG.15. Histogram of oxygen tensions calculated from 2500 simulations of model with intercapillary distance 124% of normal (dashed line), compared with experimental histogram in 222 galactose-fed rats (solid line).

124% of normal tissue cylinder radius range from 5.2 Torr at the lethal corner to 85.9 Torr at the arterial end adjacent to the capillary. Figure 15 compares histograms of experimentally measured oxygen tensions with those generated by the model. A 24% increase in tissue cylinder radius can probably explain the reduced endoneurial oxygen tension measured in experimental galactose neuropathy, provided that oxygen consumption per unit volume of tissue does not change in galactose neuropathy. In fact, oxygen consumption may be reduced because of fewer cellular (metabolically active) elements per unit volume when edema is present. However, this factor may be counterbalanced by the experimental observation that the degree of edema is significantly greater in the outer portion of fascicles than in the center, so that intercapillary distances may be even more than 24% increased in these regions.

Acknowledgments

Supported in part by grants from NINCDS (NO1 NS7 2302, R 0 1 NS2 2352, NS14302) and MDA and by Mogg and Mayo Funds. PAL is a recipient of a Jacob Javits Neuroscience Investigator award.

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INDEX

A Abnormal Involuntary Movement Scale, tardive dyskinesia and, 300 Absorption, zinc and, 152 Acetylcholine, 82-84 dopamine receptor subtypes and, 253 expression, 85-87, 90, 92-94 immunocytochemical location, 112, 113, 115, 122 neuron development, 128, 129, 131, I32 neuropathy and, 399 parkinsonism and, 5, 59 age of injury, 33 drugs, 41 future research, 48,49, 5 1 lesions, 25, 26, 28 neuropathology, 15 zinc and, 206 Acetylcholinesterase, 90, 93, 94, 120 ACTH, see Adrenocorticotropic hormone Adenylate cyclase dopamine receptor subtypes and, 244 neuropathy and, 42 1 parkinsonism and, 26, 28 Adrenalectomy, choline acetyltransferase and, 95 Adrenergic innervation, neuropathy and. 381,382 Adrenocorticotropic hormone (ACTH), angiotensin I1 and, 282, 286, 289 Advanced glycosylation endproducts, neuropathy and, 402,405-407 Age of injury, parkinsonism and, 30-33 Aging neuropathy and, 362,367 parkinsonism and, 20, 29, 58, 59 tardive dyskinesia and incidence, 3 11 morbidity, 3 15 natural history, 3 1 1-3 13

pathophysiological mechanisms, 338, 339,344 prevalence, 303-305,307,309 vulnerability, 316-327, 331, 332 zinc and, 157 a-Agonists, neuropathy and, 381 Akinesia, parkinsonism and, 3 , 4 , 57, 58 age of injury, 33-36, 38, 39 future research, 46 lesions, 24 neuropathology, 10, 11 Aldosterone, angiotensin I1 and, 257, 258, 282 Alleles, choline acetyltransferase and, 93, 123 Alzheimer’s disease choline acetyltransferase and, 84, 120, 121, 131, 132 tardive dyskinesia and, 302, 306, 308, 331,344,345 zinc and, 215 Amantidine, parkinsonism and, 46 Amino acids choline acetyltransferase and, 89, 90 neuropathy and, 426 parkinsonism and, 3, 5,23,44, 59 zinc and brain proteins, 168 CNS pathology, 2 17 histochemistry, 178 neurons, 203,204,221 vesicles, 207, 209, 2 12 r-Aminobutyric acid (GABA) choline acetyltransferase and, 112, 114 parkinsonism and, 5, 14, 15,48, 49 tardive dyskinesia and, 34 1, 342 zinc and, 209, 212, 214, 219 Amygdala choline acetyltransferase and, 11 1, 117, 121 zinc and, 148, 201

439

440

INDEX

Anastomoses, neuropathy and, 356, 357, 410,414,415 Anesthesia, neuropathy and, 383 Angiotensin 11, 257-260, 281-290 stress, 279, 280 study methods, 263-265 animals, 260-262 labeling, 263 tissue preparation, 262 study results hypertension, 271, 273-275 receptor distribution, 265-269 water balance, 276 Anorexia, zinc and, 216, 219, 220 Antibodies choline acetyltransferase and expression, 86, 88, 96- 100 future directions, 133 imrnunocytochemical location, 108 neuron development, 130 neuropathy and, 402 Anticholinergics, tardive dyskinesia and, 324,332,333 Antidepressants, tardive dyskinesia and, 328 Antigens, choline acetyltransferase and, 97-100, 108 Antioxidants, parkinsonism and, 53 Anxiety, tardive dyskinesba and, 302, 327 Apomorphine dopamine receptor subtypes and, 243, 244,246,248,249,252 parkinsonism and, 47, 56,57 Arachidonic acid, neuropathy and, 358, 417,421-423 Arousal, doparnine receptor subtypes and, see Dopamine receptor subtypes Arteries, neuropathy and diabetes, 409 ischemia, 410-417, 419, 424 ligation, 412-415,417,418 nerve blood flow, 384,386,387,389, 390,393,394 oxygen delivery, 364, 375, 378 regulation of blood flow, 379, 380, 382 Arterioles, neuropathy and nerve microvasculature, 358-362 regulation of blood flow, 379-381 Arteriovenous shunt, neuropathy and, 385,391,415

Atherisclerotic vascular disease, 403 ATPase choline acetyltransferase and, 87 zinc and, 176, 177 Atrial natriuretic peptide, 257, 281, 283-288,290 hypertension, 272-276 receptor distribution, 266-268, 270, 27 1 study methods, 263-265 animals, 260-262 labeling, 262, 263 tissue preparation, 262 water balance, 276-278 Atrophy neuropathy and, 399 tardive dyskinesia and, 330, 332 Atropine, parkinsonism and, 3,40,41, 49,59 Attention deficit disorder, parkinsonism and, 30, 31, 58 Autism, tardive dyskinesia and, 308, 31 1, 313,320 Autoinhibition, parkinsonism and, 24 Autonomic nervous system choline acetyltransferase and, 104 parkinsonism and, 3 , 4 , 4 1 Autoradiography angiotensin I1 and, see Angiotensin I1 atrial natriuretic peptide and, see Atrial natriuretic peptide neuropathy and edema, 414,430 ischemia, 414, 415 nerve blood flow, 382,383,386-391

B Basal forebrain, choline acetyltransferase and, 127, 128, 131, 132 Basal ganglia parkinsonism and, 2, 29, 34, 59 tardive dyskinesia and, 330, 342 Biochemistry choline acetyltransferase and expression, 85-92 neuron development, 124, 125 zinc and, 210,211

INDEX

Bipolar affective disorder, tardive dyskinesia and prevalence, 302, 307 vulnerability, 316, 320, 328, 329 Blood-nerve barrier, neuropathy and, 402,419,423,425-428 Blood pressure, angiotensin I1 and, 257-262,271-276,284-287,290 Brain proteins, zinc and, 164-174 Bromocriptine dopamine receptor subtypes and, 244, 245,249 parkinsonism and, 47 Bulk analysis, zinc and, 150-153 Butanol, neuropathy and, 383,384

