International Review of Neurobiology Volume 38

/nternationa/ Review

of

NEUROBIOLOGY VOLUME 38

EditoriaI Board

w. ROSS h E Y

Bo HOLMSTEDT

JULIUS AXELROD

PAULJANSSEN

Ross BALDESSARINI

KEw SEYMOUR

SIRROGER BANNISTER

KEITHK~LLAM

FLOYDBLOOM

CONANKORNETSKY

PHILLIP BRADLEY

RODOLFO PAOLETIT

Yuiu BUROV

SOLOMON SNYDER

JOSEDELGADO

STEPHEN WAXMAN

SIRJOHNECCLES

RICHARD WYATT

KJELLFUXE

International Review of

Editedby RONALD J. BRADLEY Deportment of Psychiatry LSU Medico/ Center Shreveport, louisiano

R. ADRON HARRIS Department of Pharmacology University of Colorado Health Sciences Center Denver, Colorado

VOLUME 38

ACADEMIC PRESS San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1995 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW I 7DX

International Standard Serial Number: 0074-7742 International Standard Book Number: 0- 12-366838-7 PRINTED IN THE WITTED STATES OF AMERICA 95 96 9 7 9 8 99 0 0 B B 9 8 7 6

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CONTENTS

Regulation of GABA, Receptor Function and Gene Expression in the Central Nervous System

A. LESLIEMORROW I. Introduction: The Structure and Function of GABA,/Benzodiazepine Receptors ...................................................... 11. Multiple Mechanisms of GABA,/Benzodiazepine Receptor

1 3

111.

6

Iv. 9 13 24 28

V.

VI. Endogenous Neurosteroid Regulation of GABA, Receptors. . . . . . . . . . . . VII. Developmental Alterations in GABA, Receptor Expression . . . . . VIII. Alterations in Receptor Subunit Composition: A Novel Mechanism of Regulation of Ligand-Gated Ion Channels . . . . . . . . . . . . . IX. Conclusions: New Breakthroughs, New Questions . . ... References .....................................................

32 33 34

Genetics and the Organization of the Basal Ganglia

ROBERT HITZEMA",YIFANCQIAN, STEPHEN KANES,KATHERINEDAINS, AND BARBARA HITZEMANN I. 11. 111. W. V.

Introduction .................................................... Genetic Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics and Behaviors Related to the Basal Ganglia . . . . . . . . . . . . . . . . . Genetics and the Functional Architecture of the Basal Ganglia.. . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

43 44 49 62 86 86

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CONTENTS

Structure and Pharmacology of Vertebrate GABAA Receptor Subtypes

PAULJ. WHITING, RUTHM. MCKERNAN, AND KEITHA. WAFFORD I. Introduction to the GABA, Receptor.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

. .. 11. Diversity of the GABA, Receptor Gene Family.. 111. Phosphorylation of GABA, Receptors . . . . . . . . . , . . . . . . . . . . . . . . . . . . . .

Iv. The Composition of GABA, Receptors in V i m . . . . . . . . . . . . . . . . . . . . . . . V. Drug Binding Sites o n the GABAA Receptor.. . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion ..................................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95 96 102 105 113 125 126

Neurotransmitter Transporters: Molecular Biology, Function, and Regulation

BETHBOROWSKY AND BETHJ. HOFFMAN ........................................

I. 11. 111.

Iv. Regulation of Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

V.

VI.

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

VII. VIII.

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

IX. Future Directions References . . . . . . . . . . . . .

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

139 143 165 170 180 184 186 189 190 192

Presynaptic Excitability

MEYERB.JACKSON ............................................. I. 11. Motor Nerve Terminals . . . . . . . . . . . . . . . . . . . . . . . 111.

Iv. V. Ciliary Ganglion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Goldfish Retina Bipolar Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... VII. Honorable Mention . . . . . VIII. Summary and Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I

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

20 1 203 21 1 217 232 235 239 240 243

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CONTENTS

Monoamine Neurotransmitters in invertebrates and Vertebrates: An Examination of the Diverse Enzymatic Pathways Utilized to Synthesize and Inactivate Biogenic Arnines

B. D. SLOLEY AND A. V.JUORIO I. Introduction ....................................................

253

11. A Critical Review of Methods Used for the Analysis of Catecholamines,

Indoleamines, Their Metabolites, and Related Enzymes in Biological ..... ........................... Tissues 111. Distribution of Monoamines in Invertebrates and Vertebrates.. . . . . . . . . Iv. Synthesis of Monoamines in Invertebrates and Vertebrates . . . . . . . . . .. . ........... V. Storage, Release, and Reuptake of Monoamines . . . . .

256 259 264 269

VI. 270 288 290

VII. References

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

Neurotransmitter Systems in Schizophrenia

GAVINP. REYNOLDS

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I. Introduction. 11.

111.

Iv. V.

VI. Acetylcholine . . . . VII. VIII. IX. X. XI. XII.

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

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

305 307 308 309 312 319 320 323 324 326 328 332 334

Physiology of Bergmann Glial Cells

THOMAS MOLLER AND HELMUT KETTENMANN I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. GapJunctions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Iv. Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Neurotransmitter Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

341 342 343 346 349

...

vlll

CONTENTS

VI . Ion Exchangers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Transmitter Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Summary....................................................... References .....................................................

355 355 357 357

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENT VOLUMES ...............................

361 369

REGUMTION OF GABAA RECEPTOR FUNCTION AND GENE EXPRESSION IN THE CENTRAL NERVOUS SYSTEM

A. Leslie Morrow Deportment of Psychiatry and Center for Alcohol Studies, School of Medicine, University of North Corolina at Chapel Hill, Chapel Hill, North Carolina 27599

I. Introduction: The Structure and Function of GABAA/Benzodiazepine Receptors 11. Multiple Mechanisms of GABAA/Benzodiazepine Receptor Regulation A. Homologous Desensitization and Regulation of GABAAReceptor Function 111. Benzodiazepine Regulation of GABAAReceptor Binding and Function A. Benzodiazepine Interactions with GABAAReceptors B. Effects of Chronic Benzodiazepine Administration on GABAA Receptors IV. Barbiturate Regulation of GABAAReceptor Function and Expression A. Barbiturate Interactions with GABAAReceptors B. Effects of Chronic Barbiturate Administration on GABAA Receptors V. Ethanol Regulation of GABAA Receptor Function and Expression A. Ethanol Interactions with GABAAReceptors B. Chronic Ethanol Administration Alters GMAAReceptor Function C. The Importance of GABAAReceptor Subunit Composition Following Chronic Ethanol Administration VI. Endogenous Neurosteroid Regulation of GABAA Receptors A. Neurosteroid Interactions with GABAAReceptors B. Do Neurosteroids Regulate GABAAReceptors in VznoP VII. Developmental Alterations in GABAAReceptor Expression VIII. Alterations in Receptor Subunit Composition: A Novel Mechanism of Regulation of Ligand-Gated Ion Channels IX. Conclusions: New Breakthroughs, New Questions References

1. Inhoduction: The Shuaure and Function of GAB&/Benzodiazepine Receptors

y-Aminobutyric acid (GABA), the most ubiquitous inhibitory neurotransmitter in the brain, interacts with a family of receptors containing recognition sites for the neurotransmitter GABA and the anxiolytic and sedative benzodiazepines and barbiturates as well as endogenous neurosteroids. These binding sites are linked allosterically to a GABA recognition site, and each site is involved directly or indirectly in the gating properties of integral C1- channels. GABA receptor-mediated activation of C1- conductance results in membrane hyperpolarization and decreased neuronal excitINTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 38

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Copyight 6 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MULTIPLE DISTINCT DRUG RECOGNITION SITES

CI

-

GABA recognition site Benzodiazepine agonist site Benzodiazepine inverse agonist site Barbiturate recognition site Neuroactive steroid recognition site Chloride channel blockers Anesthetics ? Ethanol ?

MULTIPLE DISTINCT SUBUNIT COMBINATIONS a1 -a6

PI44 Yl,y2S;12J+Y3,Y4

6 E

rho 1-2

FIG.1. Schematic diagram of the structure of the family of GABAAisoreceptors expressed in the central nervous system. GMAAreceptors appear to assemble as pentamers (Nayeem et al., 1994); however, the subunit composition and stoichiometry differ from this example in various brain regions and cell types. GABAA isoreceptors contain various distinct recognition sites which may be expressed on different receptor subtypes and which appear to be assemhled from the various subunits indicated.

ability (Skolnick and Paul, 1982b). Barbiturates and benzodiazepines have been shown to augment the actiiity of GABA via these specific recognition sites on the GABA receptor complex (Olsen, 1982). Ethanol also alters the gating properties of this receptor complex; however, ethanol binds with little or no affinity to recognition sites for GABA, benzodiazepines, barbiturates, and cage convulsants on GABAA receptors (Davis and Ticku, 1981; Greenberg et al., 1984). GABA, Benzodiazepine receptors comprise a family of receptors in the central nervous system (CNS) which are characterized by the presence of multiple distinct drug recognition sites and multiple distinct subunit combinations which comprise distinct GABAA isoreceptors.' GABAAreceptors are heteromeric protein complexes consisting of several homologous membrane-spanning glycoprotein subunits (Fig. 1). These subunits exist in six major classes: a,p, y, 8, and p (Schofeld et al., 1987; Levitan et al.,

'

The term isoreceptor (analogous to isozyme) refers to receptors composed of multiple protein subunits encoded by different genes which respond to the same neurotransmitter and produce the same biophysical response. albcit with different affinities, kinetics, and drug sensitivities (Hebebrand and Friedl, 1988).

REGULATION OF GABAA RECEPTOR FUNCTION AND GENE EXPRESSION

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1988; Schofield, 1989). Various isoforms within each subunit class have been isolated, including (YI-a6, &P4, 71-74, 6, p1 and p2 (Cutting et al., 1991, 1992), and additional variants are possible due to post-translational processing. [See Seeburg et al. (1990) or Macdonald and Olsen (1994) for review.] The structural features of GABAA receptor channels have been inferred to a large extent by analogy to nicotinic cholinergic and glycine receptors which are all members of the superfamily of ligand-gated ion channels (Unwin, 1989). However, the pentameric structure of native GABAA receptors has just been confirmed by electron microscopy and rotational spin analysis of isolated receptors (Nayeem et aZ., 1994). Although the actual heteroligomeric protein complexes that are expressed in brain are not yet known, it is clear that subunit composition determines the pharmacological and functional properties of GABAA isoreceptors. For example, the coexpression of different a and y subunits has been shown to result in GABA, receptors with different pharmacological properties that may account for the functional heterogeneity of GABAA receptors (Pritchett et al., 1989a,b; Levitan et al., 1988;Wafford et al., 1990; Puia et al., 1991). In transient expression systems, such as human kidney embryonic cells transfected with GABAA receptor subunits, benzodiazepine type I binding characteristics are observed with the expression of a&y2 subunits (where x indicates any /3 subunit). By contrast, the expression of a&.y2, a&y2, or cr&.y? subunits results in benzodiazepine type I1 pharmacology (Pritchett et al., 1989a). Further diversity of y2 subunits is created by an eight amino acid insertion site of the GABAA receptor yZL subunit, which encodes a sequence that can be phosphorylated by protein kinase C (Whiting et al., 1990) and appears to confer ethanol sensitivity to recombinant GABAA receptors expressed in frog oocytes (Wafford et al., 1991; Wafford and Whiting, 1992). Thus, the actions of GABA and drugs that act at GABAA isoreceptors are likely to vary depending on the subunit composition of the receptor complex in a particular brain region or cell type. Immunohistochemical studies have suggested the presence of at least five distinct GABAA receptor subtypes in the mammalian brain formed by the combination of several subunits from these classes (Fritschy et al., 1992).

II. Multiple Mechanisms of GABA/Benzodiazepine Receptor Regulation

GABAA receptors may be regulated by multiple mechanisms which are activated in different physiological states and in different circumstances. Evidence from radioligand binding studies suggests that there may be

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decreases in binding to the chloride channel which can occur within seconds of agonist stimulation (Havoundjian ut aL, 1986), while other effects on GABAA receptor function may require days or weeks (see below). Figure 2 shows a schematic diagram which suggests that GABAA receptor regulation is dependent on both the dose of agonist or drug administered as well as the time period over which these agents are administered. Several mechanisms of GABAA receptor regulation are proposed from studies that will be reviewed in this chapter. These mechanisms include rapid receptor inactivation, receptor downregulation, receptor uncoupling, posttranslational receptor modification, and transient alterations in gene expression. The relationship between dose and time of exposure may dictate the mechanism(s) which are activated in response to drug exposure and result in alterations in the function of GABAAreceptors. Alterations in GABAA receptor function are the fundamental endpoint of receptor regulation.

A. HOMOLOGOUS DESENSITIZATION AND REGULATION OF GABAA RECEPTOR FUNCTION It appears to be a general phenomena that neurotransmitter receptors containing integral ion channels are desensitized by their neurotransmitter. ACUTE

SUBACUTE

(seconds-minutes)

(hours-days)

CHRONIC (drys-weeks-months)

w 7 Receptor Desensitization

1

Altered Transcription of Receptor Genes

Post-Translational Receptor Modification (phosphorylation?) Altered mRNA Turnover

FIG.2. Regulation of GABAAreceptor function involves multiple mechanisms. The relationship between the dose and time of administration of various agents which activate or inhibit GABA, receptor function appears to insract to regulate GABA, receptor function. Evidence reviewed in this chapter suggests that several mechanisms may be involved in the biochemical regulation of these channels and that different mechanisms may be activated in different physiological conditions. (Schematic developed by A. L. Morrow and S. M. Paul, 1990, unpublished.)

REGULATION OF GABA, RECEPTOR FUNCTION AND GENE EXPRESSION

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Desensitization occurs when activation of the receptor by the neurotransmitter transforms the receptor to an inactive state in which the channel does not open. The physiological importance of this phenomenon is not well understood, but may represent an important biochemical mechanism for receptor regulation. it has been suggested that GABA receptor desensitization (progressive decrease in the GABA response) may participate in the development of epileptiform activity (Thalmann and Hershkowitz, 1985). Desensitization of GABAA receptors has been studied in hippocampal neurons (Thalmann and Hershkowitz, 1985), ganglion cells (Akaike et al., 1985), and membrane vesicle preparations from rat cerebral cortex (Cash and Subbarao, 1987; Schwartz et al,, 1986b; Wood and Davies, 1989). in mammalian cells, GABAA receptor desensitization occurs in the millisecond to second range (Cash and Subbarao, 1987) and continues for several minutes (Schwartz et aL, 1986b). Desensitization is reversible and exhibits multiphasic kinetics with high GABA concentrations (Celentano and Wong, 1994). The level of GABA in postsynaptic vesicles has also been reported to desensitize GABAAreceptors, decreasing GABA receptor-mediated chloride flux in synaptoneurosomes (Wood and Davies, 1989, 1991). The biochemical mechanism (s) that underlie desensitization of GABAA receptors has not been identified. The potential role of phosphorylation in the desensitization response is a matter of considerable controversy with evidence suggesting that phosphorylation of the receptor is either associated with (Heuschneider and Schwartz, 1989; Porter et al., 1990; Leidenheimer et al., 1991) or prevents (Gyenes et al., 1988; Stelzer et al., 1988) GABAA receptor desensitization. Artifacts related to the actions of CAMP which are independent of protein phosphorylation have been described (Heuschneider and Schwartz, 1989; Leidenheimer et al., 1990) and complicate the interpretation of the results of these studies. in addition, the heterogeneity of GABAA receptors may explain some of the conflicting results, i.e., some GABAA isoreceptors are phosphorylated by maximal GABA stimulation, while other isoreceptors are not phosphorylated. GABAA receptors contain putative phosphorylation sites on &-/33 and yZL subunits (Ymer et al., 1989; Wang and Burt, 1991), but the expression of these subunits varies in different brain regions (Zhang et al., 1991; Wang and Burt, 1991). Thus, the identification of specific mechanisms for GABAA receptor desensitization may require the isolation of a homogeneous population of isoreceptors for study. Chronic exposure of chick embryonic cultured cortical neurons to maximally activating concentrations of GABA results in a decrease in the density of GABAA receptors labeled by [3H]flunitrazepam (Tehrani and Barnes, 1988; Hablitz et al., 1989). The reduction in the expression of these receptors is accompanied by a decrease in peak current amplitudes in response

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to GABA (Hablitz et al., 1989). GABA exposure also results in a decrease in a1subunit protein on the surface of these cells (Calkin and Barnes, 1994). The binding of the benzodiazepine receptor antagonist ['H]Rol51788 is also decreased following exposure of chick brain neurons to GABA for 72 h (Roca et al., 1990a). This reduction in binding is accompanied by a 50% reduction in the levels of GABAA receptor aI subunit mRNAs, an effect which is reversed by the GABA receptor antagonist, SR 95531 (Montpied et al., 1991a). Similar effects on GABAAreceptor al subunit mRNAs have been observed following muscimol or flunitrazepam exposure in mouse cerebral cortical cultured neurons (Hirouchi et aL, 1992). These results suggest that GABA regulates the expression of its homologous receptor population by downregulation or a reduction in the expression of receptor subunits. The mechanism(s) which underlie these effects is unknown, but may involve alterations in transcription rates or mRNA stability.

111. Benzadiazepine Regulation of GAB& Receptor Binding and Function

A. BENZODIAZEPINE INTERACTIONSWITH GABAARECEPTORS It is generally agreed that the pharmacological effects of benzodiazepine agonists are mediated by selective facilitation of the postsynaptic actions of GABA (Skolnick and Paul, 1982; Choi et al., 1977). Electrophysiological studies have shown that benzodiazepines decrease neuronal excitability by increasing the frequency of chloride channel openings and thereby increasing chloride ion conductance (Study and Barker, 1981; Choi et al., 1981). Radioligand-binding studies have demonstrated that benzodiazepines bind to distinct recognition sites on GABAA receptors which are coupled allosterically to both GABA and barbiturate recognition sites (Skolnick and Paul, 1982a; Olsen, 1982). In subcellular preparations, biochemical studies have confirmed that benzodiazepine agonists have no direct effect on chloride channel function in the absence of GABA or GABA agonists (Morrow and Paul, 1988). In the presence of GABA, benzodiazepines show concentrationdependent and stereoselective potentiation of GABA receptor-mediated chloride channel function (Fig. 3) (Morrow and Paul, 1988). Benzodiazepines increase the potency of GABA, decreasing the EC5,, (concentration that produces the half-maximal effect), with no effect on the maximal response produced by GABA (Choi et al., 1977; Morrow and Paul, 1988).Thus, benzodiazepines are incapable of producing greater maximal inhibition of neuronal transmission than GABA itself. This contrasts with the effects of barbiturates, which exceed the maximal

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inhibition produced by GABA and exhibit more pronounced depressant and anesthetic effects (Macdonald and Barker, 1978; Morrow et al., 1988b). Benzodiazepines have been classified pharmacologically into several groups according to their intrinsic efficacy in the potentiation or inhibition of GABA receptor-mediated C1- currents (Haefely et al., 1990). Full and partial agonists and inverse agonists have been identified. Full agonists show the highest efficacy while partial agonists show lower efficacy, even though some partial agonists show high affinity for benzodiazepine recognition sites. The clinical utility of partial agonists has been suggested as a strategy to reduce the negative side effects of benzodiazepine therapy in humans (Haefely et al., 1990). Inverse agonists have intrinsic activity as negative modulators of GABA receptor-mediated C1- conductance, which are believed to be mediated by allosteric changes in receptor conformation. Biochemical studies have suggested that inverse agonists bind to a distinct recognition site on GABAA receptors that can be affinity purified and distinguished from benzodiazepine agonist recognition sites. B. EFFECTS OF CHRONIC BENZODIAZEPINE ADMINISTRATION ON GABAA RECEPTORS Prolonged treatment with benzodiazepines results in a decreased drug response in both humans (Greenblatt and Shader, 1978; Nutt, 1986) and

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experimental animals (File, 1982; Frey et al., 1984; Lister et al., 1984; Treit, 1985; Rosenberg and Chiu, 1985). Chronic benzodiazepine treatment reduces the ability of benzodiazepines to potentiate the inhibitory actions of GABA. This effect of chronic benzodiazepine administration has been observed using electrophysiological techniques in the raphe nucleus (Gallagher et al., 1984a, 1985), substantia nigra (Wilson and Gallager, 1987; Tietz and Fosenberg, 1988) and spinal cord neurons (Sher et al., 1983), as well as using biochemical techniques (Yu et al., 1988; Ngur et al., 1990; Li et aL, 1993;Allan et aZ.,1992a). Chronic exposure of animals to benzodiazepines results in a decrease in GABA-mediated chloride uptake (Miller et al., 1988a; Lewin et al., 1989b; Marley and Gallager, 1989), as well as a reduced ability of various benzodiazepines to enhance GABA-stimulated chloride uptake (Yu et aL, 1988; Ngur et al., 1990; Li et al., 1993). In contrast, chronic administration of benzodiazepine partial agonists, such as imidazinil, does not produce tolerance or alterations in the expression of benzodiazepine recognition sites (Giusti et al., 1993). The mechanisms that underlie behavioral and functional tolerance to the effects of benzodiazepines are complex and may be related to both the dose of drug and the method of administration. Following repeated injections of diazepam (5 mg/kg/day), there is a reduction in “low-affinity” GABAAreceptor binding sites and an increase or conversion to “highaffinity” [3H]muscimol binding sites (Marangos and Crawley, 1982; Gallagher et aL, 1984b).These low-affinitybinding sites are believed to mediate the potentiation of GABAresponsesby benzodiazepines (Burch et al., 1983), and therefore the reduction in these binding sites could be related to the development of tolerance to the anxiolytic and sedative effects of benzodiazepines following chronic treatment or administration. Chronic exposure to GABA may also produce a functional uncoupling of the recognition sites for GABA and benzodiazepines. The ability of GABA to potentiate [’HI flunitrazepam binding in the cerebral cortex is reduced following chronic administration of flurazepam or diazepam (Tietz et al., 1989; Gallager et aZ., 1984a; Mele et al., 1984). Likewise, in primary cultures of chick brain neurons, chronic exposure to flurazepam results in decreased enhancement of [’Hlflunitrazepam binding by GABA, which can be blocked by the benzodiazepine antagonist, Ro15-1788 (Roca et al., 1990b). The effects of flurazepam are not blocked by the specific GABA receptor antagonists, SR95531 or picrotoxin, suggesting that endogenous GABA does not contribute to this effect (Roca et al., 1990b). The effects of chronic repeated benzodiazepine administration on the number of benzodiazepine recognition sites have been inconsistent. There are reports of both decreases (Rosenberg and Chiu, 1981; Crawley et aL, 1982; Sher et al., 1983; Tieu et al., 1986) and no change (Mohler et al., 1978; Gallager et al., 1984a,b; Lista et aL, 1990) in the number of receptors.

REGULATION OF GABAA RECEPTOR FUNCTION AND GENE EXPRESSION

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The method of administration or the interval between treatment and sacrifice may account for these differences, since discontinuation of benzodiazepine administration is associated with an increase in benzodiazpine agonist binding and flux (Miller et aL, 198813).In addition, prolonged continuous administration of benzodiazepines, by minipumps or silastic pellet implantation, produces a consistent decrease in the density of binding sites for the antagonist ligand [3H]Ro15-1’788as well as a reduction in GABA receptor-mediated %l- uptake into cerebral cortical synaptoneurosomes (Marley and Gallager, 1989; Miller et aL, 1988a). Therefore, the chronic effects of benzodiazepines on GABAA receptor function appear to require continuous exposure to benzodiazepines, at least in rodent models. Prolonged administration of benzodiazepine antagonists and inverse agonists have opposite effects on the expression of GABAA receptors. Chronic administration of the benzodiazepine antagonist, Ro15-1788, increases the density of binding sites for various ligands that bind GABAA receptors in rat cortex (Kulkarni and Ticku, 1990) as well as chick cerebral cortical cultured neurons (Miller et al., 1990). The functional properties of GABAA receptors are also enhanced following chronic exposure to R01.51788 as well as the benzodiazepine inverse agonist, FG7142 (Miller et aL, 1990; Marley et aL, 1991). Prolonged FG7142 exposure increases in GABAA receptor subunit mRNA expression (Lewin et aL, 1994), consistent with other studies on the effects of FG7142 (Primus and Gallagher, 1992). Taken together, these data suggest that GABA receptor expression may be upregulated by chronic exposure to anxiogenic agents which block GABA and benzodiazepine receptor function. In contrast, decreases in GABA receptor-mediated chloride uptake have been observed in the brain of rats that were kindled with FG7142 (Lewin et aL, 1989; Corda et al., 1988) or pentylenetetrazol (Corda et aL, 1990).The production of chemical kindling may affect GABA receptor function and expression in a different manner compared to chronic exposure of rats to negative modulators of GABAA receptors. The physiological significance of regulation by these drugs may be considerable if the release of endogenous inverse agonists is an important factor in the discontinuation syndromes produced by benzodiazepines, barbiturates, and ethanol.

IV. Barbifuraie Regulation of GAB& Receptor Function and Expression

A. BARBITURATE INTERACTIONS WITH GABAA RECEPTORS Barbiturates have both direct and indirect effects on GABAA receptormediated chloride channel activity. The direct effects of pentobarbital are

10

A. LESLIE MORKOW

observed at concentrations between 0.1 and 1.O m M (Schwartzet al., 1986b), and there is a biphasic effect on both chloride conductance in frog sensory neurons (Akaike et al., 1985) and chloride uptake in cerebral cortical synaptoneurosomes (Schwartz et al., 1986a,b). The decrease in chloride channel function observed in the presence of high concentrations of pentobarbital may be due to desensitization of the chloride ion channel (Schwartz et al., 198613).Desensitization of GABA- and barbiturate-induced responses has been observed in numerous preparations including rat primary afferent neurons (Higashi and Nishi, 1985), hippocampal slices (Thalmann and Hershkowitz, 1985) and in dissociated hippocampal pyramidal cells (Numann and Wong, 1984).At subthreshold concentrations for direct modulation of GABA receptor-coupled chloride channels, barbiturates enhance the effects of GABA (Wong et al., 1984; Harris and Allan, 1985). This potentiation involves an increase in the maximal response to GABA as well as the apparent affinity, supporting the notion that barbiturates act via a distinct site from benzodiazepines (Morrow et al., 1988b). The effects of barbiturates are blocked by GABAA receptor antagonists, but not by benzodiazepine antagonists, indicating that there is specificity of the effects of barbiturates on GABAA receptor-mediated chloride channel activity. The effects of barbiturates on GABAA receptor-activated C1- conductance to enhance GABAergic neurotransmission represent a mechanism for their anxiolytic, sedative/hypnotic, anticonvulsant, and anesthetic properties. B. EFFECTSOF CHRONIC BAR~ITURATE ADMINISTRATION ON GABAA RECEPTORS It is well known that chronic exposure to barbiturates causes animals to become tolerant to their sedative/hypnotic effects and dependent on the drugs. Although hepatic metabolizing enzymes are induced following barbiturate administration, the role of metabolic tolerance can be separated from pharmacodynamic or functional tolerance in experiments in which the blood level of the barbiturate is measured upon awakening or regaining the righting reflex. Barbiturate-dependent animals exhibit a marked reduction in seizure thresholds which is one of the primary characteristics of the barbiturate withdrawal syndrome (Flint and Ho, 1980). The mechanisms that underlie the increased sensitivity to seizures in barbiturate withdrawal are unknown, but alterations in GABAA receptors have been postulated since the acute effects of barbiturates as well as convulsants, such as pentylenetetrazole and picrotoxin, involve receptor-gated chloride channels (It0 et al., 1989). GABAA receptor-mediated chloride flux in cerebral cortical synaptoneurosomes is significantly reduced following chronic pentobarbital administration for 2 weeks by daily injections (Morrow et al., 1990a). These effects

REGULATION OF GABA, RECEPTOR FUNCTION AND GENE EXPRESSION

11

on GABA receptor function are reversed 7 days after withdrawal from the drug, suggesting that the functional changes are related to the symptoms of withdrawal. Similar effects on GABAAreceptor-mediated chloride flux are observed after pentobarbital administration for 6weeks (Fig. 4),suggest300

1

p^n

200

CONTROL

CHRONIC PENTO 6 WEEKS 100

0 1

10

MUSCIMOL

100

(KM)

120 -l

100

CONTROL

CHRONIC PENTO 6 WKS

Vmax

-42.8%

pe.001

I'd

'

PENTOBARBITAL (mM)

FIG.4. Chronic pentobarbital administration reduces GAF5AAreceptor function in cerebral cortical synaptoneurosomes. Rats were injected with pentobarbital (30 mg/kg) once daily for 6 weeks. Control rats received saline injections. Data are from a representative experiment using pooled tissue from four animals, conducted in quadruplicate, and repeated four times (A) Chronic pentobarbital administration decreases the apparent &- of muscimol-stimulated "C1- uptake by 27.2% (P < 0.05) with no significant effect on the ECSo. (B) The apparent &a of pentobarbital-stimulated 36Cl- uptake is decreased by 42.8% ( P < 0.001) following repeated pentobarbital administration.

12

A. LESLIE MORROW

ing that longer treatment does not necessarily produce a greater decrement in GABA receptor function. However, the reversal of the effects of chronic pentobarbital administration is not observed immediately following pentobarbital withdrawal. There is incomplete reversal of the decrements in muscimol-stimulated chloride uptake 10 days following withdrawal from pentobarbital (Fig. 5). These data suggest that the duration of the chronic

A 300 200

1 -

CONTROL WITHDRAWN (10 DAYS)

100

-

V M X -22.5% p . 0 5

01

'1 100

I

10

MUSCIMOL (pM)

B CONTROL WITHDRAWN (10 DAYS) Vmax NO A

100

PENTOB'&~TAL

(

10000

FIG.5. Partial reversal of the effects of chronic pentobarbital administration 10 days following withdrawal from chronic pentobarbital administration. Rats were injected with pentobarbital (30 mg/kg) once daily for 6 weeks. Control rats received saline injections. Rats were withdrawn from pentobarbital and %C1- uptake was measured 10 days later. Data are from a representative experiment using pooled tissue from four animals, conducted in quadruplicate, and repeated four times (A) Muscimol-stimulated "Cl- uptake is reduced by 22.5% (P< 0.05) following chronic pen tobarbital administration and withdrawal with no significant effect o n the EC,,. (B) Pentobarbital-stimulated 36Cl- uptake is reversed to control levels following withdrawal from pentobarbital.

REGULATION OF GAB& RECEPTOR FUNCTION AND GENE EXPRESSION

13

administration can influence the time course of the recovery from the adaptations in GABAA receptors which are associated with dependence. Chronic phenobarbital administration has no effects on GABA-stimulated chloride flux, but reduces the ability of both phenobarbital and flunitrazepam to potentiate GABA-mediated chloride channel function in whole brain preparations of microsacs (Allan et al., 1992b). These reductions in GABAA receptor function cannot be explained by reductions in the expressions of GABAAreceptors. Chronic pentobarbital administration by pellet implantation has no effect on the density of ["S] tbutylbicyclophosphorothionate ( [35S]TBPS) (Ito et al., 1989) and increases ['HI muscimol (Sivam et aZ., 1982) binding sites to these receptors. During withdrawal from pentobarbital, however, there are increases, rather than decreases, in the density of ['5S]TBPS (It0 et aL, 1989) recognition sites on GABAAreceptors. In contrast, chronic phenobarbital administration reduces the number of benzodiazepine recognition sites in various brain regions (Liljequist and Tabakoff, 1985; Allan et aL, 1992b). These data suggest that chronic barbiturate administration may have different effects on distinct G B A Aisoreceptors or subunits that may be preferentially labeled by these ligands. Pentobarbital dependence has no effect on the levels of GABAAreceptor a,subunit mRNAs in rat cortex (Morrow et aL, 1990a), but additional subunits need to be analyzed to determine whether chronic barbiturate administration alters the subunit composition of GABAAreceptors.

V. Ethonol Regulotion of GAB& Receptor Function and Expression

A. ETHANOL INTERACTIONS WITH GAB&

RECEPTORS

Ethanol is believed to act at many sites in the brain, but GABAA/benzodiazepine receptors may be the major target responsible for its behavioral actions. GABA-mimeticdrugs, such as aminooxyacetic acid and tetrahydroisoxazolopyridinol (THIP), enhance and prolong the behavioral effects of ethanol, while antagonists shorten ethanol narcosis (Martz et aL, 1983). Likewise, the benzodiazepines and GABA mimetics ameliorate the symp toms of ethanol withdrawal (Sellers and Kalant, 1976; Frye et aL, 1983), whereas GABA antagonists potentiate these symptoms (Goldstein, 1973). Furthermore, benzodiazepine receptor inverse agonists, such as Ro15-4513 and FG7142, antagonize many ethanol-induced behaviors in the rat, including intoxication (Suzdak et aL, 1986a, 1988; Koob et aL, 1988; Lister, 1987, 1988).

14

A. LESLIE MORROW

The most direct evidence that ethanol interacts with GABAAreceptors at pharmacologically relevant concentrations has been demonstrated using subcellular brain preparations and cultured embryonic neurons in which ethanol and other short-chain alcohols have been shown to stimulate (Suzdak et al., 1986b, 1987) or potentiate GABA,receptor-mediated 96C1-uptake (Allan and Harris, 1986; Suzdak et aL, 1986b, 1987;Mehta and Ticku, 1988; Ticku et al., 1986). These effects of ethanol are observed at concentrations well within the range observed during acute intoxication (20-60 mM), while subintoxicating concentrations augment muscimol or pentobarbital stimulation of W - uptake (Allan and Harris, 1986; Suzdak et al., 198613, 1987;Ticku et at., 1986). The action of alcohols in stimulating "Cl- uptake in vitro appears to be mediated via the GABA-coupled C1- channel because its effects are blocked by the specific GABAAreceptor antagonists bicuculline and picrotoxin (Suzdak et al., 1986b; Mehta and Ticku, 1988). Moreover, the effects of short-chain alcohols in GABA-mediated C1- ion flux i n vitro are highly correlated with their intoxication potencies in rats and with their membrane/buffer coefficients (Suzdak et aL, 1987). Electrophysiological studies have confirmed that ethanol enhances GABAA receptor-mediated C1- conductance, but only in specific brain regions (Givens and Breese, 1990a,b; Celentano et al., 1988; Nestores, 1990; Mereu and Gessa, 1985) or cell populations (Aguayo, 1990; Reynolds and Prasad, 1991). We, and others, have suggested that the molecular composition of GABAA receptors determines the presence or absence of ethanol sensitivity (Morrow et al., 1990a; Montpied et aL, 1991b; Givens and Breese, 1990a; Wafford et al., 1990; Breese et ah, 1993; Criswell et aL, 1993a). This hypothesis has been supported by evidence in mammalian brain regions where ethanol sensitivity is highly correlated with the simultaneous presence of benzodiazepine type I ( [sH]zolpidem) binding sites and GABAA receptor a,,p2, and yL subunits (Criswell et al., 1993a). It is not clear from these studies whether the y2 subunit splice variants influence ethanol sensitivity in rat brain, since yZL subunits were abundant in both ethanolsensitive and ethanol-insensitive sites (Criswell et aL, 1993a). However, it is clear that ethanol potentiation of GABAA receptors may be limited to very specific subtypes of GABAA receptors that have a unique regional distribution in brain. In frog oocytes, the expression of the human ynL subunit has been reported to be required for ethanol potentiation of GABA (Wafford et al., 1991; Wafford and Whiting, 1992), but other studies have not confirmed this effect using rat subunits (Sigel et al., 1993). Recent studies on the expression of GABAA receptors from long-sleep and shortsleep mice have demonstrated genetic differences in the ethanol sensitivity of GABAAreceptors (Wafford et aZ.,1990).Ethanol enhanced electrophysiological responses to GABA in oocytes injected with mRNA from long-sleep

REGULATION OF GABAA RECEPTOR FUNCTION AND GENE EXPRESSION

15

mice but antagonized GABA responses in oocytes injected with mRNA from short-sleep mice (Wafford et al., 1990). These studies also suggest that heterogeneity of GABAA receptors and/or associated intracellular messengers may account for differences in sensitivity to ethanol.