C

Calcium neuropathy and, 381,421,422 tardive dyskinesia and, 314 zinc and, 22 1,223 brain proteins, 172, 174 CNS pathology, 210 histochemistry, 184 membranes, 175- 177 neurons, 197 vesicles, 209 Calibration, zinc and, 153, 194 Calmodulin neuropathy and, 38 1 , 4 21 zinc and, 172, 174, 209 Cannon’s Right-or-flight theory, parkinsonism and, 39,40 Capillaries, neuropathy and, 357-360 diabetes, 396, 397, 399, 400, 403, 404, 408 edema, 425-427,430-432 ischemia, 410,415-417,419-421,424 nerve blood Row, 391,392 oxygen delivery, 363-376 regulation of blood Row, 378, 379 Carbohydrate, choline acetyltransferase and, 82,85 Cardiovascular function angiotensin I1 and, 257-260,282-284, 286,290 tardive dyskinesia and, 334 Catalysis, zinc and, 164, 167, 194, 195

44 1

Catecholamines angiotensin I1 and, 282,287 dopamine receptor subtypes and, 239 neuropathy and, 399,422 parkinsonism and, 2,4, 5 , 5 8 age of injury, 32 future research, 50, 5 1 , 5 8 lesions, 28 neuropathology, 14 stress, 39, 40 zinc and, 219 cDNA, choline acetyltransferase and, 83, 89, 92, 96 Cell surface components, choline acetyltransferase and, 130 Central nervous system angiotensin I1 and, 259, 283 choline acetyltransferase and expression, 93,95, 97 function, 120-122 immunocytochemical location, 100- 103 invertebrate nervous system, 123 neuron development, 131 regional distribution, 104- 120 dopamine receptor subtypes and, 240, 244,246,250 parkinsonism and, 5, 14, 55,56,59 tardive dyskinesia and, 305 zinc and, 146, 147, 149,221-223 brain proteins, 164, 171, 172, 174 distribution in brain, 153, 156 histochemistry, 177, 178, 188, 190- 196 membranes, 176 neurons, 202 pathology, 214-220 uptake, 161, 163 vesicles, 209, 212 Centrifascicular infarction, 4 19-42 1 Cerebellum tardive dyskinesia and, 331 zinc and, 170 Cerebral cortex choline acetyltransferase and, 107-1 11, 118,120-122 zinc and, 186, 191, 204 Cerebrospinal fluid angiotensin I1 and, 283, 289

442

INDEX

tardive dyskinesia and, 34 1, 343 zinc and, 160-163,217 Choline choline acetyltransferase and, 82, 85, 86,94 parkinsonism and, 28, 29 Choline acetyltransferase (ChAT), 81, 82 expression, 84 biochemistry, 85-92 genetics, 92, 93 immunology, 96-100 pharmacology, 94-96 future directions, 132- 134 gene expression, 83, 84 history, 82, 83 imrnunocytochemical location, 100- 103 function, 120-122 invertebrate nervous systems, 123 regional distribution, 104-120 neuron development descriptive studies, 123- 129 experimental studies, 129- f 32 tardive dyskinesia and, 342 Cholinergic factor, choline acetyltransferase and, 129, 130 Cholinergic neurons, choline acetyltransferase and descriptive studies, 123- 129 experimental studies, 129-132 Choroid plexus, angiotensin I1 and, 275, 283,287-289 Chromosomes, choline acetyltransferase and, 92 Chronic obstructive pulmonary disease, 399 Chronic progressive diabetic neuropathy, 407,408 Chronic progressive experimental diabetic neuropathy, 397, 398,400, 401, 407, 408 Clones, choline acetyltransferase and, 83, 89,92,96 Coenzyme A, choline acetyltransferase and, 82, 85, 86 Cognition parkinsonism and, 13, 31, 57,59 tardive dyskinesia and, 345 pathophysiological mechanisms, 337 prevalence, 303

vulnerability, 322-325, 328, 331-333, 335,336 zinc and, 221 Compensation, parkinsonism and, 58 age of injury, 31 future research, 50 lesions, 20-29 stress, 38, 40 Contamination, zinc and, 150-152 Coronary artery, neuropathy and, 402, 403 Corticosteroids, choline acetyltransferase and, 95 Cranial nerve, choline acetyitransferase and, 119 Creatine phosphate, neuropathy and, 398 Cues, parkinsonism and, 13, 30 Cycloheximide, choline acetyltransferase and, 95 Cytoplasm, choline acetyltransferase and, 87, 100, 117

D D-1 receptors, 239, 240, 244, 246-252 parkinsonism and, 47, 48 D-2 receptors, 239, 240, 244-246, 249-253 parkinsonism and, 47,48 DA, see Dopamine Decarboxylation, parkinsonism and, 39, 41,42 DEDTC, see Diethyldithiocarbamate Dementia parkinsonism and, 3 tardive dyskinesia and, 305, 306, 324 Dendrites choline acetyltransferase and, 100, 103-109, 112, 114-1 17, 119,122 zinc and, 191 Dental status, tardive dyskinesia and, 333,334 Dentate gyrus, choline acetyltransferase and, 109, 110, 116 Deprenyl, parkinsonism and, 46,47 Depression dopamine receptor subtypes and, 248 parkinsonism and, 2 , 4 6 tardive dyskinesia and, 327,328

443

INDEX

Diagonal band, choline acetyltransferase and, 116,117 Diethyldithiocarbamate (DEDTC), zinc and, 208,216,218 Differentiation, choline acetyltransferase and, 95 Dih ydroxyphen ylacetic acid, tardive dyskinesia and, 340,341 3,4,-Dihydroxyphenylacetic acid (DOPAC), parkinsonism and, 37 2,3-Diphosphoglycerate, neuropathy and,

365,368,402,409 Dithiozone, zinc and CNS pathology, 218 histochemistry, 178,183-186,189,

Dopaminergic systems, parkinsonism and,

3,58,59 age of injury, 30-33 drugs, 41-43 future research, 44,46,47, 56 lesions, 16-20,26 neuropathology, 1 1, 13 stress, 36-38 DOPS, parkinsonism and, 39 Drosophila, choline acetyltransferase and expression, 87-90,92-94,96, 97 irnmunocytochemical location, 123 neuron development, 124 Dyskinesia, tardive, see Tardive dyskinesia

191-196 vesicles, 208 DNA choline acetyltransferase and, 133 neuropathy and, 403,404 zinc and, 167,168,174 DOCA, angiotensin I1 and, 260,261,271,