B. CHRONIC ETHANOL ADMINISTRATION ALTERSGABAA RECEPTOR FUNCTION Ethanol shares several pharmacologic actions with the barbiturates and benzodiazepines, including anxiolytic and sedative activity (Liljequist and Engel, 1984) and, in appropriate circumstances, the development of crosstolerance and cross-dependence (Boisse and Okamoto, 1980; Le et al., 1986). The similarities in the actions of ethanol with benzodiazepines and barbiturates further suggest that all three drugs may share some mechanism(s) of action. Tolerance to the sedative and intoxicating effects of ethanol has been postulated to result from a compensatory decrease in GABA-mediated inhibition in the brain (Hunt, 1983).Alterations in endogenous GABA concentrations and turnover rates following chronic ethanol administration are inconsistent (Hunt, 1983) and probably cannot account for the ethanol withdrawal syndrome. However, recent studies have suggested that alterations in the function of the GABAA receptor chloride channels may contribute to some of the signs and symptoms of the ethanol withdrawal syndrome (Fig. 6). Chronic exposure of rats to ethanol produces physical dependence and tolerance (Frye et al., 1981; Goldstein and Pal, 1971; Karanian et al., 1986; Morrow et al., 1988a, 1992a). These effects are associated with a decrease in the sensitivity of GABAA receptor-coupled C1- channels in cerebral cortex of dependent animals (Sanna et al., 1993) and when blood ethanol levels produced by the ethanol exposure were greater than 150 mg/% (Morrow et al., 1988a). Muscimol-stimulated 36Cluptake is decreased by 26% and pentobarbital-stimulated 36Cl-uptake is decreased by 25% following chronic ethanol inhalation. The ability of ethanol (20 mM) to potentiate muscimol-stimulated W l - uptake is completely lost in rat cerebral cortical synaptoneurosomes (Morrow et al., 1988a) and in mouse cerebellar microsacs (Allan and Harris, 1987) following chronic ethanol administration. Benzodiazepine enhancement of muscimol-stimulated chloride flux is reduced in the cerebral cortex of mouse microsacs, while the functional efficacy of inverse agonists is enhanced (Buck and Harris, 1990). Behavioral responses to injections of muscimol into the substantia nigra (Gonzalez and Czachura, 1989) and to subcutaneous THIP injections (Martz et al., 1983) are also reduced following chronic ethanol administration. Likewise, chronic ethanol administration reduces electrophysiological responses to GABA in the medial septa1 nu-

B

A 1

T , h

P

.A Y

El-CH

$ 5 0 m ( D

t; MUSCIMOL (pM)

C

-

z

0.1 .02 0.4 .71.0 PENTOBARBITAL (mM)

0

D .CONTROL "CHRONIC ETHANOL

...w Y

m

20 30 4050 70 100 160 ETHANOL (mM)

FIG.6. Chronic ethanol administration alters the function of the GABA,, receptor complex. Rats were administered ethanol by inhalation for 14 days. Data represent the mean 2 SEM of four or five independent experiments, each conducted in quadruplicate. (A) Muscimol stimulation of 9hCl-uptake is reduced following chronic ethanol inhalation. The apparent Emax of muscimol-stimulated 36C1-uptake is reduced 26% ( P < 0.01) with no significant change in the mean ECso.(B) Pentobarbital-stimulated % - uptake is decreased after chronic ethanol administration. The apparent F,, was decreased 25% (P < 0.05) and the ECSowas increased 39% ( P < 0.05) after chronic ethanol exposure. (C) Direct stimulation of 36Cl- uptake by ethanol is not altered in cerebral cortical synaptoneurosomes after chronic ethanol administration. Ethanol, muscimol, and pentobarbital responses were measured simultaneously in the same tissue preparation. (D) Ethanol enhancement of muscimol-stimulated uptake is abolished following chronic ethanol inhalation. Muscimol and ethanol (where indicated) were added simultaneously and uptake was terminated after 5 s. There was a significant potentiation of muscimol stimulation by ethanol in control synaptoneurosomes ( 5 5 % , P < 0.001), but there was no potentiation by ethanol in synaptoneurosomes from ethanoltreated rats. Muscimol stimulation of "CI- uptake was decreased following ethanol administration (40%, P < 0.001, n = 6). Adapted from Morrow, A. L., Suzdak, P. D., Kavanian, J. W., and Paul, S. M. (1988a). Chronic ethanol administration alters y-aminobutyric acid, pentobarbital and ethanol-mediated "C1- uptake in cerebral cortical synaptoneurosomes. J. Pharmacol. Exp. Ther. 246, 158-164.

REGUIATION OF GABA, RECEPTOR FUNCTION AND GENE EXPRESSION

17

cleus (Criswell et al., 1993b) and GABA-induced chloride flux in spinal cord-cultured neurons (Mhatre and Ticku, 1993). Following the completion of ethanol withdrawal, the decrements in muscimol-stimulated "Cl- uptake in rat cerebral cortical synaptoneurosomes (Morrow et al., 1988a) and flunitrazepam potentiation in mouse cortical microsacs (Buck and Harris, 1990) are completely reversed, as would be predicted if these neurochemical changes are related to the behavioral state of withdrawal. However, alterations in GABA receptor-mediated function have not consistently been observed in tolerant and dependent animals (Allan and Harris, 1987;Buck and Harris, 1990; Frye et al., 1993).Muscimol stimulation of chloride flux was not altered in cerebral cortex (Buck and Harris, 1990) or cerebellum (Allan and Harris, 1987) of mice who were administered ethanol for 7 days, producing blood ethanol concentrations of approximately 100 mg/%. However, this same treatment regimen produced a decrement in benzodiazepine enhancement of GABA-mediated chloride uptake as well as an increased effectiveness of inverse agonists in mice (Buck and Harris, 1990). In whole rat brain, no effect of chronic ethanol administration on muscimol-stimulated chloride uptake was found in ethanol-dependent animals (Frye et al., 1993).Taken together, these observations suggest that chronic ethanol exposure produces alterations in the function of the GABA receptor-coupled C1- channels that may be dose, time, species, and/or region dependent and may be mediated by multiple neurochemical mechanisms. Attempts to demonstrate alterations in the density or affinity of brain GABAA/benzodiazepine receptors following chronic ethanol administration have yielded conflicting results (Table I ) . For example, Ticku and colleagues (1980) as well as Unwin and Taberner (1980) reported a decrease in the density of low-affinity ['HIGABA or [3H]muscimol binding sites in brain following chronic ethanol exposure to rats and mice. DeVries et al. (1987) reported a reduction in the ability of GABA to enhance ['HI flunitrazepam binding in brain membranes prepared from ethanoltreated mice. Since the enhancement of benzodiazepine binding induced by GABA is mediated by low-affinity GABAA binding sites (Burch et al., 1983), these data also suggest an ethanol-induced reduction in the lowaffinity population of GABAA receptors. However, others have failed to find alterations in the number or affinity of GABAA receptors using radioligandbinding techniques (Volicer, 1980;Volicer and Biagoni, 1982a). Moreover, several laboratories have failed to find changes in the number of ['Hlflunitrazepam (Rastogi et al., 1986;Volicer and Biagioni, 1982b; Karobath et al., 1980) or [35S]TBPSbinding sites (Rastogi et al., 1986; Thyagarajan and Ticku, 1985) in brain tissue from rats chronically administered intoxicating doses of ethanol. However, [35S]TBPSbinding sites have also been reported

18

A. LESLIE MORROW

TABLE I BIDIRECTIONAL EFFECTS OF CHRONIC ETHANOLADMINISTRATION ON GABA, RECEPTOR FUNCTION, RECOGNITION SITES, AND SUBUNIT EXPRESSION I N RAT BRAIN Receptor property

Alteration

Source

Sensitivity to convulsant action of antagonists

Increased

Frye et al. (1981), Goldstein and Pal (1971), Karanian el al. (1986)

GABA-mediated C1channel function

Decreased

Martz et al. (1983), Morrow et al. (1988a), Gonzalez and Czachura (1989), Criswell et al. (1993b). Sanna et al. (1993). Mhatre and Ticku (1993)

GABA-mediated C1channel function

N o change

Allan and Harris (1987), Buck and Harris (1990), Frye et al. (1993)

Pen tobarbital-mediated c1- flux

Decreased

Morrow et al. (1988a)

Ethanol-enhanced C1-

Abolished

Allan and Harris (1987), Morrow et el. (1988a)

Benzodiazepineenhanced Cl- flux

Decreased

Buck and Harris (1990)

Inverse agonist modulation of C1channel function

Increased

Mehta and Ticku (1989), Buck and Harris (1990)

High-affinity [3H]muscimol binding

N o change

Volicer (1980), Volicer and Biagoni (1982a)

['HI Flunitrazepam binding

No change

Rastogi rt al. (1986), Volicer and Biagioni (1982b), Karobath el al. (1980)

["SITBPS binding

No change, increased

Rastogi et al. (1986), Thyagarajan and Ticku (1985), Sanna el al. (1993)

Low-affinity ~3H]rnuscimoIbinding

Decreased

Ticku and Burch (1980), Unwin and Taberner ( 1980)

GABA enhancement of benzo binding

Decreased

Devries ~t al. (1987)

['HI Zolpidem binding

Increased

Devaud et al. (1993). Devaud and Morrow (1994)

flux

['HI Rol5-4513 binding

Increased

Mhatre et al. (1988)

Cerebral cortical a, subunit mRNAs and polypeptides

Decreased

Morrow et al. (1990a), Montpied et al. (1991b), Mhatre and Ticku (1993), Mhatre et al. ( 1993)

Cerebral cortical a2 subunit mRNAs and polypeptides

Decreased

Morrow et al. (1990a), Montpied et al. (1991a), Mhatre and Ticku (1993), Mhatre et al. (1993)

Cerebral cortical as subunit mRNAs and polypeptides

N o change, deceased

Morrow et al. (1990a), Montpied et al. (1991a), Mhatre and Ticku (1993), Mhatre et al. (1993) ( continues)