274,275,284-287, 289 L-DOPA dopamine receptor subtypes and,

241-243,248,249,252 parkinsonism and, 4 age of injury, 32 drugs, 41-43 future research, 44,46-48 lesions, 23 neuropathology, 10,15 stress, 36,39 tardive dyskinesia and, 299,339 Dopamine (DA) choline acetyltransferase and, 84,115 parkinsonism and, 3,4,7,8,57-59 age of injury, 31-33 future research, 44-51,53,54,56 lesions, 15-21,23-27,29 neuropathology, 9-12,15 stress, 35-39 receptor subtypes, 239,240,251-253 arousal, 240,241 central receptors, 241-244 D-lreceptors, 246-25 1 D-2receptors, 244-246,249-251 tardive dyskinesia and, 338-341,343 zinc and, 219

E Edematous neuropathy, 358,424,425 blood flow, 430,43I diabetes, 399 endoneurial pressure, 429,430 mathematical modeling, 431, 432 nerve blood flow, 382 oxygen delivery, 368 pathophysiology, 425-429 regulation of blood flow, 379,380 EEG dopamine receptor subtypes and,

240-252 tardive dyskinesia and, 328,336 Electroconvulsive therapy, tardive dyskinesia and, 321 Electron microscopy choline acetyltransferase and expression, 97-99 immunocytochemical location, 100,

108,119,120 neuropathy and, 418 zinc and, 184,206 Embolization, neuropathy and, 416 Encephalitis, parkinsonism and, 30 Endoneurial oxygen tensions, neuropathy and, 363-367 edema, 430,432 ischemia, 408,409,424 Endoneurial vessels, 358,359 diabetes, 396-400,405-407

444 edema, 425-431 ischemia, 414, 418 nerve blood flow,388 oxygen delivery, 365,368,376 regulation of blood flow,379-381 Endoplasmic reticulum, choline acetyltransferase and, 100 Endothelium, neuropathy and, 403,407, 42 1,423-428 Environment choline acetyltransferase and, 128 parkinsonism and, 43, 5 1-53 Epilepsy, zinc and, 174, 216-21 9 Epinephrine angiotensin I1 and, 287 parkinsonism and, 4, 28 Epineurial vessels ischemia, 4 14 nerve blood Row,388 nerve microvasculature, 356,359 regulation of blood flow,379-381 Epitopes, choline acetyltransferase and, 96,97, 100 Erythrocytes neuropdthy and diabetes, 401, 409 oxygen delivery, 363-365, 371 regulation of blood flow,379 zinc and, 175, 176, 188 Escherichia coli, choline acetyltransferase and, 89, 97 Euglycemia, neuropathy and, 407 Experimental diabetic neuropathy, 367, 382,398-400 Extraction ratio, neuropathy and, 389, 390 Extrapyramidal disorders, Cardive dyskinesia and, 299,332, 333 Extrinsic system, neuropathy and, 356, 357,379-382

F Familial factors, tardive dyskinesia and, 335,336 Fatty acid choline acetyltransferase and, 85 neuropathy and, 400

INDEX

Feedback angiotensin 11and, 290 choline acetyltransferase and, 94 parkinsonism and, 17, 37, 38 tardive dyskinesia and, 333 Fibroblast, choline acetyltransferase and, 132 Fibroblast growth factor, choline acetyltransferase and, 131 Fluorescence, zinc and, 152, 223 histochemistry, 178, 183, 184, 186, 191, 194, 195 neurons, 198 Fluorometry, zinc and, 153, 194 Free radicals neuropathy and, 382,402,407 parkinsonism and, 8 , 5 2 tardive dyskinesia and, 344

G

GABA, see 7-Aminobutyric acid GAD, see Glutamate decarboxylase Galactitol, neuropathy and, 428 Galactose, neuropathy and, 380,425,427, 428,430-432 Ganglion cells angiotensin I1 and, 282 choline acetyltransferase and, 115 Gangliosides, parkinsonism and, 56,57 Gangrene, neuropathy and, 409,410 GDH, see Glutamate dehydrogenase Gene expression, choline acetyltransferase and, 82-84 Genetics choline acetyltransferase and, 92, 93 tardive dyskinesia and, 335, 336 Glial cells parkinsonism and, 8 zinc and, 195, 196 Globus pallidus, tardive dyskinesia and, 34 1 Glutamate, zinc and, 146, 147 neurons, 203,222 vesicles, 206, 212, 214 Glutamate decarboxylase (GAD), zinc and, 209,212 Glutamate dehydrogenase (GDH), zinc and, 212

INDEX

Glutathione, zinc and, 168, 170 Glycogen, neuropathy and, 403 Glycosylation, neuropathy and, 402, 405-407 Golgi apparatus choline acetyltransferase and, 100 zinc and, 221 Growth factors, parkinsonism and, 54-56

H Haloperidol, tardive dyskinesia and, 343, 344 Heat clearance, neuropathy and, 390 Hematocrit, neuropathy and, 379,402 Hemoglobin, neuropathy and diabetes, 402 oxygen delivery, 363, 365, 368, 371 regulation of blood flow, 378, 379 Hemorheologic mechanisms, neuropathy and, 397,398,400,401 Hexachlorophene, neuropathy and, 426-428,43 1 Hippocampus choline acetyltransferase and, 103, 108-111,116, 118, 122 zinc and, 148, 149 brain proteins, 170 CNS pathology, 216, 218 distribution in brain, 155, 159 histochemistry, 193, 195 neurons, 197-200,203,204 uptake, 161, 162 vesicles, 207, 208, 212 Histochemistry, zinc and, 147, 149, 177, 178 CNS distribution, 190-196 methods, 178-186 neurons, 198-200,203 reactive pool, 186-190 uptake, 162, 163 vesicles, 204 Homeostasis angiotensin I1 and, 288, 290 parkinsonism and, 58 lesions, 16-19, 29 stress, 37-40 zinc and, 169, 170

445

Homology, choline acetyltransferase and, 90 Homovanillic acid (HVA) parkinsonism and, 37 tardive dyskinesia and, 340, 341 Hormones angiotensin I1 and, 289, 290 choline acetyltransferase and, 95 Human diabetic neuropathy, 396-398, 400,408 HVA, see Homovanillic acid Hybrids, choline acetyltransferase and, 92, 125 Hydrogen peroxide choline acetyltransferase and, 99 parkinsonism and, 5, 8, 53 Hydrogen polarography, neuropathy and, 36 1 diabetes, 398 edema, 43 1 ischemia, 414 nerve blood flow, 382-386,391 regulation of blood flow, 380, 381 Hydrogen washout, neuropathy and, 391-396

6-H ydroxydopamine dopamine receptor subtypes and, 241 parkinsonism and, 4-7, 57-59 age of injury, 31-34 drugs, 4 1-43 future research, 46,47, 54-56 lesions, 15, 16, 21 neuropathology, 11, 12, 15 stress, 34-36, 38, 39 Hyperactivity dopamine receptor subtypes and, 244 parkinsonism and, 30,58 Hyperbaric oxygenation, neuropathy and, 400 Hyperglycemia, neuropathy and, 404, 405,407 Hyperinsulinemia, neuropathy and, 402 Hypertension, angiotensin 11, 257-260, 271-276,284-287,290 Hypotension, neuropathy and, 357,415 Hypothalamus angiotensin I1 and, 265, 267,268, 288 choline acetyltransferase and, 115 parkinsonism and, 10, 11, 24,30 zinc and, 219