REGULATION OF GABAA RECEPTOR FUNCTION AND GENE EXPRESSION

19

TABLE I (Continued) Receptor property

Alteration

Source

~~~~

Cerebral cortical p2 subunit mRNAs

No change

Devaud et al. (1994)

Cerebral cortical yPs subunit mRNAs

Decreased

Devaud et al. (1994)

Cerebral cortical ynL subunit mRNAs

Increased

Devaud et al. (1994)

Cerebellar a,subunit mRNAs and polypeptides

Decreased

Morrow et al. (1992a), Mhatre et al. (1993)

Cerebellar a6subunit mRNAs and polypeptides

Increased

Morrow et al. (1992a), Mhatre and Ticku ( 1993)

Cerebellar p2 subunit mRNAs

Increased, no change

Morrow et al. (1992b), Devaud et al. (1994)

Cerebellar apsubunit mRNAs

Not detected

Morrow et al. (1992a), Mhatre and Ticku (1993)

Cerebellar assubunit mRNAs

Not detected

Morrow et al. (1992a), Mhatre and Ticku (1993)

to increase in the same animals that showed reductions in GABAA receptor function (Sanna et aL, 1993). Recently, Mhatre et aZ. (1988) reported an increase in the density of specific binding sites for [3H]Ro15-4513in the rat cerebellum following chronic ethanol administration as well as increased sensitivity to its behavioral effects (Mehta and Ticku, 1989). Conceivably, the use of different experimental protocols (species, doses of ethanol, routes of administration, etc.) may contribute to the conflicting radioligandbinding data following chronic ethanol administration. Alternatively, chronic ethanol administration may have differential effects on the expression of individual GABAA receptor subunits, accounting for the diverse effects of ethanol on different radioligand binding sites. It is tempting to speculate that the alterations in GABAA receptor function observed following chronic ethanol administration (vide supra) are the result of a decrease in the synthesis and expression of subpopulation(s) of GABAAisoreceptors. If ethanol alters the expression of specific populations of GABAAisoreceptors, radioligands which measure all GABA receptors may lack the selectivity to detect changes in one or more receptor subunits or subpopulations of G B A Aisoreceptors.

20

A. LESLIE MORROW

C. THEIMPORTANCE OF GABAA RECEPTOR SUBUNIT COMPOSITION FOLLOWING CHRONIC ETHANOLh M I N I S T R A T I O N Recombinant expression of functional GABAA receptors has been demonstrated using various combinations of subunits; however, the expression of different combinations of these subunits results in GABAA receptors with distinct gating and pharmacological properties (Levitan et aL, 1988; Pritchett et al., 198913;Verdoorn et aL, 1990). Studies of the developmental expression of a subunit mRNAs in rat and chick brain have suggested that the expression of various GABAA receptor subunits is developmentally regulated and that distinct a subunits may be substituted in adult brain compared to fetal brain as well as in different species (see below) (Montpied et al., 1989). This phenomena of subunit substitution or replacement has also been reported for the nicotinic cholinergic receptor in which substitution of the y subunit by the E subunit produces functional alteration of the receptor during development (Imoto et al., 1986; Mishina et al., 1986). Therefore, in both the GABA and the nicotinic receptor classes in the family of ligand-gated channels, there is precedence for the concept that substitution of receptor subunits in vivo may underlie alterations in the function of the receptor. This notion is consistent with the observation that substitution of GABAAreceptor subunits in transient expression systems in vitro also results in alterations in the function of the receptor (Levitan et aL, 1988; Pritchett et aL, 1989b; Verdoorn et al., 1990). Thus, alterations in the subunit composition of GABAA receptor subunits following chronic ethanol exposure could underlie the alterations in the function of the GABAA receptors in brain that are associated with ethanol dependence (vide supra). Studies on the effects of ethanol on individual GABAAreceptor subunit mRNA levels were conducted in order to determine whether ethanol differentially alters the expression of various GABAA receptor subunits (see Table I). Chronic ethanol administration resulted in a substantial reduction in the levels of G B A Areceptor a1 subunit mWAs measured in the cerebral cortex of individual rats (Morrow et al., 1990a; Montpied et al., 1991b; Mhatre and Ticku, 1993).The level of the 4.8-kb mRNA species was reduced 47 2 5.0% compared to control animals, while the 4.4kb species was reduced 43% (Fig. 7). Using polyA+ RNA pooled from groups of control and ethanol-treated animals, the levels of a1 subunit mRNAs (4.4 and 4.8 kb) were reduced by 39 and 5376, respectively, in ethanol-treated rats compared to controls. In addition, there was a significant reduction in the a2subunit (8.0 kb) mRNA levels. In contrast, no alteration in the level of the agsubunit (3.1 kb) transcript was detected. The same Northern blots

REGIJIATION OF GABA, KECEPTOK FUKCTION AND GENE EXPRESSION

Control

21

Ethanol- Treated

G A B 4 Receptor a Subunlt mRNAs 4.8 Kb 4.4 Kb

G DmRNA

3.5 Kb

BActln mRNA 1.9 Kb

FIG.7. Chronic ethanol inhalation reduces the level ofGABAAreceptor a,-subunit mRNA in the rat cerebral cortex. Total RNA was prepared from individual rats and Northern blot analysis was conducted on two or three blots for each experiment. The 4.8- and 4.4kb species of CY subunit mRNA were detected in cerebral cortex from these animals. The same blots were rehybridized with the P-actin ripoprobe. Densitometric measurements were made without knowledge of the treatment group. Chronic ethanol administration reduced the level of the 4.8-kb species of CY subunit mRNA by 47% ( P < 0.01) and reduced the level of the 4.4kb species by 43% ( P < 0.01). The levels of p-actin mRNA and polyA' RNA were not altered by chronic ethanol administration. Data are representative of experiments in Morrow et al. (1990a) and Montpied el al. (1991a).

rehybridized with a riboprobe for @-actinshowed no alteration in the level of b-actin mRNA following chronic ethanol administration. PolyA' RNA

levels were also measured in order to control for a possible nonspecific reduction of all mFWA resulting from repeated ethanol administration. Chronic ethanol inhalation had no effect on the levels of polyA' RNA in the cerebral cortex of ethanol-treated rats compared to control rats (Montpied et al., 1991b). Similar effects have been observed on a subunit polypeptide levels in rat brain; however, downregulation of all three a subunit polypeptides was observed following chronic ethanol administration (Mhatre et al., 1993). The hypothesis that these alterations in receptor structure are related to the development of ethanol dependence (Montpied et al., 1991b; Morrow et al., 1992a) is supported by studies in withdrawal seizure-prone (WSP) vs withdrawal seizure-resistant (WSR) mice in which chronic ethanol administration resulted in reductions in a1subunit RNAs in the WSP mouse line but not the WSR line (Buck et al., 1991).

22

A. LESLIE MORROW

1. Chronic Ethanol Exposure Diffentially Alters GABAA Receptor Subunit mRNA Leueh in Cerebellum Rats were administered alcohol by liquid diet for 2 weeks using a pairfed design. The rats drank 8-10 g/kg/day of 5-7.5% ethanol diet (BEC = 248 2 35 mg/dl on sacrifice). Additional rats treated concurrently were tested for ethanol dependence following ethanol withdrawal. Tremors (100%) and audiogenic seizures (50%) were observed in these rats 8 h following ethanol withdrawal. GABAA receptor a subunit mRNA levels were quantified by Northern analysis using subunit-specific cRNA probes. Cerebral cortical GABAA receptor al subunit mRNA levels were reduced by about 30-40% (Morrow et aL, 1992a), similar to the effects of chronic ethanol inhalation (Morrow et al., 1990a; Montpied et al., 1991b). This is an interesting finding because it demonstrates ethanol regulation of GABAA receptor a subunit mRNA levels using a different method of ethanol administration that controls for the effects of dietary stress. Similar data have been obtained using the intragastic intubation technique for ethanol administration (Mhatre and Ticku, 1993; Mhatre et al., 1993). These data strengthen the interpretation that the effects of chronic ethanol exposure are due to ethanol itself and not due to artifacts of the method of ethanol administration. No effect of chronic ethanol administration on GABAA receptor p2subunit mRNA levels was detected in three independent experiments (Morrow et al., 1995). In the cerebellum, chronic ethanol administration by liquid diet reduced the levels of GABAA receptor a, subunit mRNAs (4.8 and 4.4 kb) by 30-40% and increased the levels of GABAA receptor (Ye subunit mRNA (2.7 kb) by 30% (Fig. 8) ( P < 0.05, n = 16) (Morrow et al., 1992a). There was no significant effect on glutamic acid decarboxylase (GAD) and Pactin mRNA levels or polyA' RNA levels following chronic ethanol exposure by liquid diet. Mhatre et al. (1993) have also shown an increase in GABAA receptor (Ye subunit mRNA and polypeptide levels in cerebellum. The effects of chronic ethanol administration on f12 subunit mRNA levels in cerebellum were detected in a preliminary study (Morrow et al., 1992b),but these effects have not been reproducible in subsequent experiments (Morrow et al., 1995). These data suggest that chronic ethanol exposure regulates GABAA receptor gene expression by differential effects on the synthesis of specific subunits of GABAA receptors. This could result in alterations in subunit composition that could underlie alterations in GABAA receptor function following chronic ethanol administration (Morrow et al., 1990a, 1992a; Montpied et al., 1991b; Mhatre et al., 1993). The cellular mechanism(s) resulting in the alteration(s) in GABAA receptor subunit mRNAs in the cerebral cortex and cerebellum of rats sub-

REGULATION OF GABAA RECEPTOR FUNCTION AND GENE EXPRESSION

GABAA Receptor a 6 Subunit

2.7 kb

P-Actin

1.9 kb

GAD67

3.7 kb

23

FIG.8. Chronic ethanol exposure by liquid diet for 2 weeks increases GABAAreceptor a6 subunit mRNA levels in rat cerebellum with no effect on GAD or P-actin mRNA levels. The same rats exhibited decreases in GABAAreceptor a, subunit mRNA levels (see text). Rats were fed ethanol in a liquid diet and Northern blots were prepared using polyA+ RNA purified from pooled tissue samples. The data shown are representative blots using four RNA concentrations (0.2,0.5, 1.0, and 2.0 pg) from each group, respectively.Lanes 1-4 are control samples from pair-fed rats and lanes 5-8 are samples from ethanol-fed rats. The same blot was hybridized sequentially with each probe as a control for the specificity of the effects of ethanol on GABAA receptor (Y6 subunit mRNA levles. Chronic ethanol ingestion increased the levels of GABAAreceptor (Ye subunit mRNAs to 145.9 ? 19.4%of control levels (P< 0.01). There was no effect on GAD or Pactin mRNA levels. (Adapted from Morrow et al. (1992b) with permission.)

jected to chronic ethanol exposure could involve a decrease in transcription of a1subunit gene(s) or mRNA stability. The fact that the brain concentrations of polyA+ RNA, GAD mRNA, and Pactin mRNA were unaffected by chronic ethanol exposure shows that the alterations in a1and afisubunit mRNAs were relatively selective and not due to a generalized change in transcription or mRNA processing. The observation that GABAA receptor a,and a2subunit mRNAs are reduced following chronic ethanol administration suggests that chronic ethanol administration may reduce the synthesis of GABAAisoreceptors containing these subunits. Similarly, the increase in as subunit mRNAs suggests that isoreceptors containing afi subunits are increased. This interpretation is supported by the increases in ['H]Ro154513 binding which have been observed (Mhatre et al., 1988). Since different combinations of GABAAreceptor subunits result in receptors with different gating properties (Schofield et al., 198'7; Blair et al., 1988; Ymer et al., 1989; Pritchett et al., 1989b; Levitan et al., 1988), the exact mechanism(s) by which ethanol alters GABAA receptor function may involve the regulation of genes encoding the various GABAAreceptor subunits. Clearly, further

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studies are required to determine whether alterations in the structure of GABAA receptors account for the regulation of the function of the receptor.

2. Chronic Ethanol Exposure Increases PH]Zolpidem Binding in Select Brain Regions The effect of chronic ethanol administration on ['H]zolpidem binding was measured in rat brain because [ 'H]zolpidem selectively labels GABAA-benzodiazepine type I receptors that are highly correlated with ethanol-sensitive GABA responses in brain (Criswell et aZ., 1993a). This subtype of GABAAreceptor requires the expression of a1subunits (Pritchett et al., 1989a; Luddens and Wisden, 1991) and is not selectively labeled by benzodiazepine ligands. Since chronic ethanol administration reduces GABA receptor function and a1subunit mRNA levels, we investigated the effect of chronic ethanol administration on [ 'H]zolpidem binding. Surprisingly, chronic ethanol administration did not reduce ['HI zolpidem binding. Moreover, when single-point measurements were analyzed by paired comparisons, small (10-12%) but significant increases in maximal ['Hlzolpidem binding were detected in medial septum, cortex, and cerebellum (Devaud et aL, 1993; Devaud and Morrow, 1994). No alterations in ['Hlzolpidem binding to striatum and hippocampus were observed. These findings suggest that chronic ethanol administration may have differential effects on aI subunit expression and ['H]zolpidem binding sites. These data are consistent with the hypothesis that chronic ethanol exposure alters the function and subunit composition of GABAA receptors without having a pronounced effect on the total number of any particular GABAAreceptor subtype.

VI. Endogenous Neurosteroid Regulation of GAB& Receptors

A. NEUROSTEROID INTERACTIONSWITH GABAARECEPTORS Several major metabolites of progesterone and deoxycorticosterone, including 3a-hydroxy-5a-pregnan-2O-one (allopregnanolone or 3a,5aTHP) and 3a,21dihydroxy-5a-pregnan-2O-one (tetrahydrodeoxycorticosterone or THDOC) , have been shown to interact with the G B A Areceptor chloride channel complex in the rat central nervous system (Majewska et al., 1986; Gee et aL, 1988; Harrison et al., 1987; Morrow et al., 1987; Turner et al., 1989; Peters et al., 1988). In the presence of low concentrations of GABA, these steroids compete with ["SITBPS binding to the GABAA receptor complex with very high affinity ( K s in the low nanomolar range) (Gee

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25

et al., 1987), and with somewhat lower affinity for [35S]TBPSbinding in the absence of GABA (Y = 200-500 nM) (Majewska et al., 1986; Gee et al., 1987).Allopregnanolone and THDOC also enhance benzodiazepine recep tor binding in vitro in a manner similar to that of barbiturates (Majewska et al., 1986), suggesting that these steroids bind to sites on the GABAA receptor complex which interacts allostericallywith benzodiazepine as well as GABA recognition sites. In electrophysiological experiments, both allopregnanolone and THDOC have been shown to augment GABA receptormediated chloride ion conductance in cultured rat hippocampal neurons (Majewska et al., 1986). Using chloride flux assays, we have demonstrated that both allopregnanolone and THDOC enhance GABAreceptor-mediated chloride ion uptake into rat cerebral cortical synaptoneurosomes at concentrations as low as 25-1000 nM (Morrow et al., 1987,1988b). These neurosteroids are 20 times more potent than benzodiazepines and 200 times more potent than barbiturates in augmenting GABA receptor function. In behavioral studies, THDOC possesses anticonflict (Crawley et al., 1986) and hypnotic effects when administered parenterally to rats (Mendelson et al., 1987). THDOC also has antiaggressive properties in mice (Kavaliers, 1988).Allopregnanolone has been shown to produce analgesic effects in mice when administered intracerebroventricularly(Wiebe and Kavaliers, 1988).Since allopregnanolone and THDOC are endogenous steroid metab olites (Holzbauer et al., 1985; Schambelan and Biglieri, 1972) and potently modulate GABA receptor-mediated inhibitory neurotransmission in the central nervous system, we have proposed that these steroids may be naturally occurring anxiolytic and/or hypnotic agents (Majewska et al., 1986; Morrow et al., 1987,1988b). The importance of understanding the pharmacological properties and functional interactions of these steroid metabolites with the GABA receptor complex is supported by evidence that levels of progesterone and deoxycorticosterone and their metabolites are altered in various physiological states, including stress (Schambelan and Biglieri, 1972;Fenske, 1986; Ladisich, 1975; Holzbauer, 1975; Ichikawa et al., 1972). Functional studies show that neurosteroids enhance the potency of muscimol in stimulating 36Cl-uptake in a concentrationdependent and stereospecific manner. Structure activity experiments demonstrate that a 3a configuration, in either the 5a- or the 5D-reduced metabolites, is essential for pharmacological activity (Purdy et al., 1990b; Morrow et al., 1990b). Computer modeling of the concentration-response curves for steroidenhanced muscimol-stimulated 36Cl-uptake suggests that the endogenous steroid metabolites interact with multiple binding sites or conformational states (Purdy et al., 1990b). Pseudo-Hill coefficients for steroid potentiation of muscimol-stimulated '%l- uptake ranged from 0.5 to 0.7, further suggesting heterogeneous receptor interactions with the GABA receptor complex

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by these steroids. Moreover, THDOC 21-mesylate appears to interact selectively with one of the steroid binding sites, producing a smaller maximal response, monophasic concentration-response curves, and a Hill slope approximating 1.0. These data suggest the presence of multiple distinct steroid recognition sites or conformational states on GABAA receptor( s) (Morrow et al., 1990b). There is increasing evidence that steroids and barbiturates interact with the GABAAreceptor at different sites. Comparisons of steroid and barbiturate binding to the ["SITBPS recognition site on the GABAAreceptor complex have suggested that steroids and barbiturates interact with distinct binding sites labeled by [35S]TBPS.Turner et al. (1989) suggested that the steroid anesthetic, alphaxalone, and the barbiturate, pentobarbital, inhibit [35S]TBPS binding via separate sites since the effects of the steroid and the barbiturate are additive. Likewise, Gee et al. (1988) demonstrated that saturating concentrations of allopregnanolone and pentobarbital additively accelerate the dissociation of ["SITBPS, which would not be expected if both compounds acted at a single site. Further, the enhancement of [3H]muscimol binding to GABA receptors by pregnanolone and secobarbital is differentially inhibited by antagonists (Kirkness and Turner, 1988). Perhaps the simplest model to account for these data would suggest the presence of two steroid binding sites on GABAA receptors, one of which is sensitive to barbiturates as well as neurosteroids. The heterogeneous interactions of steroids and barbiturates with [35S]TBPS binding and/or GABA receptor-mediated chloride ion flux may be due to the existence of multiple GABAA isoreceptors. Different populations of GABAAreceptors, composed of homologous but unique subunits, appear to have different affinities for steroids (Sapp et al., 1992). However, there is evidence for heterogeneous interactions of neurosteroids with recombinant GABAA receptors composed of a l ,PI,and yn subunits or p2 subunits alone (Puia et aL, 1990), supporting the hypothesis for multiple steroid recognition sites on individual subtypes of GABAA receptors.

REGULATE GABAARECEPTORS in Vim? B. Do NEUROSTEROIDS The high potency of neuroactive steroids as well as the presence of both 5a-reductase and 3a-hydroxysteroid oxidoreductase in brain and many peripheral tissues have prompted suggestions that neurosteroids may act as endogenous modulators of GABAA receptors (Holzbauer et al., 1985; Majewska el al., 1986). The biosynthetic pathways for the formation of allopregnanolone and THDOC are shown in Fig. 9. Allopregnanolone can be formed from progesterone in mixed cultures of neurons and glia (JungTestas et al., 1989a,b) and THDOC can be formed in brain from deoxycorti-

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-

c-

no Cholesterol

Pregnenolone

Progesterone

5wDihydroprogesterone

/

Allopregnanolone

Deoxycorticosterone

Allotetrahydrodeoxyconicosterone

SaOihydrodeoxycorticosferone

lallotetrahvdroDOC)

FIG.9. Biosynthetic pathways for the formation of the neurosteroids, allopregnanolone, and THDOC. These steroids are formed by reduction of the 3a-and 5a-hydroxyl groups by nonspecific reductases which are abundant in brain and peripheral tissues.

costerone metabolized from progesterone in the adrenal cortex (Kraulis et al., 1975). Allopregnanolone has been measured by radioimmunoassay in rat brain and plasma where its levels have been shown to fluctuate in response to stress (Purdy et aL, 1991) and during the estrous and menstrual cycle (Paul and Purdy, 1992). It is also detectable in human female plasma and male urine (Purdy et aZ., 1990a; Hiemke et al., 1991). These levels fluctuate in females in a manner highly correlated with plasma progesterone levels (Purdy et al., 1990a), while in males levels do not vary over a 48-h sampling period (Hiemke et al., 1991). Both progesterone and allopregnanolone can be detected in plasma and brain. Female rats show higher levels of both steroids in plasma and brain than male rats and these levels fluctuate across the estrous cycle (Paul and Purdy, 1992; CorpCchot et aL, 1993). Allopregnanolone is highest during estrous and proestrous and is lowest at diestrous (Paul and Purdy, 1992). The concentrations measured in brain of female rats reach levels sufficient to produce physiological effects at GABAA receptors (Purdy et al., 1990a; Finn and Gee, 1994). In addition, allopregnanolone, but not THDOC, is detectable in the brain of adrenalectomized and gonadectomized rats (Purdy et al., 1991; Paul and Purdy, 1992; CorpCchot et aL, 1993) suggesting that this neuroactive steroid is generated de novo in brain. In male rats, both endogenous steroids can be detected in plasma and brain, but their levels are rather low under most conditions. Following exposure to brief swim stress in ambient temperature water, significant