446 Hypovalemia, neuropathy and, 362,379 Hypoxemia, neuropathy and, 364, 368 H ypoxia neuropathy and, 362 diabetes, 397-40 1, 405-408 edema, 425,427,428 ischemia, 416, 418 oxygen delivery, 363-368 regulation of blood flow, 380 tardive dyskineska and, 335,343

I Immunochemistry, choline acetyltransferase and, 96,97 Immunocytochemistry, choline acetyltransferase and, 82 expression, 84,92,96-100 function, 120-122 location in CNS, 100-103, 123 neuron development, 125-128 regioiial distribution, 104- 120 Irnmunofluorescence, choline acetyltransferase and, 98 Immunohistochemistry, zinc and, 149 Immunology, choline acetyltransferase and, 96-100 Immunoperoxidase, choline acetyltransferase and, 100, 101 Indicator dilution, neuropathy and, 389, 390 Inhibition angiotensin I1 and, 258, 285, 286, 290 choline acetyltransferase and expression, 85, 88, 92-94 immunocytochemical location, 1 12, 119, 122 dopamine receptor subtypes and, 242, 248 neuropathy and diabetes, 401, 402,404, 407 edema, 428 ischemia, 42 1-423 regulation of blood flow, 381 parkinsonism and, 5 age of injury, 32, 33 drugs, 41 future research, 44,46,47, 51, 53

INDEX

lesions, 19, 23, 25 stress, 38 zinc and brain proteins, 167, 172, 174 CNS pathology, 2 17 membranes, 176 vesicles, 209, 2 12 Inositol, neuropathy and, 404 Insulin neuropathy and, 397,40'2-404 zinc and, 206, 221 Insulin-dependent diabetes niellitus, neuropathy and, 402 Insulin-like growth factors, neuropathy and, 403,404 Intercapillary distance, neuropathy and, 358 diabetes, 399,400,409 edema, 430-432 oxygen delivery, 368 Interpeduncular nucleus, choline acetyltransferase and, 119 Intrinsic system, neuropathy and, 356-358,378-380 Iodoantipyrine autoradiography, neuropathy arid edema, 430 ischemia, 414 nerve blood flow, 382,383,386-390 Ischemic neuropathy, 409,410 centrifascicular infarction, 4 19-42 1 diabetes, 396,397, 399-404,406-408 early observation, 41 1, 4 12 edema, 430,43 1 experimental models, 4 12-4 17 mathematical modeling, 424 molecular mechanisms, 42 1-424 nerve blood flow, 382 nerve conduction, 418, 419 nerve microvasculature, 356-358,360 oxygen delivery, 367, 368 pathology, 417,418 regulation of blood flow, 380

K Kinetics, neuropathy and, 366, 367, 373, 375

INDEX

L Lamina choline acetyltransferase and, 105- 107, I23 neuropathy and, 359 Laser Doppler velocimetry, neuropathy and, 381,388 Lead, neuropathy and, 427-429 Lesions angiotensin I1 and, 286 choline acetyltransferase and, 107, 110, 116 dopamine receptor subtypes and, 241 parkinsonism and, 15-20, 57, 58 age of injury, 30-33 drugs, 40-42 future research, 43, 46, 50-52, 54, 56 neuropathology, 10- 15 recovery of function, 24-29 stress, 33-36, 38-40 subtotal injury, 20-24 zinc and, 188, 196, 203 Leukotrienes, neuropathy and, 421, 422 Ligands angiotensin I1 and, 259, 260, 279 choline acetyltransferase and, 94, 96 zinc and, 147 distribution in brain, 159 membranes, 176 uptake, 160, 162 vesicles, 207, 212 Light microscopy choline acetyltransferase and, 97-99, 109, 117 zincand, 184, 191, 194 Lipid choline acetyltransferase and, 82 neuropathy and, 383, 398,401,407, 423 tardive dyskinesia and, 343, 344 zinc and, 175 Lipid hydroperoxides, neuropathy and, 407,423 Lithium, tardive dyskinesia and, 327 Locus coeruleus, parkinsonism and, 14, 21,27 Low-density lipoproteins, neuropathy and, 405,406 Lysosornes, zinc and, 175,204

447

M Macromolecules, zinc and, 204-207 Macrophages, neuropathy and, 405,406 Medial habenula, choline acetyltransferase and, 118, 120 Medial septum, choline acetyltransferase and, 116 Membranes, zinc and, 175-177 Memory choline acetyltransferase and, 132 zinc and, 216 Metalloenzymes, zinc and, 148, 220 brain proteins, 164-167, 170 CNS pathology, 216 distribution in brain, 159 histochemistry, 188 membranes, 175 vesicles, 208 Metalloproteins, zinc and, 161, 170 Metallothionein, zinc and, 163, 168-170 Microembolization, neuropathy and, 415-417 Microligands, zinc and, 159, 160, 163 Microsomes, zinc and, 176 Microspheres, neuropathy and, 391, 415 Microtubules, zinc and, 171, 172 Microvascular disease, neuropathy and, 400-404,410,415,416 Mimicry, zinc and, 209-213 Mitochondria choline acetyltransferase and, 86 neuropathy and, 399,418 zinc and, 159, 164, 188 Mitosis, choline acetyltransferase and, 128 Monoamine oxidase dopamine receptor subtypes and, 242 parkinsonism and, 8,46,47,53 Monoaminergic projections, parkinsonism and, 11-14 Monoaminergic systems, parkinsonism and, 18, 19, 31, 39 Monoclonal antibodies, choline acetyltransferase and, 83 expression, 96, 99, 100 immunocytochemical location, 108, 123 Morbidity, tardive dyskinesia and, 3 14, 315 Morphology, choline acetyltransferase and expression, 97, 98

448

INDEX

immunocytochemical location, 104, 105, 107, 112, 113, 120, 122 Mortality, tardive dyskinesia and, 314,315 MPP', parkinsonism and, 8, 53 MPTP, parkinsonism and, 4, 7, 8, 57-59 drugs, 41 future research, 52, 56 lesions, 15, 23, 24, 29 neuropathology, 11, 12, 15 mRNA choline acetyltransferase and, 123, 125, 130 expression, 89, 90, 92, 95, 96 immunocytochemistry. 123 neuron development, 125, 130, 131 parkinsonism and, 32 Multiplication-stimulating activity, neuropathy and, 403,404 Muscarinic receptors, parkinsonism and, 40,4l Mutation, choline acetyltransferase and, 92,93, 123 Myelin, neuropathy and, 422,425-427 Myoinositol, neuropathy and, 397,404, 405,407-409

N Necrosis, neuropathy and, 413,416,418 Neocortex, choline acetyltransferase and, 107, 108, 110, 111, 117, 121 Neostriatum, choline acetyltransferase and, 111-113, 122 Nephropathy, 396 Nerve blood flow diabetes, 397, 399, 407,408 edema, 430,43 1 ischemia, 412, 414-417, 419, 423 measurements hydrogen washout, 391-396 methods, 382-391 nerve microvasculature, 356-362 regulation, 378-382 Nerve crush, neuropathy and, 428,429 Nerve growth factor choline acetyltransferase and, 95, 129, 130 parkinsonism and, 55 Nerve microvasculature, 356-362