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increases in both allopregnanolone and THDOC are observed in cortex, hypothalamus, and plasma. The brain levels of these neurosteroids increase to approximately 3-10 ng/g (10-30 nM) after swim stress (Purdy et al., 1991), which is within the range of concentrations that potentiate GABA receptor-mediated chloride uptake in synaptoneurosomes (Morrow et al., 1987, 1990b). In adrenalectomized rats, neither progesterone nor the neurosteroids could be detected in the plasma before or after swim stress; however, allopregnanolone was still detectable in the cerebral cortex in both groups (Purdy et al., 1991). Acute stress increased brain levels of allopregnanolone prior to the elevation in circulating levels. These results show that the adrenal gland is the major source of progesterone and THDOC following exposure to stress, but that allopregnanolone may be synthesized in brain. There is only indirect evidence to suggest that neurosteroids may regulate GABAA receptor function and expression in v i v a Stress activates the hypothalamic-pituitary-adrenal axis (HPA), eliciting increases in adrenocorticotropin and corticosterone which result in the alterations in steroid and neurosteroid levels described previously. Stress has been shown to produce many effects on GABA,, receptors which mimic the effects of neurosteroids on various recognition sites and chloride channel function (Serra et al., 1991; Marazziti et al., 1990; Schwartz et al., 1987; Andrews et al., 1992; Drugan et al., 1989). It is possible that some of these responses are mediated via interactions of neurosteroids with G B A Areceptors. Additional evidence suggests that gender elicits differential response to stressors, and that variations in the HPA response to stress are observed across the estrous cycle in the rat (Viau and Meaney, 1991). For example, there are sex differences in the effects of acute swim-stressresults on GABAAreceptorbinding characteristics (Akinci and Johnston, 1993). Furthermore, repeated swim stress over 2 weeks reduces [3H]Ro15-1788binding as well as GABAA receptor a subunit mRNA levels in the mouse hippocampus (Weizman et al., 1989;Montpied et al., 1993). Thus, alterations in neurosteroid levels in both male and female brain may be an important factor which controls the expression of GABAAreceptors.

VII. Developmental Alterations in GAB& Receptor Expression

GABA,., receptors develop in spatiotemporal synchrony with GABAergic pathways in the rat embryo (Cobas et al., 1991;Schlumpf et al., 1989).These receptors appear to be functional, as judged by electrophysiologic criteria in acutely dissociated cells (Fiszman et al., 1990; Serafini et al., 1993;Walton

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et al., 1993a,b) and the ability of GABA to stimulate C1- influx in fetal synaptoneurosomes (Kellogg and Pleger, 1989). In the human embryo, GABAAreceptors are detectable by ['H]flunitrazepam binding at 7 weeks postconception and increase sharply between 8 and 11 weeks (Hebebrand et al., 1988). GABA enhances the binding of benzodiazepine ligands to these receptors, suggesting that these are functional receptors. The temporospatial association of GABAergic innervation with the expression of functional GABAAreceptors suggests afferent innervation may stimulate the expression of these receptors. The expression of specific GABAAreceptor subtypes may be associated with trophic functions or the formation of synapses in early brain development. This possibility is suggested by the finding that GABAAreceptor a4, P I , and y1 subunits are coexpressed in the ventricular zone (neuroepithelium) of the El3 rat spinal cord, which consists primarily of proliferating cells (Ma et al., 1993; Serafini et al., 1993). These subunits continue to be expressed through E l 7 during the period of active neuronal differentiation from proliferating progenitors. Whether this subunit combination is expressed in other CNS germinal zones is not yet known. In the mantle zone, where neurons are beginning to differentiate, GABAAreceptor a p ,as,a5, p2,p3,y2,and y 3subunits are expressed by E13. GABA., receptors constituted by these subunits appear to be functional, since cells acutely isolated from El3 spinal cord exhibit C1- channel activity in response to GABA (Serafini et al., 1993). Benzodiazepines modulate such responses in all of these cells, whereas bicucuiline and picrotoxin have variable effects. These receptors could mediate trophic effects of GABA on cell proliferation, migration, or the onset of neuronal differentiation during early embryogenesis (Ma et al., 1993), although the mechanism of these actions is still unclear. The mechanisms which underlie developmental regulation of GABAA subunit expression may involve both intrinsic mechanisms and local extrinsic factors (e.g., afferent activity). Coordinate development of GABAergic axons and GABAAreceptors in uiuo and evidence for trophic functions of GABA raise the possibility that this transmitter could regulate (modulate) expression of its own receptors in developing brain. Recent studies in the cerebellum provide evidence supporting this hypothesis. Meinicke and Rakic (1990) demonstrated that GABAergic axons innervate recently migrated granule cells prior to the appearance of GABAAreceptors, and have suggested that GABA released from these afferents could regulate receptor expression. The importance of afferent influences for GABAA receptor development is further illustrated by the finding that increases in al and a6subunit transcripts found in granule cells of postnatally developing cerebellum in uiuo are not found in granule cell cultures prepared prior to the migratory phase, which includes the onset of dendritic outgrowth and

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innervation by afferents (Bovolin et al., 1992; Zheng et aL, 1993; Beattie and Siegel, 1993). Further evidence suggests that P2 and y 2 subunits may be similarly regulated by granule cell afferents (Beattie and Siegel, 1993). Purkinje cells, however, appear to express a1subunits that are not regulated by afferent input (Frostholm et al., 1991; Kato, 1990). Merents that might regulate expression of GABAA receptors in cerebellar granule cells include GABAergic axons from Golgi neurons and glutamatergic mossy fibers. Trophic effects of GABA on cultured cerebellar granule cells include enhanced expression of GABAA receptor P subunits and induction of low-affinityGABAA receptors of unknown subunit composition (Belhage et al., 1986, 1987; Meier et al., 1985;Hansen et al., 1988, 1991). Recent evidence suggests that glutamate may also regulate expression of GABA, receptors in cerebellar granule cells. Chronic treatment with NMDA receptor agonists has been reported to promote expression of GABAA receptor a, and a5subunits in cerebellar granule cell cultures (Memo et nL, 1991). Recent studies have shown that a single treatment of these cells with NMDA (10 p M ) increases the absolute amounts of a1and a5, but not a6 subunit mRNAs and polypeptides (Harris et al., 1994). These changes in subunit expression are accompanied by increased responsiveness to flunitrazepam potentiation of GABA-mediated C1- currents and an increased affinity for GABA (W.J. Zhu, S. Vincini, B. T. Harris, and D. R. Grayson, 1994, personal communication).The relationship between the development of GABAAand NMDA receptors in cerebellar granule cells is unknown. It is possible that expression of GABAAreceptors could be sequentially regulated by GABAergic and glutamatergic afferents. Thus, GABA and glutamate could act together to regulate developmental expression of specific GABAA receptor subunits in cerebellum and other regions of the CNS. The subunit composition of GABAA receptors in various brain regions appears to change during normal ontogencsis. Transient mRNA expression patterns for transcripts encoding various GABAA receptor subunits have recently been described in developing rat brain (Fig. 10). The typical embryonic/neonatal pattern involves expression of a?,a%, a5,PI, p3,and y1-y3 subunit transcripts, whereas during postnatal development a,, a4, afi, P2, y2, and 6 predominate (Montpied et al., 1989; Gambarana et al., 1990, 1991; Laurie et al., 1992; Bovolin et al., 1992; Zdilar et al., 1992; Poulter et al., 1992, 1993; Ma et al., 1993; Serafini et nl., 1993). Specific coexpression patterns differ by brain region. For example, at E13-14, only a3is expressed in cortex and cerebellum, whereas aq,a3,and a5are found in diencephalon. In brain stem and spinal cord, multiple subunits are present at this age (e.g., CQ-CY~, Pl-P3, and yI-y3 (Laurie et al., 1992; Ma et al., 1993; Poulter et al., 1992, 1993; Serafini et al., 1993). These expression patterns appear to confer specific functional characteristics to developing GABAA receptors.

REGULATION OF GABA, RECEPTOR FUNCTION AND GENE EXPRESSION

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Rat

9.0 8.0

4.7 4.5

3.5

FIG.10. Alterations in GABAAreceptor subunit mRNA expression during normal ontogenesis in rat and chick brain. PolyA+ RNA was prepared from whole brain embryonic (15 day), newborn, and adult rat and chicken brain. In embryonic brain, the 8.0-kb a subunit RNA was about 8-fold more abundant than that in adult brain, while the 4 . 4 and 4.8-kb transcripts (rat) or the 4 . 5 and 4.7-kb transcripts (chick) were more than 50- and 15-fold higher in adult compared to embryonic brain. Reprinted by permission of the publisher from: Multiple GABA, receptor (Y subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain, by Montpied, P., Ginns, E. I., Martin, B. M., Stetler, D., O'Carroll, A,-M., Lolait, S. J., Mahan, L. C., and Paul, S. M.; FEES Letl. 258, 94-98. Copyright 1989 by Elsevier Science Inc.

Several studies have demonstrated clear functional differences in responses of embryonic and postnatal cells to GABA agonists. Responses of early developing cells are typically depolarzing and involve calcium influx (Segal, 1993), which may be involved in trophic cellular responses. These cells switch to hyperpolarizing responses at later ages (Fiszman et aL, 1990; Cherubini et aL, 1991). There is a temporal correspondence between this functional switch and the change from the early to the adult-like pattern of subunit expression in neuroepithelial cells (Ma et aL, 1993) and cultured spinal cord neurons (Walton et aL, 1993b). The functional significance of the switch in GABAA receptor subunit expression is not known in the cerebral cortex, but changes in a,and a6subunit expression in the cerebellum are associated with alterations in the sensitivity of GABAA receptors to benzodiazepine agonists and inverse agonists (Bolos et aL, 1993). Given the evidence that benzodiazepines, barbiturates, and ethanol regulate the function and expression of GABAA receptors in adult brain, it seems likely that prenatal exposure would alter the expression of GABAA receptors during development. Administration of benzodiazepines to preg-

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nant rats produces behavioral and physiological abnormalities in offspring (especiallyfollowing pharmacologic challenge), suggesting that alterations in GABAAreceptor function and/or expression have occurred (Kellogg, 1988; Schlumpf et al., 1989). In vitro studies provide further evidence that prenatal exposures to GABAergic ligands could alter expression of GABAA receptors. In cell cultures derived from embryonic chick or mouse brain, chronic exposure to GABA or muscimol downregulates expression of mRNA transcripts encoding crl and a2subunits. These effects are reversed by GABA antagonists (Montpied et al., 199la; Hirouchi et al., 1992; Calkin and Barnes, 1994). The benzodiazepine agonist, flunitrazepam, causes a similar decline in a, transcripts in rat brain and cultures neurons (Damschoder-Williamet al., 1990; Hirouchi et aL, 1992). Therefore, results of both in vivo and in vitro studies support the hypothesis that prenatal exposure to drugs or environmental neurotoxins that target GABA or benzodiazepine recognition sites on GABAA receptors could alter the developmental expression of GABAA receptor subunits and produce long-lasting behavioral abnormalities in offspring.

VIII. Ahemtions in Receptor Subunit Composition: A Novel Mechanism of Regulation of Ligand-Gated Ion Channels

The molecular cloning of ligand-gated ion channel receptors, including GABA, receptors, has elucidated several important concepts. Receptorgated ion channels are composed of multiple distinct subunits, derived from different genes, which are expressed in different combinations resulting in pharmacologically distinct isoreceptors. The subunit composition of these isoreceptors differs in various brain regions and during the course of normal ontogenesis. Recombinant expression studies for all of the ligand-gated ion channel receptors have clearly shown that the functional properties of these channels are determined by the subunit composition and that alterations in subunit composition can result in alterations in the biophysical or pharmacological properties of these receptors (Betz, 1990; Boulter et al., 1987).Regional studies using electrophysiology and in situ hybridization confirm the heterogeneity of neuronal nicotinic receptors (Mulle et al., 1991),GABAAreceptors(Seeburg et al., 1990),glutamate receptors (Boulter et al., 1987; Moriyoshi et al., 1991), and glycine receptors (Betz, 1991) in brain. Several investigators have suggested that the heterogeneity of GABAA receptor subunits accounts for the functional heterogeneity of GABAAreceptor binding and function (Schofield et al., 1987; Levitan et al., 1988). We have proposed that alterations in GABAAreceptor subunit composition,

REGULATION OF GABAA RECEPTOR FUNCTION AND GENE EXPRESSION

33

in vim, may result from adaptations to chronic drug exposure (Morrow et al., 1992a). This could result in alterations in the functional properties of receptors with no change in the total number of receptors expressed. Hence, GABAA receptors may be regulated by alterations in subunit expression which result in alterations in the function of receptors. This mechanism appears to play a role following chronic exposure to ethanol and possibly other drugs that modulate GMAAreceptors. There is ample evidence for alterations in GABAA receptor subunit composition during development (vide supra). Similarly, alterations in the subunit expression of nicotinic cholinergic (Imoto et al., 1986; Mishina et al., 1986), glutamate (Sheng et al., 1994), and glycine receptors (Kuhse et al., 1990; Malosio et al., 1991) have been observed. Therefore, we propose that ligand-gated ion channels may be subject to alterations in subunit composition which regulate receptor function as well as upregulation and downregulation, like other neurotransmitter receptors.

IX. Conclusions: New Breakthroughs, New Questions

Recent studies on the molecular biology of GABAAreceptors in the CNS have demonstrated remarkable heterogeneity in the expression of unique GABA isoreceptors (Olsen and Tobin, 1990). Given that multiple ( ( Y I - ~ Y ~ PI-& , 71-74, 6, p1 and p2) distinct polypeptide GABAA receptor subunits have been isolated and identified, the theoretical potential for multiple pentameric receptor subtypes is enormous. At the present time, the actual subunit composition and stoichiometry of GABAA isoreceptors expressed in the brain is not known. Detailed studies on the distribution of GABA,, receptor subunits throughout the brain will eventually elucidate the subunit composition of isoreceptors present in various cell populations. Furthermore, studies on the effects of various neuropharmacological agents on the expression of these subunits may elucidate how each of the subunits are regulated and how alterations in the expression of various subunits affect the function of various GABAA isoreceptors. The complexity of GABAA isoreceptors in the mammalian CNS may explain the diverse effects of chronic benzodiazepine, barbiturate, and ethanol administration on GA13AAreceptor binding sites. Identification of the actual binding sites for GABAA/benzodiazepineligands will help iden ti@ the GAB& isoreceptors/ subunits are labeled by each of the avaiIable radioligands. The application of pharmacological techniques for the classification of receptor subtypes will become increasingly important, once again, since the molecular cloning studies have revealed so much diversity in the structure of GABA,, receptors.

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Understanding the mechanisms which control the expression of various GABAA isoreceptors or their subunits may provide the most innovative approach to therapeutics in the next decade. Anxiety, epilepsy, depression, and other mood disorders may be ameliorated by interventions in the structure, function, and regulation of GABA,, receptors. The challenge of the next decade will be to identify both genetic aberrations and regulatory mechanisms for GABAA receptor genes which are correlated with disease states, the fundamental basis for potential gene therapy.

Acknowledgments

The author thanks Drs. Jean Lauder, Leslie Devaud, Dennis Grayson, and Rochelle Schwartz for critical review of the manuscript as well as helpful comments and suggestions. Drs. Steven Paul, Pascale Montpied, Peter Suzdak, and Robert Purdy are gratefully acknowledged for collaborations and permission to use figures. Support for the preparation of this chapter was provided by NIAAA Grants AA09013 and AA09122, the Pharmaceutical Manufacturers Association Foundation, and the UNC Center for Alcohol Studies.

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Schwartz, R. D., Skolnick, P., Seale, T. W., and Paul, S. M. (1986a). In “Advances in Biochemical Pharmacology” (G. Biggio and E. Costa, eds.), pp. 33-49. Raven, New York. Schwartz, R. D., Suzdak, P. D., and Paul, S. M. (1986b). Mol. Phannacol. 30, 419-426. Schwartz, R. D., Wess, M. J., Labarca, R., Skolnick, P., and Paul, S. M. (1987). Brain Res. 411, 151-155. Seeburg, P. H., Wisden, W., Verdoorn, T. A., Pritchett, D. B., Werner, P., Herb, A., Luddens, H., Sprengel, R., and Sakmann, B. (1990). Cold SpingHarbor Symp. @ant. Biol. 55,29-38. Segal, M. (1993). Hippocampus 3, 229-238. Sellers, E. M., and Kalant, H. (1976). N. En@. J. Med. 294, 757-760. Serafini, R., Ma, W., Tang, K., and Barker, J. L. (1993). Neurosci. Abstr. 19, 1146. Serra, M., Sanna, E., Concas, A,, Foddi, C., and Biggio, G. (1991). Neurocha. Res. 16, 17-22. Sheng, M., Cummings, J., Roldan, L. A,, Jan, Y. N., and Jan, L. Y. (1994). Nature (London) 368, 144-147. Sher, P. K., Study, R. E., Mazzetta,J., Barker, J. L., and Nelson, P. G. (1983). Brain Res. 268,171. Sigel, E., Baur, R., and Malherbe, P. (1993). FEES Lett. 324, 140-142. Sivam, S. P., Nabeshima, T., and Ho, I. K. (1982).J Neurosci. Res. 7, 37-47. Skolnick, P., and Paul, S. M. (1982a). In “International Review of Neurobiology” (J. R. Smythies and R. J. Bradley, eds.), pp. 103-140. Academic Press, New York. Skolnick, P., and Paul, S. M. (1982b). Int. Rev. Neurobiol. 23, 103-140. Stelzer, A., Kay, A. R., and Wong, R. K. S. (1988). Science 241, 339-341. Study, R. E., and Barker, J. L. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 7180-7184. Suzdak, P. D., Glowa, J. R., Crawley, J. N., Schwartz, R. D., Skolnick, P., and Paul, S. M. (1986a). Science 234, 1243-1247. Suzdak, P. D., Schwartz, R. D., Skolnick, P., and Paul, S. M. (198613).Proc. Natl. Acad. Sci. U.S.A. 83, 4071-4075. Suzdak, P. D., Schwartz, R. D., and Paul, S. M. (1987). Brain Res. 444,340-344. Suzdak, P. D., Paul, S. M., and Crawley, J. N. (1988).J. Pharmacol. Exp. Ther. 245, 880-886. Tehrani, M. H. J., and Barnes, E. M. (1988). Neurosci. Lett. 87, 288-292. Thalmann, R. H., and Hershkowitz, N. (1985). Brain Res. 342, 219-233. Thyagarajan, R., and Ticku, M. K. (1985). Brain Res. Bull. 15, 343-345. Ticku, M. K., and Burch, T. (198O).J.Neumchem. 34, 417-423. Ticku, M. K., Lorimore, P., and Lehoullier, P. (1986). Brain Res. Bull. 17, 123-126. Tietz, E. I., and Rosenberg, H. C. (1988). Bsain Res. 438, 41-51. Tietz, E. I., Rosenberg, H. C., and Chiu, T. H. (1986).J. Pharmacol. Exp. Ther. 236, 284-292. Tietz, E. I., Chiu, T. H., and Rosenberg, H. C. (1989). Eur.J Pharmacol. 167, 57-65. Treit, D. (1985). Pharmacol. Biocha. Behav. 22, 383-387. Turner, D. M., Ransom, R. W., Yang, J. S.-J., and Olsen, R. W. (1989).J. Pharmacol. Exp. Ther. 248, 960-966. Unwin, J. W., and Taberner, P. V. (1980). Neurophannacolugy 19, 1257-1259. Unwin, N. (1989). Neuron 3, 665-676. Verdoorn, T. A,, Draguhn, A,, Ymer, S., Seeburg, 1’. H., and Sakmann, B. (1990). Neuron 4,919-928. Viau, V., and Meaney, M. J. (1991). Endocrinology (Baltimore) 129, 2503-2511. Volicer, L. (1980). Brain Res. Bull. 5, 809-813. Volicer, L., and Biagioni, T. M. (1982a). Neurophanacology 21, 283-286. Volicer, L., and Biagioni, T. M. (198213). Subst. Alcoh,ol Actions/Mzsuse 3, 31-39. Wafford, K. A., and Whiting, P. J. (1992). FEBSLett. 313, 113-117. Wafford, K. A., Burnett, D. M., Dunwiddie, T. V., and Harris, R. A. (1990). Science249,291-293. Wafford, K. A., Burnett, D. M., Leidenheimer, N. J., Burt, D. R., Wang, J. B., Kofuji, P., Dunwiddie, T. V., Harris, R. A., and Sikela, J. M. (1991). Neuron 7, 27-33.

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GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

Robert Hitzemann, Yifang Qian, Stephen Kanes, Katherine Dains, and Barbara Hitzemann Department of Psychiatry and Behavioral Medicine, Pharmacology and Neurobiology and Behavior, SUNY at Stony Brook, New York 1 1794 and Psychiatry and Research Services, Veterans Administration Medical Center, Northport, New York 11768

I. Introduction 11. Genetic Strategies A. Inbred Strains B. Crosses and Backcrosses C. Recombinant Inbred Strains and Quantitative Trait Loci Analysis D. Selected Lines 111. Genetics and Behaviors Related to the Basal Ganglia A. Introduction B. Dopamine Agonists (Direct) C. Dopamine Agonists (Indirect) D. Dopamine Receptor Antagonists E. Morphine and Related Compounds F. Other Behaviors lV. Genetics and the Functional Architecture of the Basal Ganglia A. Number of Midbrain Dopamine Neurons: Midbrain and Striatal Tyrosine Hydroxylase (TH) Activity B. Steady-State Dopamine and Serotonin Levels and Turnover C. Neuropeptides D. Dopamine Receptors E. Neurotransmitter Transporters F. Striatal Cholinergic System G. Striatal GABA System(s) H. Enkephalins, Endorphins, and Opiate Receptors I. Weaver ( w u ) Mutant V. Conclusions References

1. Introduction

This chapter reviews the evidence for genetic factors contributing to variability in the functional, neurochemical, and neuroanatomical organization of the basal ganglia. Most of the citations reviewed involve comparisons INTERNATIONAL REVIEW OF NEUROBIOLOCY, VOL. 38

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Copyright 0 1995 by Academic Press. Inc. All rights of reproduction in any form rrsrrvrd.

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among inbred strains of mice or rats. Some of these studies simply ask the question of whether or not genetic factors have a significant influence on the phenotype of interest. For this work the strategy most often used is to compare the phenotypic means between two or more genetically distinct inbred strains. Some but not all phenotypes will show a difference. For example, BALB/c and LP mice have a significant difference in striatal D2 dopamine receptor density but the pattern of receptor organization (e.g., the rostral-caudal gradient) in these strains is identical (Kanes et al., 1993a). Other studies cited in this chapter have a more quantitative/mechanistic focus. Questions addressed include (1)What is the size of the genetic effect? (2) How many genes are involved? (3) Is there evidence of dominance and epistasis or is the heritability entirely additive? The methods used to answer these questions include selective breeding, large strain studies, or quantitative trait loci (QTL) analysis. Some background to these strategies is found under Section 11. The phenotypes reviewed in this chapter (e.g., receptor density, drug responses, dopamine neuron number) are quite complex. When such complex phenotypes are characterized in alarge panel of inbred stains or in genetically segregating population, the phenotypic values are almost always continuously distributed. To explain this observation, quantitative genetic theory argues that multiple genes, each with a modest effect, combine to produce the phenotypic variance. The number of genes need not be large; three or four genes, coupled with some environmental smoothing can produce a pseudonormal distribution (Lush, 1945). In comparison to the number of genes associated with most phenotypes (see, e.g., Edwards et al., 1987), this would be considered a relatively “simple” genetic picture; fortunately (from an experimental perspective) some of the phenotypes discussed in this chapter appear to be under the control of relatively few genes. As we began to survey the literature, it became clear that a review acknowledging every contribution would be difficult. For example, the search statement “inbred strain and dopamine” yielded >300 citations for the period of 1966-1994. Therefore, we decided to present a solid sampling of the literature which would illustrate the major advances. The choices of which citations to include and which to exclude were, of course, to some degree subjective. To any author whose work was not cited, we apologize. II. Genetic Strategies

A. INBRED STRAINS

The majority of the studies cited in this chapter involve comparisons among inbred strains of mice and rats. The advantage of inbred strain

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comparisons is that the results are cumulative and one can build upon results obtained years earlier. Inbred strains are generally formed by brother-sister mating for 20 generations, at which point the animals are nearly homozygous at all autosomal loci, such that all animals of the same sex are identical. Since different inbred strains will become homozygous for different alleles, differences among strains reflect genetic effects (assuming environmental effects are equal). When comparing different inbred strains, the usual strategy is to select the most genetically different. For many inbred mouse strains, the degree of relatedness or kinship has been calculated (see, e.g., Seale and Carney, 1991; Atchley and Fitch, 1992; Dietrich et aL, 1992). The data in Table I show the polymorphism rates for >300 simple sequence repeats (microsatellites) among seven commonly used inbred mouse strains. Both the C57BL/6 (B6) and LP strains differ 50% or more from the outer six strains. Thus, either of these strains would be appropriate for any two strain comparison. In contrast, the C3H strain is closely related to the DBA/2 (D2), A, and BALB/c (C) strains and, thus, is a less ideal candidate. The most common comparisons are between the B6 and D2 strains and the B6 and C strains. Frequently, for comparisons between B6 and C, two substrains are used, the BSBy and CBy, as these are the progenitors of the CXB recombinant inbreds (RIs) (see below). Although the B6 and B6By and the C and CBy are both closely related pairs, the differences are not trivial. For example, the polymorphism rate between the B6 and B6By strains is only about 1% (Roderick and Guidi, 1989); however, these strains differ markedly in some of their drug responses, e.g., to cocaine (Seale and Carney, 1991). The purpose of many inbred strain studies is not only to establish the existence of genetic differences but also to test hypotheses about relationships between traits, e.g., apomorphine-induced stereotypy and D2receptor

TABLE I POLYMORPHISM RATEOF SIMPLE SEQUENCE REPEATS AMONGSEVENINBRED MOUSESTRAINS (Mus MUSCULLJS)~

Strain B6 D2 A C3H C AKR LP

B6

D2

A

C3H

C

AKR

LP

38 44 54

47 49

57

-

51 53 51 49 52 57

46 35 45 48 53

32 46 51 55

“Adaptedfrom Dietrich et al. (1992).

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ROBERT H I T Z E M A ” et al.

density. Strain similarities on multiple traits suggest a common genetic influence. The power of these studies is determined by the number of different strains examined, not the total number of animals. Eight strains are necessary to detect a 50% shared variance, 25 strains are necessary to detect a 18% shared variance, and so on.

B. CROSSES AND BACKCROSSES When two strains are found to differ on the phenotype of interest, the next line of inquiry frequently addresses questions such as what is the heritability of the phenotype or what is the mode of inheritance? The strategy generally used builds upon Menclel’s approach of characterizing the parental, F1, and F4 generations for the phenotype of interest and is frequently termed the classical analysis (Plclmin et al., 1991b). The determination of heritability is instructive. All animals within the parental and F1 populations are genetically identical. The phenotypic variance within these populations provides an estimate of the environmental variance. Because of recombination which occurs during meiosis in the F1 population, each F2 animal is genetically unique. Phenotypic variance in the F2 population is due to both environmental and genetic factors. Since the environmental variance is known from the parental and F, animals, the genetic contribution in the F2 population can be calculated. Heritability is defined as the poportion of phenotypic variance due to genetic factors. This is heritability in the broad sense since it includes both additive and nonadditive components. The mode of inheritance can also be investigated using a classical design which frequently also includes backcrosses from the F1 generation to the parental strains. For a qualitative trait (present/absent) agreement is sought between the proportion of animals in each population showing the trait and the expected proportions for the model adopted, e.g., recessive, single locus. Collins and Fuller (1968) provided a particularly useful example of this strategy in their study of audiogenic seizures. The classical analysis can also be applied to quantitative traits, e.g., EDE0 values or enzyme activity, for which one assumes the involvement of multiple genes. In this context, one is attempting to determine the extent to which dominance or epistatic effects cause a deviation from a purely additive genetic model (see Falconer, 1989).

C. RECOMBINANT INBRED STRAINS AND QUANTITATIVE TRAITLOCIANAI.YSIS The formation of RI strains is illustrated in Fig. 1.The progenitor inbred strains are crossed to form F1animals. In this example, the progenitors are

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA Responder - DBAIPJ

47

Non-Responder - C57B1/6J

n

F2

n'n

D

R Rlll

F2 Generation Followed by 20 Generations of Brother-Sister Mating

Allele D' is Associated with Non-Response

n'n

m~*

NR RllZ

FIG.1. Schematic illustration of the formation of BXD recombinant inbred strains. In this example, the D2 strain is considered a responder for some phenotype and the B6 strain is considered a nonresponder.

the D2 and B6 strains. All F, animals of the same sex are genetically identical and heterozygous at all alleles where the B6 and D2 strains differ. As noted previously (Table I), the polymorphism rate between these strains is approximately 50%. Families are formed from the F2 animals, followed by brother-sister mating for at least 20 generations. On average, there are approximately four recombinations per chromosome during this process. Overall, this strategy results in the formation of RI strains. In this chapter, citations will be made to two RI series: the CXB series, formed from B6By and CBy strains, and the BXD series, formed from the B6 and D2 strains. Currently, there are 7 strains in the CXB series and 26 strains in the BXD series. These and other RI series were originally developed to detect single gene effects (Bailey, 1971, 1981; Taylor, 1978). The influence of a single major gene is inferred whenever a bimodal distribution among the strain means is observed, with one progenitor strain in each mode (Crabbe and Belknap, 1992; Bailey, 1971; Gora-Maslek et al., 1991). The expected ratio

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ROBERT HITZEMAh'N et al.

of the two modes is 1 : 1 since all animals are homozygous. In the example, RI strain 1 has the D allele and is a responder like the D2 strain, while RI strain 2 has the D' allele and is a nonresponder like the B6 strain. When there are sufficient RI strains, the trait distribution pattern can be used for linkage detection in gene mapping. For both the CXB and the BXD series, the strain distribution patterns (SDPs) are known for a large number of marker loci previously mapped to a particular chromosome region and known to show allelic differences between the progenitor strains. For the BXD series, there are currently >1300 such markers. The SDP for the trait is compared with the SDPs for the marker loci. A match suggests that the gene of interest is closely linked to the marker locus. RI series can also be used to map the multiple loci associated with quantitative traits (those which will show a continuous and essentially normal distribution among the RI strains) (reviewed in Plomin and McClearn, 1993).For this analysis, it is assumed that there are multiple genes involved, each with a relatively modest effect size. Thus, a relatively large RI series is required to obtain the necessary statistical power. For example, the full BXD series (26 strains) can detect a QTL associated with 16% of the phenotypic variance. Although one makes hundreds of comparisons in generating QTL data because of linkage disequilibrium caused by the inbreeding to form the RI strains, the actual number of independent observations is probably <100 (Belknap, 1992). Still, some of the QTL will be significant due to chance. This problem has been discussed in some detail by Neumann (1992) and Belknap (1992). Although these authors have a somewhat different perspective, both are in general agreement that if the RI-QTL approach is used alone, a Pvalue of at least 0.001 will be required. For the full BXD series, this would require a QTL associated with nearly 40% of the variance! Since detecting such a QTL is unlikely, an alternative use of RI data has been suggested by Belknap (1992), Plomin and McClearn (1993), and Buck et al., (1994). These authors argue that the BXD data could serve as a screen to identify candidate map locations which are then confirmed in other RI series, standard inbred (non-RI) strains (Goldman et al., 1987), congenic lines (Bailey, 1981), or by linkage analysis in F2 or backcross populations (Lander and Botstein, 1989;Rise et al., 1991);Neumann and Collins, 1991). When such confirmation testing is done, then P < 0.01 (single test) is probably appropriate for the RI results, since it yields lower type I1 error rates (failing to detect important map sites). Assuming the number of independent tests is
GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

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will be P < 0.0001. This level of significance is similar to that used for classical linkage analysis in human genetics.

D. SELECTED LINES Selective breeding is a technique as old as agriculture and animal husbandry. For issues of neurobiological interest, selective breeding has been widely used in the area of pharmacogenetics, in particular to study the mechanisms of ethanol action (e.g., McClearn and Kakihana, 1981; Crabbe et al., 1985; Phillips et al., 1989, 1994; Crabbe and Belknap, 1992). Most selections begin with a genetically heterogenous stock, formed by crossing several inbred strains. The most widely used is the heterogenous stock (HS) maintained at the Institute for Behavioral Genetics in Boulder, Colorado. The usual practice for selective breeding studies is to change the population mean in opposite directions, i.e., for both high and low extremes concurrently (bidirectional selection). When selection studies are properly done and inbreeding is minimized, the differences between selected lines can be assumed to be due to differences only in the allelic frequency of those genes relevant to the phenotype of interest. Furthermore, selection line differences in other phenotypes (correlated responses) should be due to the effects of the same genes which regulate the selection phenotype. Selected lines have been particularly useful in demonstrating the important role(s) of cerebellar Purkinje cell sensitivity, the GABA/CI-/benzodiazepine complex (GBRC) and the T~~ subunit of the GBRC in the mechanisms of ethanol action (Harris and Allan, 1989; Palmer et al., 1982; Wafford et al., 1991). Further details of the use of selected lines are found in Falconer (1989) and Plomin et al. (1991b).

111. Genefics and Behaviors Related to the Basal Ganglia

A. INTRODUCTION A significant proportion of the citations cited in this chapter focus on the role of genetic factors in behaviors thought to be generated in part from the basal ganglia. Not surprisingly, given the function of the basal ganglia, it is some aspect of motor behavior that is most frequently studied. Further, many of the studies use a pharmacological challenge to elicit the behavioral response. A major advantage of this approach is that one can easily generate the large number of animals required for genetic studies.

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ROBERT HITZEMA" et al.

However, a major disadvantage is that most drugs have multiple sites of action and a precise interpretation of the results may be difficult. In, addition, one must pay attention to the possibility that genetic differences may relate to differences in drug metabolism rather than differences in the sensitivity of the basal ganglia (see, e.g., Weiner and Reith, 1990).

ACONISTS(DIRECT) B. DOPAMINE In mice, the most frequently observed behavioral response to the administration of dopamine (DA) receptor agonists is a decrease in spontaneous locomotor activity. Some investigators have termed this hypolocomotion, catalepsy. However, the term catalepsy is more generally reserved for the behavioral response to DA receptor antagonists. In comparison to the B6 strain, the D2 strain is more sensitive to the hypolocomotion induced by apomorphine (a mixed DI/D2 receptor agonist)' (Vetulani et al., 1982; Seale et al., 1984a;Skrinskaya et al., 1992),quinpirole (a specific D2 receptor agonist) (Puglisi-Allegra et al., 1990a; Skrinskaya et al., 1992), and SKF 38393 (a specific D, receptor agonist) (Skrinskaya et al., 1992). The C strain resembles the D2 strain for the response to apomorphine and quinpirole but is similar to the B6 strain in response to SKF 38393 (Skrinskaya et al., 1992). A discrimination along the genetic dimension between Dl and D:, receptor effects was also observed by Nikulina et al. (1991a); these authors noted that in a genotype-dependent manner, SKF 38393 increased activity in the forced swimming test while quinpirole decreased activity. Fink and Reis (1981) found the CBA strain more sensitive than the C strain to apomorphine-induced stereotypy. Seale et al. (1984a) noted the behavioral specificity to the strain differences. In contrast to the hypolocomotion response, the B6 strain was more sensitive than the D2 strain to apomorphine-induced gnawing and there was no significant difference between strains in the sniffing response. For quinpirole-induced defensive behavior, the rank order of decreasing sensitivity is B6 > C > D2 (Cabib and Puglisi-Allegra, 1989). Cabib et al. (1985) found that repeated stressful experiences sensitized D2 but desensitized B6 mice to apomorphineinduced climbing. Genetic analysis involving F1 and F2 hybrids and the backcross populations indicated complete dominance of the B6 phenotype. Jinnah et al. (1992) found that HPRTdeficient mutant mice (a putative model for the Leysch-Nyhan syndrome) are not significantlydifferent from normal littermates in their response to apomorphine and SKF 38393.

' Beginning here and throughout the chapter, refers to the family of D1 receptors (D, and D5) and D4 refers to the family of DYreceptors (D2, D,, and D4) unless otherwise noted.

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Among rats, the F344 strain is more sensitive than the Buffalo strain to apomorphinerinduced locomotion (Helmeste, 1983). The Roman highand low-avoidance rate lines (RHA/Verh and RLA/Verh) were selectively bred on the basis of rapid acquisition of the two-way conditioned avoidance response versus the failure to acquire that response (see Driscoll et al., 1986 and references therein). The RHA/Verh rats have a higher turnover of striatal DA and are more sensitive than the RLA/Verh line to apomorphineinduced stereotypes.

C. DOPAMINE AGONISTS (INDIRECT) Genotype-dependent effects of cocaine, amphetamine, and related compounds are well described. Satinder et al. (1970) noted marked differences among four inbred strains ( A , C, SJL, and SWR) in their response to & amphetamine as measured on factors of emotionality. Studying the locomotor hyperactivity induced by a relatively high dose of &hetamine (5 mg/kg), Moisset and Welch (1973) found the C57BL/10 (B10) strain significantly more sensitive than the C strain; the C strain responded with hyperactivity only after 5 days of treatment, probably reflecting drug sensitization. Different from the C strain, the A strain showed a robust locomotor response to acute amphetamine administration but no sensitization with chronic drug treatment (Short and Schuster, 1976). Remington and Anisman (1976) noted that there were developmental differences in the amphetamine response among A, D2, and B6 strains. However, by Day 28, the responses to a 5 mg/kg challenge were relatively similar. Castellano et al. (1976) found the B6 more sensitive than the D2 strain to amphetamineinduced increases in locomotor activity. Moisset (1977a,b) examined the ambulatory and rearing responses to &hetamine in three closely related inbred strains, B6, B10, and B6By. The B6 strain showed the typical inverted U dose-response curve to amphetamine with a peak response at 5 mg/kg; the B10 strain appeared similar, although this strain was not tested at the highest dose (10 mg/kg). In contrast, the B6By strain showed a decrease in locomotor activity at 2 mg/kg and only a modest increase at 10 mg/kg. Rearing activity was depressed by amphetamine with the B6 strain the most sensitive, the B6By the least sensitive, and the B10 strain intermediate. Analysis of F, crosses and backcrosses among these strains suggested that different gene (s) regulate increased ambulation and decreased rearing. Helmeste and Seeman (1982) examined amphetamine responses in eight inbred strains. At low doses (0.5 and 1 mg/kg, ip) amphetamine increased activity in the B6 and SEC/lRe strains but had no effect on the D2 and CBA strains; all four strains showed a similar increase of activity at 5 mg/

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kg. In contrast, the C, A, and C3H strains showed a decrease of activity at the low doses and an increase at the 5 mg/kg dose. These authors noted that the strains exhibiting the hypolocomotion response had a higher density of D2 receptors. Fink and Reis (1981) found the C strain more sensitive than the CBA strain to both locomotor and stereotypy responses over a wide range of doses (2-20 mg/kg); it was suggested that these differences may be associated with the higher density of DA neurons in the C strain. Kitahama and Valatx (1979a,b) found that 1 mg/kg methamphetamine induced hyperactivity in B6, C57BR, and SEC strains, but it induced hypoactivity in three albino strains (A, C, and AKR). Oliverio et al. (1973) examined the response to damphetarnine (0.52.0 mg/kg) in the seven RI strains of the CXB series, the reciprocal F, hybrids, and the B6By and CBy parental strains. Locomotor activity was measured in a modified “jiggle box” apparatus. Under these conditions and over the dose range studied, amphetamine increased activity in the B6By strain but decreased activity in the CBy strain. The reponse of the RI strains was not distributed in a bimodal fashion and a polygenetic model was suggested. Using an open-field apparatus and an amphetamine dose of 5 mg/kg, Moisset (1977a,b) obtained results which differed significantly from those of Oliverio et al. (1973). This author found that, among the entire CXB RI series, the parent strains, and the reciprocal F, hybrids, the acute administration of damphetarnine had no significant effect on locomotor activity. In contrast, this dose of damphetarnine decreased rearing activity in the B6By strain, both F1 crosses, and three of the seven RI strains. Moisset (1977a,b) concluded that, while other explanations are possible, these data are consistent with the effect of a single major gene, where the B6By allele is autosomal dominant. Using a similar experimental design, Jeste et al. (1984) examined the effects of phenethylamine (PEA) on ambulation. The BGBy, but not the CBy, strain showed a decrease in ambulation after PEA administration; four of the seven RI strains showed the hypolocomotion response, while three were unaffected. Like Moisset (1977a,b),Jeste et al. (1984) concluded that the PEA response was largely under the control of a single gene. Methamphetamine-induced locomotor stimulation has also been examined in the large (26 strains) BXD RI series (Belknap, 1993; C. Cunningham and Duerr, unpublished observations, 1994). The QTL analysis of these data has been summarized (Crabbe et aL, 1994). The results suggest the behavior is under the control of multiple genes; interestingly, significant QTL ( P < 0.01) are found near the tyrosine hydroxylase (7%)and D4receptor (Drd4) genes on chromosome 7 and the D2 receptor gene (Drd2) on chromosome 9. Among rats, genotype-dependent responses have also been noted. Driscoll et al. (1986 and references therein) rioted that the RHA- compared to

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

53