Neural cell adhesion molecule, choline acetyltransferase and, 130 Neuroleptics dopamine receptor subtypes and, 243-245,250,251 parkinsonism and, 34, 36, 38,46 tardive dyskinesia and, see Tardive dyskinesia Neuromodulators, zinc and, 203, 206, 209 Neuropathology choline acetyltransferase and, 84 parkinsonism and, 3, 8, 57 future research, 43,48 lesions, 29 symptoms, 9-15 Neuropathy diabetes endoneurial ischemia, 396, 397 hypoxia, 397-400 mathematical modeling, 408, 409 microvascular ischemia, 400-404 pathogenesis, 404-408 edema, 424,425 blood flow, 430,431 endoneurial pressure, 429,430 mathematical modeling, 431,432 pathophysiology, 425-429 ischemia, 409, 410 centrifascicular infarction, 419-42 1 early observation, 4 1 1, 4 12 experimental models, 412-417 mathematical modeling, 424 molecular mechanisms, 42 1-424 nerve conduction, 418,419 pathology, 417,418 nerve blood flow hydrogen washout, 391-396 methods, 382-391 nerve microvasculature capillary density, 359, 360 double blood supply, 356-358 nerve vascular anatomy, 358,359 physiology, 360-362 oxygen delivery, 362-367 mathematical models, 368-378 reduction, 367, 368 regulation of blood flow extrinsic mechanisms, 379-382 intrinsic mechanisms, 378, 379

INDEX

Neuropil, zinc and histochemistry, 191-195 neurons, 198-200 vesicles, 208 Neuropsychiatric disorders, tardive dyskinesia and natural history, 313 prevalence, 305-310 vulnerability, 317, 318, 322, 332 Neurosecretory zinc, 146, 147 Neurotoxins choline acetyltransferase and, 94 dopamine receptor subtypes and, 241 neuropathy and, 425,426,430 parkinsonism and, 4,5, 8,57-59 future research, 53 lesions, 28, 29 neuropathology, 10- 12, 15 Neurotransmitters choline acetyltransferase and, 8 1, 82,84 expression, 85, 86, 88, 89, 93-95, 97 future directions, 132, 133 immunocytochemical location, 123 neuron development, 125, 127, 129, 130, 132 dopamine receptor subtypes and, 239, 241,253 neuropathy and, 381 parkinsonism and, 39 tardive dyskinesia and, 338 zinc and, 146 brain proteins, 171, 174 CNS pathology, 217, 218 histochemistry, 178 neurons, 203 vesicles, 206, 209, 212 Neutron activation, zinc and, 151-154 Nigrostriatal bundle (NSB), parkinsonism and, 3, 8, 9, 57-59 age of injury, 30-33 drugs, 40-43 future research, 43,46-53, 55, 56 lesions, 15, 16, 18-29 neuropathology, 10- 15 stress, 33-40 NOR-insulin-dependentdiabetes mellitus, 402-404 Noradrenergic nerve, neuropathy and, 359, 381, 382

449

Noradrenergic projections, parkinsonism and, 14,39 Noradrenergic systems, parkinsonism and, 26,28 Norepinephrine angiotensin I1 and, 282 dopamine receptor subtypes and, 239, 24 1 neuropathy and, 380-382,401 parkinsonism and, 3-5,59 drugs, 42 future research, 55, 56 lesions, 27, 28 neuropathology, 13, 14 stress, 39 zinc and, 219 NSB, see Nigrostriatal bundle Nucleic acid choline acetyltransferase and, 90, 96, 130, 132, 133 zinc and, 164, 166, 167, 215 Nucleotides, choline acetyltransferase and, 94,95 Nucleus accumbens, tardive dyskinesia and, 341 Nucleus basalis, choline acetyltransferase and, 117 Nucleus of the solitary tract, 265

0

Olfactory bulb angio:ensin I1 and, 268, 27 1, 274 choline acetyltransferase and, 1 14, 117, 118, 121 Organic brain disease, tardive dyskinesia and morbidity, 315 pathophysiological mechanisms, 344 prevalence, 309 vulnerability, 320, 322, 326, 328 Organon vasculosum laminae terminalis, 265,267,276,281,288 Osmotic edema, neuropathy and, 428 Oxygen neuropathy and delivery, 362-368 diabetes, 397-400, 402, 407-409 edema, 430-432

450

lNDEX

ischemia, 41 1-413, 416, 417, 419, 420,424 mathematical models, 368-378 nerve blood flow, 383,391,392 nerve microvasculature, 360, 362 parkinsonism and, 52 Oxygen free radical, neuropathy and, 382 diabetes, 402, 407 ischemia, 421-424 Oxyhemoglobin, neuropathy and, 365, 368

P PAP, see Peroxidas-antiperoxidase Parabigeminal nucleus, choline acetyltransferase and, 118, 119 Paradoxical kinesia, parkinsonism and, 34, 35, 39 Parkinsonism choline acetyltransferase and, 84 dopamine receptor subtypes and, 24 1 tardive dyskinesia and, 299, 332, 333, 339,340 Parkinsonism, animal models of, 2, 57-60 age of injury attention deficit disorder, 30 dopaminergic neurons, 30-33 atropine, 40.4 1 L-DOPA, 41-43 future research cure, 52-57 diagnosis, 43, 44 treatment, 44-52 6-hydroxydopamine, 4-7 lesions dopaminergic systems, 16-20 recovery of function, 24-29 residual DA neusons, 15, 16 subtotal injury, 20-24 MPTP, 7, 8 neuropathology, 3, 9, 10 monoaminergic projections, 12- 14 nigrostriatal bundle, 10-12, 14, 15 pharmacotherapy, 3, 4 stress, 33, 34 fight-or-flight theory, 39,40 impairments, 35-39

paradoxical kinesia, 34, 35 symptoms, 2, 3 unanswered questions, 8 , 9 Partition neurons, choline acetyltransferase and, 105 Pathophysiology, tardive dyskinesia and, 336,341-344 dopamine receptor, 338-340 topography, 337,338 Peptidergic nerves, neuropathy and, 359, 381,382 Peptides, see also Atrial natriuretic peptide choline acetyltransferase and, 88 zinc and, 147, 221 Pergolide, doparnine receptor subtypes and, 245 Perikarya, zinc and, 194, 195 Perineurial vessels, neuropathy and, 356, 359 edema, 428-431 regulation of blood flow, 379, 380 Peripheral bridges, parkinsonism and, 54,55 Peripheral nerve, neuropathy and, 356-362 diabetes, 396-399,407 edema, 425-428 ischemia, 409,410,415,417-419, 422, 423 nerve blood flow, 382, 383,385, 390, 39 1 oxygen delivery, 368, 37 1 regulation of blood flow, 379-381 Peripheral nervous system, parkinsonism and, 26,39, 56 Permeability, neuropathy and, 423,427 Peroxidase-antiperoxidase(PAP), choline acetyltransferase and, 98.99 Pharmacotherapy, parkinsonism and, 3, 4,42,43, 58 future research, 47, 50 Phenothiazines, tardive dyskinesia and, 343,344 Phenotypes, choline acetyltransferase and, 83,84 expression, 88,92, 93 future directions, 132, 133 neuron development, 123, 125, 128- 132

INDEX

Phosphates, neuropathy and, 359,381, 382 Phospholipases, neuropathy and, 381, 407,421,422 Phospholipids choline acetyltransferase and, 85 neuropathy and, 382,399,407,421, 422 Phosphorylation, choline acetyltransferase and, 95 PIXE, see Proton-induced X-ray emissions Plasma membrane choline acetyltransferase and, 86, 130 zinc and, 171 Polarography, neuropathy and, see Hydrogen polarography Polypeptides, choline acetyltransferase and, 88,96 Pontomesencephalic reticular formation, choline acetyltransferase and, 117, 118 Prostacyclin, neuropathy and, 381, 401, 402,407,421-423 Prostaglandin, neuropathy and, 381, 382, 401,421,422 Protein angiotensin I1 and, 263, 264, 279 choline acetyltransferase and, 83 expression, 86-90, 95, 96 neuron development, 131 neuropathy and, 378,383,403,405, 425 parkinsonism and, 27 zinc and, 146-148, 222 brain proteins, 164- 174 CNS pathology, 2 15 distribution in brain, 149, 157, 159 neurons, 22 1 uptake, 159 vesicles, 206 Proteolysis, choline acetyltransferase and, 87,811 Proton-induced X-ray emissions (PIXE), zinc and, 151-154 Psychiatric disorders, tardive dyskinesia and, 345 morbidity, 3 15 natural history, 3 13 prevalence, 302 vulnerability, 332, 334

45 1

Psychopathology, tardive dyskinesia and, 325-328 Psychosis, tardive dyskinesia and, 344 prevalence, 306, 307, 309 vulnerability, 331, 332 Pyridoxal phosphate, zinc and, 209

Q Quinoline fluorescence, zinc and, 178, 183, 184, 223 Quinpirole, dopamine receptor subtypes and, 245,249

R Receptors angiotensin I1 and, 283, 284, 286, 287, 290 distribution, 265-269 atrial natriuretic peptide and, 283, 284, 286,287,290 distribution, 266-268, 270, 271 Regeneration, parkinsonism and, 53-57 Renin, angiotensin I1 and, 257, 259, 285, 289 Reperfusion, neuropathy and, 406,421, 423,424 Reserpine, parkinsonism and, 4 Resistance to ischemic conduction failure, 399,400 Retina, choline acetyltransferase and, 1 15 Retinoic acid, choline acetyltransferase and, 95 Retinopathy and, 396, 404 RNA choline acetyltransferase and, 89, 95, 96 zinc and, 167, 174

S Salt, angiotensin I1 and, 260, 26 I , 274, 284-287 Schizophrenia tardive dyskinesia and, 345 incidence, 31 1 morbidity, 315

452

INDEX

natural history, 312, 313 pathophysiological mechanisms, 337, 340-342 prevalence, 302,306, 307, 309 vulnerability, 3 16,320-336 zinc and, 215 Sciatic nerve, neuropathy and, 360, 361, 363 diabetes, 398, 40 1, 408 edema, 43 1 ischemia, 4 1 1-4 16, 4 19 nerve blood flow, 392 oxygen delivery, 374 regulation of blood flow, 380 Second messengers angiotensin I1 and, 283, 285 choline acetyltransferase and, 94 parkinsonism and, 19 Senile dementia, tardive dyskinesia and, 305,306 Serotonergic nerves, neuropathy and, 359 Serotonin dopamine receptor subtypes and, 246 neuropathy and, 428 parkinsonism and, 3, 5, 7, 59 age of injury, 32, 33 drugs, 41,42 future research, 49 lesions, 28 neuropathology, 13-15 zinc and, 197 Sex, tardive dyskinesia and, 3 18,321, 322 SIDMS, see Stable-isotope dilution mass spectrometry Silver amplification, zinc and histochemistry, 178, 184-186, 195 neurons, 198, 199 Simpson-Rockland Scale, tardive dyskinesia and, 300 Sleep, dopamine receptor subtypes and, 239,244,246-248,252,253 Smoking, tardive dyskinesia and, 334, 335 Sodium, angiotensin I1 and, 257,258 Somatostatin choline acetyltransferase and, 109 parkinsonism and, 15 tardive dyskinesia and, 342 Sorbitol, neuropathy and, 428

Spinal cord choline acetyltransferase and immunocytochemical location, 104-107, 115 neuron development, 125-128, 130 zinc and, 155, 192, 193, 202 Stable-isotope dilution mass spectrometry (SIDMS), zinc and, 158 Steroid hormones angiotensin I1 and, 289 choline acetyltransferase and, 95 Streptozotocin, neuropathy and, 397, 398 Stress angiotensin I1 and, 259,262,279,280, 289,290 parkinsonism and, 2, 9, 58, 59 future research, 44 neuropathology, 13, 14 symptoms, 33-40 Striatum parkinsonism and, 3, 7, 8, 59 age of injury, 32, 33 drugs, 41,42 future research, 47-52,54, 56 lesions, 16, 18, 19, 21, 23, 25, 26, 29 neuropathology, 9-13, 15 stress, 35-38 tardive dyskinesia and, 338 zinc and, 191 Structural brain pathology, tardive dyskinesia and, 329-331,333 Subcellular compartmentalization, zinc and, 157-159, 163 Substantia nigra parkinsonism and, 3, 7 age of injury, 30, 32 future research, 49, 53, 54, 56 lesions, 19, 21 neuropathology, 9, 12 stress, 34, 35 tardive dyskinesia and, 341 Subtotal injury, parkinsonism and, 20-24 Sulfide, zinc and, 185, 194, 198, 208, 216 Superior colliculus, choline acetyltransferase and, 120 Sympathetic innervation, neuropathy and, 381 Sympathetic nervous system, parkinsonism and, 39-41

INDEX

Synapses choline acetyltransferase and expression, 85-87, 97, 98 immunocytochemical location, 100, 101, 103, 121, 122 regional distribution, 107-1 10, 112, 114-117, 119 dopamine receptor subtypes and, 246 parkinsonism and, 16-19, 21, 27 tardive dyskinesia and, 343 zinc and, 224 brain proteins, 171 CNS pathology, 218 histochemistry, 188, 193 neurons, 199,221,222 vesicles, 204, 207-214 Synaptosomes, zinc and distribution in brain, 159 membranes, 176 neurons, 197 uptake, 161, 162 vesicles, 209, 212