the RLA-selected rat lines are more sensitive to the effects of amphetamine on maze exploration, shuttle box performance, and stereotyped behavior. George et al. (1991) examined the locomotor response to amphetamine among four inbred rat strains, ACI, F344, LEW, and NBR. At doses less than 10 mg/kg, the LEW strain was considerably less responsive than the other three strains. Importantly, this difference in sensitivity was not due to a decrease in ambulation resulting from stereotypy confounds. Shuster et al. (1977) examined the effect of cocaine (20 mg/kg) on locomotor activity in the CXB RI strains, the reciprocal F1hybrids, and the B6By and CBy parental strains. These authors found that, compared to the CBy strain, the B6By strain was more sensitive to the effects of cocaine. This observation has been confirmed by Weiner and Reith (1990) and Seale and Carney (1991). Shuster et al. (1977) also noted that the sensitization response to four daily injections of cocaine was greater in the B6By strain. Analysis of the reciprocal F1 crosses suggested the presence of maternal effects. The BXCFl cross was more sensitive than the CXBFl cross to the acute effects of cocaine but showed no drug sensitization, while the CXBF1. cross showed a marked sensitization response. Among the RI strains, there was significant variability in both the acute response and the degree of sensitization, suggesting the influence of multiple genes. Interestingly, there are marked differences in the cocaine and amphetamine strain distribution patterns for the CXB RI series (see Oliverio et al., 1973; Moisset, 1977a,b). Ruth et al. (1988) examined the effects of cocaine among four inbred strains (C, B6, C3H, and D2). Locomotor activity was measured on a Ymaze apparatus. Cocaine produced a dose-related increase of activity in the C3H, B6, and D2 strains, with the C3H strain being the most sensitive. The B6 and D2 strains were relatively similar in their responses and no response was noted in the C strain. Ruth et al. (1988) also examined ['HIcocaine uptake among these strains and concluded that brain uptake did not account for the genotypic differences. In contrast, Weiner and Reith (1990) found a generally good correlation between locomotor response and uptake in four different mouse strains (cBy, BGBy, CXBH, and CXBK) . Seale and Carney (1991) found, compared to the B6 strain, the D2 strain was somewhat more responsive to cocaine-induced motor activity. However, the rewarding effects of cocaine as measured by conditioned place preference (CPP) were significantlygreater in the B6 strain. A comparison among four strains (B6, BGBy, CBy, and D2) suggests that CPP and locomotor activity are under the control of different genes. The QTL for cocaineinduced locomotion have been mapped using the SDP obtained from the BXD Rl series (Tolliver et al., 1994; Crabbe et al., 1994). The distribution of these QTL differs markedly from those for methamphetamine (see above

54

ROBERT HITZEMANN el al.

and Crabbe et al., 1994); interestingly, significant QTL are found near the D3 DA receptor gene (Drd3) on chromosome 16 and near the serotonin l b receptor gene (Htrlb) on chromosome 9. Between the NBR and F344 rat strains, both George (1990) and Witkin and Goldberg (1990) have shown that the NBR strain is more sensitive to the stimulant effects of cocaine. George (1990) also found the NBR strain to be more sensitive to the lowdose, depressant effects of cocaine. In a subsequent study, George et al. (1991) extended these investigations to include two additional strains, ACI and LEW. Again, over the dose range studied (5-60 mg/kg), the NBR strain was the most sensitive and the F344 was the least sensitive; the sensitivity of the ACI and LEW strains was somewhat intermediate. Furthermore, these authors noted that among those four rat strains there was no correlation between the cocaine- and amphetamine-induced locomotor stimulant effects. Kosten et al. (1994) also noted the difference in locomotor stimulant effects between LEW and F344 rats. Further, this and other groups (see references in Guitart et al., 1993a) have found that LEW rats self-administer cocaine to a greater extent than F344 rats.

D. DOPAMINE RECEPTOR ANTAGONISTS Both D, and D2 DA receptor antagonists induce an immobilization response in mice and rats that is generally termed catalepsy. Evidence that this response is functionally equivalent t.0 the clinical extrapyramidal symptoms induced by neuroleptic administration is summarized elsewhere (Hitzemann el al. 1991, 1993). In some but not all strains of mice, e.g., D2 but not B6, the catalepsy is preceded by marked hyperexcitability (R. Hitzemann, Y. Qlan, S. Kanes, K. Dains, and B. Hitzemann, unpublished observation, 1994). Fink et al. (1982) noted that the C strain was more sensitive than the CBA strain to haloperidol-induced catalepsy. In contrast, Severson et al. (1981a) found the CBA strain to be more sensitive. Messiha (1991a) observed that the C strain was more haloperidol sensitive than the B6 strain and noted that this difference was associated with a higher turnover of DA in the C strain. Dudek and Fanelli (1980) found that in comparison to the long sleep (LS) selected mouse line, the short sleep (SS) line is more haloperidol sensitive. Kinon and Kane (1989) observed that the F344 rat is haloperidol catalepsy sensitive while the Brown Norway rat is catalepsy resistant. In contrast to the acute differences, there was no difference between strains in the hypersensitivity which develops after chronic haloperidol administration. Hitzemann et al. (1991, 1994) have reported in two independent studies

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

55

that HS mice (formed from a cross of eight inbred strains) can be selectively bred to produce neuroleptic responsive (NR) and neuroleptic nonresponse (NNR) lines. The original selection of the NR and NNR lines was rapid, such that by the sixth selected generation, the lines differed 10-fold in their haloperidol ED50. Although haloperidol was the selection agent, these lines differed similarly in their response to all typical neuroleptics (e.g., fluphenazine and raclopride) with a high D, to D1receptor affinity ratio. In contrast the lines differed less (2 to %fold) in their response to neuroleptics, such as chloropromazine, with a low D2 to D1 receptor affinity ratio. The lines showed no difference in their response to the specific D1 receptor antagonist, SCH 23390. Correlated responses to the selection of the NR and NNR lines include increased D2somatodendritic receptor density in the NNR line and increased striatal cholinergic cell number in the NR line (Hitzemann et al., 1991, 1993; Qian et aL, 1992). Kanes et al. ( 1993a) found that there are marked differences in sensitivity to haloperidol-induced catalepsy among 8 inbred strains. The C strain was the most sensitive (ED50= 0.3 mg/kg), while the LP strain was the least sensitive (ED,o = 9.5 mg/kg). There was no significant difference among the strains in brain haloperidol levels at the time after injection that catalepsy was measured (15 min). Further, the lines showed no significant difference in response to SCH 23390. Preliminary haloperidol EDSodata have been obtained on 7 additional strains. A summary of the data for all 15 strains is provided in Fig. 2. Overall, these data show that among inbred strains of mice the range of variation in the haloperidol EDs0is approximately 30-fold. The data in Fig. 3 show the haloperidol EDSovalues for the 26 strains of the BXD RI series. These data are the average of three or four separate determinations for all strains except strains 8, 13, and 18 (two determinations) and strain 20 (one determination). Our current estimate of overall sample reliability is 0.8. No BXD strain was more sensitive than the D2 strain but 3 strains (2, 23, and 29) appear to be less sensitive than the B6 strain. The frequency distribution of the strain means was not significantly different from normal = 2.36, DF = 3, P > 0.50) and, thus, multigenetic control is inferred. The estimated heritability (additive) for haloperidol-induced catalepsy for this RI series is 0.63 and the estimated number of effective loci is four (see DeFries et al., 1989). Thus, on average each loci could account for 16% of the variance in response. Fifteen or 16% is the lower limit of effect size that can be mapped in a 26-strain RI series. The strain means for catalepsy were compared with the strain distribution patterns of >1300 loci whose chromosomal location is known and which show allelic differences between the B6 and D2 strains. The candidate QTL generated from this analysis are summarized in Table 11.

(a

56

ROBERT HITZEMANN et al.

11

10 9 8

*

m 7 F 6 I

zn 5 w 4

3

2t------AKWcrl

WJ

SJUJ

AN

CS7B1/6crl CBUJ

LP/J

Strains FIG.2. Average EDSovalues for haloperidol-induced catalepsy among 15 inbred mouse strains. Details of the experimental procedures are found in Hitzemann et al. (1991). Data are taken from Kanes el al. (1993a) and unpublished observations. The authors thank J. Belknap and C. O'Toole for the data o n the PL/J, SJL/J, CE/J, A/HeJ and C57L/J strains. J, Jackson Laboratory; crl, Charles Rivers Laboratoiy.

Significant QTL ( P < 0.01) were found on six chromosomes. Using 88 F2 B6D2 hybrids phenotyped for haloperidol response and genotyped for the markers of interest, QTL on chromosomes 4 and 15 were confirmed at P < 0.05 and a QTL on chromosome 9 was confirmed at P < 0.01. A QTL on chromosome 6was not confirmed. A QTL on chromosome 9 is important in that it appears to be either near or part of the D2 receptor gene (Drd2). No significant QTL were found near the other DA receptors, the DA transporter, or tyrosine hydroxylase (see Fig. 4). Other behaviors which map to the region of chromosome 9 near Drd.2 include several alcoholrelated behaviors (consumption of 10% ethanol, ataxic sensitivity and tolerance, tolerance to ethanol hyperthermia, and conditioned place preference) and two methamphetamine-related behaviors (consumption in saccharine and locomotion) (Crabbe et aL, 1994).

57

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

FIG.3. Strain distribution pattern for haloperidol-induced catalepsy in the BXD RI series. For the BXD strains, data are the average of three or four determinations except for strains 8, 13, and 18 (twodeterminations) and strain 20 (one determination). Data for the parental D2 and B6 strains are provided for comparison.

TABLE I1 SIGNIFICANT ASSOCIATIONS BETWEEN BXD/Ty MARKERAND HALOPERDIOL~NDUCED CATALEPSY" Locus

Chr(cM)

r

P

D2Mit6 I2yB2, LyB4, D4McC2, D4Bir5, D4Mit17, lapls 1-10, lJa D6Mit9 D2dr2, D9Bir1, Ncam, D9Mit2I Ms6-5 DISMitl, D15Mit2

2 ~ 3 ) 4(23-43) 6 ( 39) 9(28-31) 14(20) 15(45-48)

-0.54 -0.59 -0.61 0.55 0.60 -0.65

0.006 0.002 0.003 0.004 0.002 0.0004

The BXD RI strain means for haloperidol-induced catalepsy were correlated with the SDPs of 1300 marker gene loci, each mapped to a chromosome region and showing allelic differences between the B6 and D2 strains. Only correlations significant at P < 0.01 are presented. On chromosomes 4, 9, and 15 there were multiple significant-markers. Multiple regression analysis revealed only a single QTL on each of these chromosomes. Results are presented for the underlined marker.

58

ROBERT HITZEMA” el al.

Cchl2a

Atpla3

Obcam Dat-1

Gabra-5 c, Tyr

Acra-4 Acra-5

Htrlb Acra-3 Gnai-2 Gnai-1

Hras 7

Htla

Scn5a

Drd4 Ache

5

7

9 13 Chromosome

FIG. 4. Murine location of genes for the dopamine receptors DM, the dopamine transporter-1, and tyrosine hydroxylase. Other genes of neurobiological interest have also been included, e.g., Htrlb (chromosome 9) and Wu (chromosome 16).

E. MORPHINEAND RELATED COMPOUNDS The genotype dependence of the morphine-induced “running fit” has been widely studied. The B6 strain shows a significant response to morphine, while the D2 strain is either unresponsive or shows a dose-related decrease in activity (Castellano et al., 1975; Kempf et aL,1976; Collins and Whitney, 1978; Racagni et al., 1979; Sansone and Oliverio, 1980; Frischknecht et al., 1988; Gwynn and Domino, 1984; Muraki and Kato, 1985; Belknap et al., 1989). Interestingly, morphine increases DA release in the B6 but not in the D2 strain (see, e.g., Trabucchi et al., 1976; Racagni et al., 1979). Kempf et al. (1976) noted that, in comparison to the D2 strain, the C strain is relatively morphine responsive. Sansone and Oliverio (1980) found that chlordiazepoxide enhances morphine-induced stimulation in both the B6 and C strains and reverses the depression activity in the D2 strain. Belknap et al. (1989) both summarized and extended observations on the behavioral specificity of the genotypedependent effects. Comparing the B6, D2, and C3H/He strains, these authors found the D2 strain most sensitive for analgesia, antidiuresis, and hypothermia (see also Muraki and Kato, 1985). In addition, the D2 strain had lower concentrations of morphine in the brain 30 min after injection and the lowest & and the highest &,,, for specific naloxone binding. The B6 strain was the most sensitive

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

59

when locomotor activity, Straub tail, and constipation were measured. The C3H/He strain was generally intermediate in sensitivity. Belknap et al. (1990) subsequently noted that the increased sensitivity of the D2 strain for analgesia is dependent on the technique for measuring this response. Suzuki et al. (1991) noted the genotype dependence of the precipitated withdrawal “jumping” response. The B6 strain was more sensitive than the D2 and C3H/He strains; however, on other dimensions of withdrawal, such as ptosis and diarrhea, the strains were not different. The drug specificity of the “narcotic” response is of genetic interest. Middaugh and Zemp (1976) found that methadone decreased locomotor activity in the D2 strain and slight4 increased activity in the B6 strain. After seven daily injections, increased activity in the B6 strain became more pronounced, while activity in the D2 strain returned to normal. Frigeni et al. (1978) noted that the icv injection of met-enkephalin or D-Ala2met-enkephalin induced marked analgesia in both the B6 and D2 strains but no locomotor response. Michael-Titus et al. (1989) examined the effects of acetorphan, a parentally active enkephalinase inhibitor on the B6 and D2 strains. Acetorphan induced dose-dependent, naloxone-reversible analgesia in the D2 but not the B6 strain. However, acetorphan induced locomotion in both strains, although the D2 strain was more sensitive than the B6 strain. Gwynn and Domino (1984) compared the effects of morphine and ethylketocyclazocine (EKC) in B6 and D2 mice. Unlike morphine, EKC (a K receptor agonist) induced a biphasic catalepsy-stereotypy in both strains. These authors further noted that there was no cross-tolerance between the morphine and EKC effects. Gorris and Van Abeelen (1981) examined the effects of naloxone (4 and 8 mg/kg) on behavioral responses to novelty induced by placing mice in an open field. Four inbred strains were examined, SRH, SRL, B6, and D2. In general, all four strains showed a similar naloxone response on nine different behavioral measures. However, the D2 and SRH strains were more sensitive to the decrease in leaning and sniffing frequency. Through the use of congenic lines, Katz (1980) examined the effects of the c, Tyr locus on the running response. Albino lines were consistently less responsive than the closely related pigmented lines to the effects of morphine. The pleiotropic effects of the c locus on motor activity and other phenotypes have been well established (see references in Katz, 1980; also Plomin et al., 1991a). Shuster et al. (1975) examined the running fit in the CXB RI series. These authors noted that between the parent strains, the B6By strain was more sensitive than the CBy strain. Analysis of the data among the RI strains and the reciprocal F, hybrids suggested to these authors that a single gene controls the running response. Interestingly, Belknap and Crabbe (1992)

60

ROBERT HITZEMA” et al.

reached a similar conclusion in their analysis of morphine-induced running activity the BXD RI series. In this RI series, it is depression of locomotor activity that is most commonly seen. From both the analysis of the D2 and B6 strains (reviewed in Belknap et al. (1989) and the CXB RI series, many authors have concluded that the running fit and analgesia are genetically dissociated. However, among the BXD RI series, the data suggest that analgesia and the running fit have common genetic influences, resulting in a significant negative genetic correlation (-0.62, P < 0.01) (Belknap and Crabbe, 1992). In contrast to the results of Shuster et al. (1975) and Belknap and Crabbe (1992), Oliverio and Castellano (1975) concluded from their analysis of the running response in the CXB RI strains that more than two loci were involved. Further, for 10 mg/kg of morphine, there was no significant correlation between the running response and analgesia ( r = 0.25). The correlation was based on the data obtained for the seven RI strains, the two parental strains, and the reciprocal F1hybrids.

F. OTHER BEHAVIORS Oliverio et al. (1973) examine the locomotor response to scopolamine in the CXB RI series, the parental strains, and the reciprocal F1 hybrids. Over the dose range studied (2-6 mg/kg) the animals segregated into two groups. One in which locomotor activity increased and included the C/By parent and one in which activity decreased and included the B6/By parent and both of the F, crosses. These data suggest that the scopolamine response is under the control of a single gene with a dominant allele. Seale et al. (1984b, 1986) examined the effects of caffeine and other methylxanthines on locomotor activity among inbred strains of mice. B6, SWR, D2, and A strains were markedly sensitive to the caffeine-induced inhibition of locomotor activity. C3H and C strains were significantly less sensitive, and in the CBA strain, locomotor activity was actually simulated at the lowest doses (94 and 125 mg/kg:) tested. An analysis of the SWR and CBA strains and their F1 hybrids revealed that the caffeine-induced locomotor responses were probably under the control of multiple genes (Seale et al., 1986).Jarvis and Williams (1988) found that between the SWR and CBA strains there is no difference in striatal A, receptors but in both the striatum and olfactory tubercle, A2 receptor density is significantly higher in the CBA strain (59 and 70%, respectively). Kulikov et al. (1993) examined “pinch” induced catalepsy among nine inbred strains of mice, a behavior the authors suggested was related to the striatal DA system. Four strains showed no response: B6, AKR,DBA/1, and CC57BR. In four strains there was a modest percentage (10-30%) of

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

61

cataleptic animals: C3H >A = C > DD. In one strain, CBA/lcg, the percentage of cataleptics was high (56%).Examination of crosses between the CBA strain and the AKR strain suggested to these authors that the catalepsy response is controlled by a single recessive allele. It has been suggested that the pronounced cataleptic response in the CBA/lcg strain is in part related to high tryptophan hydroxylase (TPH) activity in this strain. S u p porting this argument, Kulikov et al. (1992) found that in animals selectively bred for pinch induced catalepsy, TPH activity was significantly higher in the striatum of the responsive line. This increase in enzyme activity was thought to be associated with an increase in local phosphorylation. The coordinated increase in locomotor activity induced by phencyclidine (PCP), and ketamine has long been associated with changes in DA synthesis and release (e.g., Hitzemann et al., 1973). Filibeck and Castellano (1980) examine the effects of ketamine on locomotor activity in C, B6, and D2 inbred strains. In the C and B6 strain ketamine increased activity at doses below those necessary to induce antinociception; in the D2 strain, activity was depressed at doses higher than those necessary for antinociception. Freed et al. (1984) examined the PCP response in the CXB RI panel, the parent strains, and the reciprocal F, hybrids. The distribution of the strain means suggested the involvement of multiple genes. Further, there was no correlation between the PCP strain distribution pattern and that previously established for amphetamine or scopolamine. Liljequist (1991) compared the effects of PCP, MK-801, and ketamine (noncompetitive NMDA receptor antagonists) with the competitive antagonists (CGP 39551, CGS 19755, and NPC 12626) in CBA, B6, and the outbred NMRI strain. The CBA strain was most sensitive to the locomotor effects of both the competitive and noncompetitive NMDA antagonists followed by the NMRI strain and then the B6 strain. The loss of the D2DA receptors and the accompanying loss of motor control are among the most consistent and best characterized manifestations of central nervous system senescence (see, e.g., Mesco et al., 1993, and references therein). Numerous studies have documented an age X inbred strain interaction for cognitive and motor performance (e.g., Goodrick, 1975a,b, 1978; Wax and Goodrick, 1975; Sprott, 1975; Sprott and Eleftheriou, 1974; Elias et al., 1975; Everett, 1977; Ingram et al. 1981; Fuhrmann et al., 1986a). Lhotellier and Cohen-Salmon (1989) and Lhotellier et al. (1993) collected nine measures of exploratory and ambulatory activity for C, B6, and D2 female mice at three ages (150, 400, and 750 days). For five of the nine measures there was a significant age X strain interaction. However, these authors also noted that within each of the groups, the traits show differential age effects. Some behaviors are markedly affected by age while

62

ROBERT HITZEMA” et al.

others show little or no effect. The authors conclude that these data “argue convincingly for the rejection of a general factor of aging. . . .” There is an extensive genetic literature on the locomotor-activating effects of low-dose ethanol administration. Brain DA systems are thought to have an important role in this response (e.g., Dudek and Fanelli, 1980; Kiianmaa and Tabakoff, 1983). In general, ethanol has been found to increase activity in both D2 and C mouse strains but decreases activity in the B6 strain. A comparison of this lowdose ethanol effect among 19 inbred mouse strains has appeared (Crabbe, 1986). In addition, lines of mice have been selectively bred (FAST/SLOW) for the ethanol locomotor response (Crabbe et al., 1987). Interestingly, the genes which regulate sensitivity to the stimulating effects of ethanol appear to be at least in part different from those which regulate the sedative/intoxicating effects (see, e.g., Phillips and Dudek, 1991; Crabbe et al., 1994).

IV. Genetics and the FunctionalArchitecture of the Basal Ganglia