T Tardive dyskinesia history, 298 incidence, 310, 3 1 medicolegal issue , 299 methodology, 29 -301 morbidity, 314, 315 natural history, 31 1-314 paradigms, 344, 345 pathophysiological mechanisms, 34 1-344 dopamine receptor, 338-340 topography, 337,338 prevalence, 301-310 vulnerability, 315, 336 age, 316-318 cognitive dysfunction, 322-325 dental status, 333, 334 exposure to neuroleptics, 3 18-322 extrapyramidal side effects, 332, 333 familial aspects, 335, 336 neurological features, 328, 329 onset age, 33 1, 332

453

psychopathology, 325-328 sex, 3 18 smoking, 334, 335 structural brain pathology, 329-33 1 Tetrahydrobiopterin, parkinsonism and, 44,46 Thalamus, choline acetyltransferase and, 119 Thromboxane, neuropathy and, 407,422 Thymidine choline acetyltransferase and, 125, 128 neuropathy and, 404 Thyroid hormone, choline acetyltransferase and, 95 Torpedo, choline acetyltransferase and, 87 Transcription choline acetyltransferase and expression, 84, 89, 90,95 future directions, 133 neuron development, 124, 130 zinc and, 166, 174 Translation choline acetyltransferase and, 84,95, 130, 133 zinc and, 166 Translocation, choline acetyltransferase and, 88, 89 Transmitters choline acetyltransferase and, 98 dopamine receptor subtypes and, 243 parkinsonism and, 58 future research, 46-49,51 lesions, 16-19,23, 25, 26, 28 stress, 40 zinc and, 178,209,222 Transplantation, parkinsonism and, 59 future research, 47,49-54,56 lesions, 29 neuropathology, 12 Triethyltin, neuropathy and, 426, 427 Tubulin, zinc and, 171, 172, 174 Tumor, zinc and, 175 Turnover, zinc and, 161, 166, 197, 199 Twitcher mouse, neuropathy and, 429 Tyrosine hydroxylase, parkinsonism and, 4 future research, 44.46, 56 lesions, 18, 23,25-28

454

INDEX

V Vasoactive intestinal polypeptide (VIP) choline acetyltransferase and, 12 1 tardive dyskinesia and, 342 Vasopressin, angiotensin I1 and, 257, 258, 261,282,286-289 Vasoregulatory abnormalities, 40 1 Ventral striaturn, choline acetyltransferase and, 113, 114 Vesicles choline acetyltransferase and, 87, 100, 101, 114 neuropathy and, 418,426 zinc and, 147 brain proteins, 174 CNS pathology, 216 histochemistry, 178, 188, 191-193, 195 neurons, 197-199,204,221,222 storage of macromelecules, 204-207 synaptic receptors, 207-2 14 Viscosity, neuropathy and, 378, 379, 401 Vitamin E parkinsonism and, 53 tardive dyskinesia and, 344

W

Wallerian degeneration, neuropathy and. 413,417 Water balance, angiotensin I1 and, 276-278,287,288 Withdrawal, tardive dyskinesia and, 3 12, 338, 340, 342

X X-ray angiotensin I1 and atrial natriuretic peptide and, 264 neuropathy and, 387 zinc and, 152

Xenapw, choline acetyltransferase and, 89

2

Zinc brain proteins, 164 metalloenzymes, 164- 167 nonenzymatic zinc-binding proteins, 167-170 zinc-protein complexes, 174 zinc-sensitive proteins, 170- 174 CNS pathology adult brain function, 215-220 brain development, 2 14, 2 15 distribution in brain analysis, 149 instrumental assays, 150- 153 regional, 153-357 subcellular compartmentalization, 157- 159 histochemistry, 177, 178 CNS distribution, 190-196 methods, 178- 186 reactive pool, 186-190 history, 148, 149 membranes ion channels, 176, 177 stabilization, 175, 176 metalloenzymes, 220 in nervous system, 146, 147 neurons, 22 1,222 anatomy of pathways, 200-203 colocalization, 203, 204 definition, 196, 197 turnover, 197-200 pools, 147, 148 signal, 222-224 turnover, 159- 163 uptake, 159-163 vesicular zinc storage of macromolecules, 204-207 synaptic receptors, 207-214

CONTENTS OF RECENT VOLUMES

Volume 21

Relationship of the Actions of Neuroleptic Drugs to the Pathophysiolugy of Tardive Dyskinesia Ross J . Baldessarini and Daniel Tarsy Soviet Literature on the Nervous System and Pychobiology of Cetacea Theodore H . Bullock and Vladimir S. Gurevich Binding and Iontopheretic Studies on Centrally Active Amino Acids-A Search for Physiological Receptors F . V. DeFeudis Presynaptic Inhibition: Transmitter and Ionic Mechanisms R. A . Nicoll and B . E . Alger Microquantitation of Neurotransmitters in Specific Areas of the Central Nervous System Juan, M . Saavedra Physiology and Glia: Glial-Neuronal Interactions R. Malcolm Stewart and Roger N . Rosenberg Molecular Perspectives of Monovalent Cation Selective Transmembrane Channels Dan W . Urry Neuroleptics and Brain Self-Stimulation Behavior Albert Wauquier

Brain Intermediary Metabolism in Vzvo: Changes with Carbon Dioxide, Development, and Seizures Alexander L. Miller N,N-Dimethyltryptamine: An Endogenous Hallucinogen Steven A. Barker, John A. Nonti, and Samuel T. Christian Neurotransmitter Receptors: Neuroanatomical Localization through Autoradiogaphy L. Charles M u m ' n Neurotoxins as Tools in Neurobiology E . G. McCeer and P . L. McGeer Mechanisms of Synaptic Modulation William Shain and David 0. Carpenter Anatomical, Physiological, and Behavioral Aspects of Olfactory Bulbectomy in the Rat B . E . Leonard and M . Tuite T h e Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Mapping of Local Functional Activity in the Central Nervous System Louis Sokloloff INDEX

Volume 23

Volume 22

Chemically Induced Ion Channels in Nerve Cell Membranes David A. Mathers and Jeffe? L. Barker

Transport and Metabolism of Glutamate and GABA in Neurons and Glial Cells A r n ~Schoucboe

Fluctuation of Na and K Currents in Excitable Membranes Berthold Neumcke

455

456

CONTENTS OF RECENT VOLUMES

Biochemical Studies of‘ the Excitable Membrane Sodium Channel Robert L. Barchi Benzodiazepine Keceptors in the Central Nervous System Phil Skolnack and Steven M. Paul Kapid Changes in Phospholipid Metabolism during Secretion and Receptor Activation F. T. Crews Glucocorticoid Effects on Central Nervous Excitahility and Synaptic Transmission Eduard D. Hall Assessing the Functional Significance of Lesion Induced Neuronal Plasticity Oswald Steward Dopamine Receptors in the Central Nervous System Ian Cresse, A. Leslie Morrow, Stuart E. LtfJ David R. Sibley, and Mark W . Hamblin Functional Studies of the Central Catecholamines T. W . Kobbins and B . J . Eueritt

Studies of Human Growth Hormone Secretion in Sleep and Waking Wallace B. Mendebon Sleep Mechanisms: Biology and Control of REM Sleep Dennis J . McGinty and Rent R. Drucker-Colin INDEX

Volume 24

Antiacetylcholine Receptor Antibodies and Myasthenia Gravis Bernard W . Fulpiur Pharmacology of Barbiturates: Electrophysiological and Neurochemical Studies Max Willow and Graham A. R. Johnston

Irnmunodetection of Endorphins and Enkephalins: A Search for Reliability Alejandro Bayon, William J . Shoemaker, Jacqueline F . McCznty, and Floyd Bloom On the Sacred Disease: The Neurochemistry of Epilepsy 0. Carter Snend I l l Biochemical and Electrophysiological Characteristics of Mammalian GABA Receptors Salvatore ,J.Enna and Joel P. Gallagher Synaptic Mechanisms and Circuitry Involved in Motorneuron Control during Sleep Michael H . Chase Recent Developments in the Structure and Function of the Acetylcholine Receptor F. J . Barrantes Characterization of a,-and cY2-Adrenergic Receptors David 5.Bylwnd and David C . U’Prichard Ontogenesis of the Axolemma and Axoglial Relationships in Myelinated Fibers: Electrophysiological and Freeze-Fracture Correlates of Membrane Plasticity Stephen G.Wnxman, Joel A . Black, and Robert E . FoAter INDEX

Volume

25

Guanethidine-Induced Destruction Sympathetic Neurons Eugene M . Johnston, Jr. and Pamela Toy Manning

of

Dental Sensory Receptors Margaret R. Byers Cerebrospinal Fluid Proteins in Neurology A . Lawmthal, R. Crols, E . De Schutter, J Gheuens, D. Karcher, M . Noppe, and A. Tasnaer

457

CONTENTS OF RECENT VOLUMES

Muscarinic Receptors in the Central Nervous System Mordechai Sokolovsky Peptides and Nociception Daniel Luttinger, Daniel E. Hernandez, Charles B . Nemeroff, and Arthur J . Prange, Jr.

Effect of Tremorigenic Agents on the Cerebellum: A Review of Biochemical and Electrophysiological Data V . G . Long0 and M . Massotti INDEX

Opioid Actions on Mammalian Spinal Neurons W . Zieglgansberger

Volume 27

Psychobiology of Opioids Alberta Oliverio, Claudio Castellano, and Stefan0 Publisi-Allegra

The Nature of the Site of General Anesthesia Keith W . Miller

Hippocampal Damage: Effects on DO- The Physiological Role of Adenosine in the Central Nervous System paminergic Systems of the Basal Ganglia Robert L. Isaacson Thomas V . Dunwiddie Neurochemical Genetics V . Csdnyi The Neurobiology of Some Dimensions of Personality Marvin Zuckerman,James C. Ballenger, and Robert M . Post

Somatostatin, Substance P, Vasoactive Intestinal Polypeptide, and Neuropeptide Y Receptors: Critical Assessment of Biochemical Methodology and Results Anders Undkn, Lou-Lou Peterson, and Tamas Bartfai

INDEX

Eye Movement Dysfunctions and Psychosis Philip S. Halzman

Volume 26

Peptidergic Regulation of Feeding J . E. Morley, T. J . Bartness, B . A . Gosnell, and A . S . Levine

The Endocrinology of the Opioids Mark J . Millan and AlbeTt Herz Multiple Synaptic Receptors for Neuroactive Amino Acid Transmitters-New Vistas Najam A . Sharif Muscarinic Receptor Subtypes in the Central Nervous System Wayne Hoss and John Ellis

Calcium and Transmitter Release Ira Cohen and William V a n der Kloot Excitatory Transmitters and Related Brain Damage John W . Olney

Epilepsy-

Potassium Current in the Squid Giant Axon John R. Clay INDEX

Neural Plasticity and Recovery of Function after Brain Injury John F. Marshall From Immunoneurology to Immunopsychiatry: Neuromodulating Activity of AntiBrain Antibodies Branklav D. JankloviC

Volume 28

Biology and Structure of Scrapie Prions Michael P. McKinley and Stanley B . Prusiner

458

CONTENTS OF RECENT VOLUMES

Different Kinds of Acetylcholine Release from the Motor Nerve S . 7'he.tleff Neuroendocrine-Ontogenetic Mechanism of Aging: Toward an Integrated Theory of Aging V . M . Dilrnan, S. Y. Revskloy, nnd A . G. Golubev

The Interpeduricular Nucleus Rarbara ,J. Morley Biological Aspects of Depression: A Review of the Etiology and Mechanisms of Action and Clinical Assessment of Antidepressants S . I. Ankier and R . E . Leonard Does Receptor-Linked Phosphoinositide Metabollism Provide Messengers Mobilizing Calcium in Nervous Tissue? John N . Hawthorne Short-Term and Long-Term Plasticity and Physiological Differentiation of Crustacean Motor Synapses H . L. Atwood and J . M . Wojtowicz Immunology and Molecular Biology of the Cholinesterases: Current Kesults and Prospects Stephen Brimzj'oin and Zoltan Rakonctay INDEX

Pathobiology of Neuronal Storage Disease Steven U . Walkley Thalamic Amnesia: Clinical and Experimental Aspects Stephen G . Waxman Critical Notes on the Specificity of Drugs in the Study of Metabolism and Functions of Brain Monoamines S. Garattini and Z. Mennini Retinal Transplants and Optic Nerve Bridges: Possible Strategies for Visual Recovery as a Result of Trauma or Disease Jarneb E . Turner, Jerry R. Blair, Magdalene Seiler, Robert Aramant, Thomas W . Laedtke, E . ?'hornas Chappell, and Lauren Clarkson Schizophrenia: Instability in Norepinephrine, Serotonin, and y-Aminobutyric Acid Systems Joel Gelernter and Daniel P. van Kammen INDEX

Volume 30

Biochemistry of Nicotinic Acetylcholine Receptors in the Vertebrate Brain Jakob Schmidt The Neurobiology of N-Acetybaspartylglutamate Randy D. Blakely and Joseph T. Coyle

Volume 29

Molecular Genetics of Duchenne anti Becker Muscular Dystrophy Ronald G. Worton und Arthur H . M . Burghes Ratrachotoxin: A Window on the Allosteric Natuie of the Voltage-Sensitive Sndium Channel George B. Brown Neurotoxin-Binding Site on the Acetylcholine Receptor Thomos L. Lentz and Paul T. Wilson Calcium and Sedative-Hypnotic Drug Actions Peter L. Carlen and Peter H . Wu

Neuropeptide -Processing, -Converting, and -Inactivating Enzymes in Human Cerebrospinal Fluid Lars Terenizls and Fred Nyberg Targeting Drugs and Toxins to the Brain: Magic Bullets Lance L. Simpson Neuron-Glia Interrelations Antonia Vernadakis Cerebral Activity and Behavior: Control by Central Cholinergic and Serotonergic Systems C. H . Vandenuolf INDEX