A. NUMBER OF MIDBRAIN DOPAMINE NEURONS:MIDBRAIN AND STRIATAI, TYROSINE HYDROXILASE (TH) ACTMTY It is well established that there are marked differences among inbred strains of mice in the activity of the catecholamine-synthesizing enzymes (see, e.g., Ciaranello and Boehme, 1981, and references therein). For example, Ciaranello et al. (1974) observed that in comparison to the CBA strain, the C strain has significantly higher TH activity in both the adrenals and brain. Ross et al. (1976) found that the ratio of TH activity between these strains is 0.55,0.76,and 0.73 in the substantia nigra, caudate nucleus, and olfactory tubercle, respectively. In contrast, no strain difference in TH activity was found in the locus coeruleus, demonstrating the genetic independence of the brain DA and norepinephrine (NE) systems. Ross et al. (1976) also observed that the number of DA neurons in the substantia nigra of the CBA strain was only 50%of the number found in the C strain; thus, there was no difference in cellular specific activity between strains. In a series of subsequent papers, Reis and colleagues continued their investigations on the genetic control of midbrain DA neurons in the CBA and C strains (Baker et al., 1980, 1982; Sved el al., 1984; Vadasz et al., 1987). Summarizing some of the salient points from these articles: (1) the difference in midbrain TH activity is restricted along the rostral-caudal axis to the medial one-third; this difference in TH activity is attributable entirely to differences in the number of DA neurons; (2) strain differences in TH

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

63

activity were not present at birth but first appeared at 9 days in the substantia nigra and 11 days in the striatum. Once established, the differences persisted suggesting that the strain differences are the likely result of differences in postnatal neuron survival; (3) striatal DA synthesis is higher in the C compared to the CBA strain; however, DA metabolite levels were higher the CBA strain; (4) some of the same genes which regulate TH activity also regulate DA-related behaviors, e.g., exploratory activity; and (5) there are significant maternal effects on the regulation of TH activity. Vadasz et al. (1982, 1987, 1992a,b) examined midbrain and striatal TH activity in the seven CXB RI panel, the parent strains, the reciprocal F, hybrids, and a CB6F2generation. Summarizing some major points: (1) TH activity is 17% higher in the CBy compared to the B6By strain. TH activity in the B6by strain is similar to that of the CBA strain; however, TH activity in the CBy strain is only 84% of that found in the C strain; (2) TH activity in the RI strains is not bimodally distributed; (3) the estimates of genetic determination for midbrain TH activity in the RI strains and F2hybrids are 0.63 and 0.89, respectively; (4) the estimates of genetic determination for striatal TH activity in the RI strains and F2 hybrids are 0.49 and 0.79, respectively; (5) there is a strong correlation between striatal and midbrain TH activity for all strains and crosses examined; and (6) among CB6F2 hybrids, there is no significant correlation between midbrain TH activity and spontaneous motor activity. German and colleagues investigated other features that might be associated with genetic differences in TH activity or the number of DA neurons. Mattiace et al. (1989) conducted a quantitative retrograde tracing study of mesostriatal projections in the C and CBA strains. These authors found no qualitative or quantitative strain difference in the labeling pattern of the midbrain DA neurons. Although the strains differ in the number of DA neurons, both strains give rise to a comparable axonal branching within the striatum. Bernardini et al. (1991) examined the firing rate and autoreceptor sensitivity of A10 dopmaminergic cells among C, C3H/He, CBA, and D2 strains. There was no significant difference among strains in basal firing rate or sensitivityto avariety of agonists, including DA and quinpirole. However, Crespi et al. (1989) using differential pulse voltammetry found evidence of greater DA turnover in C compared to the CBA strain. We recently completed an analysis of the number of midbrain THpositive cells among 10 inbred mouse strains. Experimental details, including the analysis strategy, are found in Hitzemann et al. (1993, 1994). The data in Table I11 summarize the data for the number of TH cells over the entire rostral-caudal axis of the SNZc and also include data from the rttrorut;al fidd (RRF). The RRF and the SNZc overlap in sections 8 and 9; data for sections 10 and 11 in Table I11 are largely from TH-positive

TABLE I11 TYROSINE HYDROXMA~E (TH)-POSITIVE CELL NUMBER IN THE SUBSTANTlA NICRA ZONA COMPACTA (SNL)

OF

10 INBRED MOUSESTRAINS‘

Mean TH cell number t SE normalized to C swain Swain or line

c D2 AKR

C3H A B6 D

CBA 129 LP

Section 1 100 f 6(89)’ 103 f 3 127 2 i n 107 2 14 99 t 9 108 t 5 92 f 15 124 t 2 96 2 2 126 t 1

Section 2

Section 3

Section 4

Section 5

Section 6

Section 7

Section 8

Section 9

Section 10

Section 11

100 -t 6(131)

ion 2 3(25fi) 75 2 4 69 t 5 70 t 3 74t 1 70f 1 III in

100 ? 6(174) 108 2 8 83 t 2 79 f 2 87 f 5 82 2 2

100 t 9(165) 99 f 7 98 t 7 98 t 5 107 -t 5 99Z6 87 t 11 I13 t 5 81 2 8 85 f 7

1 O O t 81148) 98 t 5 95 2 2 95 2 5 84 2 5 92 f 4 101 5 9

100 -C 5(151)

115 2 7 I08 t 4 106 t 4 9927 in8 2 3 !64 t !! 109t4 x7 2 i 11n-t4

100 2 7(149) 91 f 9 76 f 5 I04 f 4 97 t 3 75 % 2 111 Z 13 103 3 76 f 4 87 f 4

I00 2 lO(158) 77 t 5 65 f 4 72 t 2 66 2 2 72 f 3 83 f 3

I 0 0 2 6(113) 76 2 5 74 f 4 82 f 6 75 t 3 72 f 3 95 f 7 89 f 9 72 f 4 74 t 6

100 Z 16(68) 72 f 7 9627 100 c 7 88 2 10 72 f 4 I13 f 6 71 t 7 96 t 7 82 f 9

+

6Rt2 73 c 9 77 t 4

in5 t R 100 t 3 74 f 10 91 t 3

10023 71 t R 76 t 7

84 f 4 80 t 3 101 I 6 86 It 3

9022 96 c 3 10029 6R 2 13 97 t 4

93 f 4 60f4 69 f 4

“Data for TH cell number are the normalized averages obtained from five to seven animals/strain. Sections are arranged in rostra1 to caudal order. Section 1 corresponds to section No. 321 from the Sidman Atlas. In general, each section is separated by 60 pm. However, for accurate alignment, a complete midbrain section series, strained with cresyl violet was used. Data adapted from Hitzemann et al. (1994). bAveragecell per section for C strain.

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

65

neurons of the REW. All data have been normalized to the C strain. Over sections 1-9, the strain X section interaction was significant (F= 1.86, df = 63,345, P < 0.0001).A comparison of the P/J and 129/J strains, which is representative of the range of variation in the number of TH cells number among all strains, is shown in Fig. 5. The difference between these strains was greatest in section 2 (89%).Within the SNZc, the highest number of cells is generally found in section 3 which is immediately rostra1 to the point where the medical lemniscus begins to intrude into the TH cell cluster. Only in this section was the number of TH cells for the C strain different from that of the CBA strain, paralleling the results described previously. The data in Table N summarize the data for the number of TH-positive cells in the ventral tegmental area (VTA). For the VTA, the strain X section interaction was significant (F= 1.66, df = 45,224, P < 0.01). As with the SNZc, the P/J and 129 strains were representative of the range of variation in the number of TH-positive cells within the VTA. However, there was no significant correlation between cell number in the SNZc and VTA. Using the BXD RI strategy, several behavioral QTL map near Th and Drd4 on mouse chromosome 7 (see Fig. 4). These include ethanol locomotor activation and methamphetamine locomotor stimulation, temperature sensitivity, and conditioned place preference (see Crabbe et al., 1994, and references therein).

FIG.5. Tyrosine hydroxylase cell number in the substantia nigra zona compacta of the P/ J and 129/J inbred mouse strains. Data are taken from Hitzemann et al. (1994).The differences noted between these strains in general define the extreme range of variation noted among 10 inbred mouse strains. Data are the average ? SE offive to seven determinations/section. See the legend to Table I11 for additional experimental details. *Significantlydifferent at P < 0.01.

TVROSlNE

TABLE N HYDROXWE(TH)-POSITrvECELL NUMBER IN THE VENTRAL TECMENTAL AREA OF 10 INBRED MOUSESTRAINS'

Mean TH cell number Strain or line

C D2 AKR

C3H A B6 P CBA 129 LP

2 SE normalized to

C strain

Section 4

Section 5

Section 6

Section 7

Section 8

Section 9

100 2 3(139) 78 2 3 6025 71 2 4 65 2 3 73 2 4 97 2 6 88 ? 3 65 2 7 58 2 4

100 2 Z(142) 77 2 5 61 2 5 74 2 3 65 2 3 79 2 3 119 ? 4 90 2 3 66 2 5 80 2 3

100 2 4(147) 98 2 3 73 2 3 67 2 2 76 t 3 70 2 3 116 2 8 99 2 5 59 2 3 89 ? 3

100 2 4(147) 10024 85 2 4 83 2 5 76 2 3 88 2 5 123 2 9 99 ? 5 80 2 5 94 2 4

100 2 5(147) 121 2 4 90 2 1 88 2 3 79 2 3 87 2 2 115 2 7 100 2 3 82 ? 6 95 2 2

100 2 3(147) 126 2 7 99 2 5 92 2 4 92 t 3 90 2 3 119 ? 7 90 2 5 76 2 3 91 2 5

See legend to Table 111.

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67

As noted previously, some genetic data are available for striatal tryptophan hydroxylase (TrH) (see Kulikov et al., 1992). In addition, Natali et al, (1980) found that TrH activity is significantly higher in the B6 strain compared to that in the C strain. Furthermore, these strains show significant differences in the circadian rhythm of TrH activity. Nestler and colleagues (Beitner-Johnson and Nestler, 1991; Beitner et al., 1991,1993; Guitart et al., 1992, 1993a,b) examined various biochemical differences between LEW and F344 rats in the VTA and nucleus accumbens (NAc), including TH activity. These data are summarized in Table 5 of Guitart et al. (1993a). These authors have noted that TH activity is higher in the VTA of the LEW strain, but in the NAc, TH activity is higher in the F344 strain. Compared to the LEW strain both adenylat cyclase and CAMPdependent protein kinase show higher activity in the NAc of the F344 strain. Significant strain differences were also found in G protein levels. Within the NAc, the LEW strain had significantly lower levels of GilolI2, Gio3, and Go. There were no significant strain differences in Gial12and Gp in either the caudate-putamen or the VTA. Buzsaki et al. (1990) found that, compared to the Buffalo strain, TH activity in the F344 strain was significantly higher in the substantia nigra, corpus striatum, and olfactory tubercle; the higher TH activity was paralleled by higher D,-binding values in the striatum and olfactory tubercle. In addition, the F344 strain was more sensitive on several EEG measures to the DA antagonist, acerpromazine. B. STEADYSTATE DOPAMINE AND SEROTONIN LEVELS AND TURNOVER Kempf et al. (1974) examined brain DA levels and DA turnover in B6, D2, and SEC/lRe mouse strains. The DA content per gram whole brain is significantly greater in the D2 strain (+ 13%) and less in the SEC strain (- 13%)compared to the B6 strain. However, when these data are corrected for the differences in brain size (the brain weights in the D2 and SEC strains are respectively 14 and 5% smaller than the B6 brain weight) only the lower content in the SEC strain remained. Compared to the B6 strain, DA turnover was significantly slower (-10%) in the D2 strain but significantly faster (+23%) in the SEC strain. In comparison to the differences in DA content and turnover, the differences in NE and serotonin (5-HT) content and turnover among strains were significantly more modest. Messiha (1990) found no significant difference in striatal DA, DOPAC, or HVA among B6, C, and the hybrid CDF-1 strains. The most marked differences among these strains were in 5-HT and 5-HIAA content, particularly in the medulla. Although the B6 and C do not differ greatly in DA or DA metabolite levels, Kempf et al. (1990) found that during ethanol withdrawal, there

68

ROBERT HITZEMA" st al.

is a marked increase of DA turnover in the C but not the B6 strain. A genotypic-dependent response is also noted when animals are given ethanol chronically (4 weeks) in a free-choice paradigm. The ethanolpreferring B6 strain showed no difference in brain DA levels while the ethanol-nonpreferring D2 and C3H strains showed significant increases in both DA and 5-HT (Yoshimotoand Komura, 1989). Messiha (1991a) found that haloperidol increased striatal DA turnover to a greater degree in the C compared to the B6 strain; in contrast, pimozide caused a greater decrease of striatal 5-HT turnover in the B6 strain. The B6 and C strains differ in their developmental DA profile (Daszuta et al., 1982). Daszuta et al. (1992) noted that before Week 3, striatal DA levels are higher in the B6, but after Week 5 the reverse is true. The B6 and C strains also differ in their response to aging. Ebel et al. (1987) found that dopaminergic mechanisms in the striatum become particularly defective where both DA release and turnover are affected. Also, striatal cholinergic activity and somatostatin levels are more affected in the C strain. Messiha (1991b) examined the effects of amitriptyline and nortriptyline treatment on DA turnover in C and B6 mice. Amitriptyline, and to lesser degree nortiptyline, increased DA turnover in the C strain to a significantly greater extent than the B6 strain. Skrinskaya et al. (1992) examined DA, DOPAC, and HVA levels among eight inbred mouse strains, including B6 and D2. Data were obtained for both the caudate-putamen (CPu) and the NAc. The largest variation among strains was in HVA levels. Within the CPu the rank order was DD > C3H > C > AKR > B6 > D2 > CBA > CC57BR. Within the NAc the rank order was B6 > CC57Br = D2 > C > DD = C3H = AKR > CBA. Within the striatum there was no significant correlation between DA/ DOPAC, DA/HVA, and DOPAC/HVA. The situation was similar in the NAc except for a significant negative correlation between DOPAC/HVA. There was no significant correlation in any parameter between the CPu and NAc. Ishikawa et al. (1989) measured the levels of DA in discrete brain regions of the mouse mutant, wriggle mouse sagami (WMS), and the normal C strain littermate controls. DA, DOPAC, and HVA levels were higher in the striatum of the WMS mutant but the data suggested DA turnover was not increased. In the transgenic (Hprt-) mouse model of Lesch-Nyhan syndrome, the mutant mice manifested a 20-30% decrease in forebrain DA content and a corresponding increase of DA turnover. Vriend et al. (1993) examined amino acid and monoamine concentrations in striatal tissue extracts prepared from C substrains that were audiogenic seizure sensitive or resistant. Significant decreases in both excitatory (glutamate and aspartate) and inhibitory [y-aminobutyric acid (GABA) and

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69

taurinel amino acids were observed in the seizure-prone mice. Significant decreases in both DA synthesis and release were also noted in the seizureprone animals. In contrast, 5-HT synthesis was not different between the substrains. Mice are not equally susceptible to the neurotoxic effects of Nmethyl4phenyl-l,2,3,t%tetrahydropyradine(MPTP). In general, colored mice are less susceptible than albino mice to the decrease in striatal DA level (see, e.g., Bradbury et al., 1986; Riachi and Havik, 1988). Hoskins and Davis (1989)examined the effects of MPTP among eight inbred mouse strains, including three albino strains. These authors confirmed that the depletion of DA is greatest in the colored mice. The comparison between the C57BL/ lOScSn strain and the albino C57BL/lOScSn-cc strain is especially interesting since the congenic albino strain differs by a maximum of 22 cM on chromosome 7 between the p and Hbb loci. Howes et al. (1984)examined DA and DOPAC levels at 6, 14,28,and 36-40 weeks of age in the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. No difference in striatal DA was found but DOPAC levels were elevated in the SHR strain suggesting a more rapid DA turnover. The authors suggest that this may be related to the hyperactivity of the SHR strain. Between the Dahl salt-sensitive (DS) and salt-resistant (DR) rat lines, striatal DA and 5-HT are higher in the DS line (Dawson and Oparil, 1986).High salt diets had no effect on monoamine or metabolite levels except to increase cortical but not striatal5-HIAA levels. Orosco et al. (1992 and references therein) examined the effect of insulin on striatal DA metabolism in the genetically obese Zucker rat. The recent microdialysis study (Orosco et al., 1992) illustrates the complex genetic relationships. Lean homozygous Fa-Fa rats responded to insulin as expected with increases in 3-MT and DA levels and decreases in DOPAC and HVA. Lean heterozygous Fa-fa rats responded with decreases in DA and all metabolites suggesting that insulin had decreased synthesis. The data for the obese fa-fa rats fell into two modes. One mode was similar to the data for the heterozygous animals. The second mode was characterized by an increase in all DArgic parameters, a pattern distinctly different from the Fa-Fa results. The author conclude that 16 weeks of age may be a critical transition period in the development of genetic obesity with regard to brain monoamine disturbances and the reponse to insulin. Nervous pointer dogs have been suggested to be a genetic model for anxiety. Gurguis et al. (1990)compared eight nervous and six normal dogs and found that within the striatum there was a trend for lower HVA and DOPAC levels and a significantly lower DOPAC/DA ratio in the nervous dogs, suggesting decreased dopaminergic function.

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ROBERT H I T Z E M A " et al.

C. NEUROPEPTIDES The B6 and C strains differ in the effects of age and the age X alcohol interaction on striatal somatostatin (SRIF) levels (Fuhrmann et al., 1986b). Fuhrmann et al. (1986b) noted that at 3 months of age, SRIF levels were modestly lower ( - 12%) in the B6 strain. Age produced a progressive decrease of SRIF levels which was significant at 12 (- 18%),18 (-2'7%), and 27 months (-20%). There were no agedependent changes of SRIF in the B6 strain. Chronic alcohol treatment increased SRIF levels in the C strain at the three ages studied but had no effect on the B6 strain. Erwin and colleagues (Erwin and Jones, 1989, 1993; Erwin and Korte, 1988; Erwin and Su, 1989) found that the L S and SSselected lines and the RIs derived from these lines differ in their responsiveness to icvadministered neurotensin. Comparing the LS and SS lines, it was found that the density of [3H]neurotensin binding sites was higher in the striatum, cortex, and cerebellum of the SS line.

D. DOPAMINE RECEPTORS An excellent review of the genetic aspects of DA receptor binding in the mouse and rat brain has recently appeared (Vadasz et al., 1992c) and should be consulted by the interested reader. As noted elsewhere in this chapter, five subtypes of DA receptors hiwe been described. These have been divided into two families, the D1 family containing the D1 and D5 receptor subtypes, and the D2family containing the DP,Dg, and D4receptor subtypes (see, e.g., Sunahara et al., 1990; Civelli et al., 1991; O'Malley et al., 1990; Van To1 et al., 1991). In addition, numerous splice variants of the receptor subtypes have been described (see, e.g., Neve et al., 1991; Tiberi et al., 1991; Seeman and Van Tol, 1994), although the functional/pharmacological significance of these variants is not entirely clear (see, e.g., Liu et al., 1992). For both the D1 and D2 receptor families relatively specific ligands are available (e.g., SCH 23390 and epidepride); however, within the two families, ligands which show sufficient discrimination between receptor subtypes to be useful for receptor binding studies have generally not been available. Recently it has been suggested that 7-OH-DPATmay be a specific DS receptor ligand; however, this ligand appears to also bind to non-DA receptors (McGonigle et al., 1993; Schoemaker, 1993). There are marked quantitative and regional differences in mRNA distribution for the various receptor subtypes. For example, within the caudate-putamen, D2 mRNA levels are one or two orders of magnitude higher than those for D3 and/or D4 mRNA. Dg mRNA levels are enriched

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in the ventral and ventromedial striatum, including the nucleus accumbens, and in the midbrain (Sokoloff et aL, 1990). The apparent distribution of D3 receptors parallels this mRNA distribution (McConigle et aL, 1993). D4 receptor mRNA is enriched in the midbrain, amygdala, and frontal cortex. Given similar mRNA stability and to the extent that mRNA levels and receptor density are proportional at some level, one could conclude that “ apparent” D2 receptor binding in the caudate-putamen, especially the dorsomedial and lateral aspects, is indeed to D2 receptors. In contrast, binding to midbrain somatodendritic D2 receptors is less assured and is more likely to include confounds from D3and D4receptors. mRNA distribution for the D1 receptor family shows some parallels to that for the D2 receptor family. In areas where both D1 and D5mRNA are coexpressed, e.g., the caudate-putamen, D1mRNA is present with an order of magnitude higher density (Sunahara et aL, 1990). D5mRNA is present in the ventral striatum, including the nucleus accumbens, the olfactory tubercle, and olfactory bulb. The question arises as to what extent D1 and D2 receptors are found in the same or different neurons within the striatum. Gerfen (1992) suggested that D1 receptors are principally associated with the striatonigral GABA output neurons which contain substance P and dynorphin; on the other hand, D2 receptors are suggested to be principally associated with the striatopallidal GABA ouput neurons which contain enkephalin (see also Le Moine et aL, 1990). Gerfen (1992) further noted that a smaller subset of striatopallidal GABA output neurons that contain neurokinin B appear to express both receptor types (Burgunder and Young, 1989). Segregation of the D, and D2 receptors has clear functional consequences. Striatonigral neurons provide inhibitory input to the SNZr and entopeduncular (EP) nucleus. SNZr/EP GABA output neurons inhibit neurons in the thalamus, superior colliculus, and pedunculopontine nucleus (Gerfen, 1992). Thus, activation of the striatonigral pathway results in enhanced activity in these regions. The striatopallidal output system has the opposite effect. The GABA/enkephalin neurons provide inhibitory output to the globus pallidus which in turn provides inhibitory output to the subthalamic nucleus (STN). Excitatory neurons project from the STN to the SNZr/EP; thus, activation of the GABA/enkephalin output neurons has the downstream effect of increasing inhibitory output from the SNZr/EP. The release of DA will stimulate the striatonigral pathway (D1 receptor effect) but inhibit the striatopallidal pathway (D2 receptor effect). This relatively simple design explains how both D, and D2receptor antagonists cause catalepsy. Others have suggested that D1 and D2 receptors are colocalized to a greater extent than suggested by Gerfen (1992). For example, suing immunofluorescence techniques, Ariano et al. (1992) estimated that 60% of

72

ROBERT HITZEMA”

d

al.

the striatonigral output neurons contain D2 receptors. Lester et al. (1993) observed the colocalization of D1 and D2 mRNAs in striatal neurons. Surmeier et al. (1993) reviewed the molecular and cellular evidence for and against colocalization, including new information from single-cell mRNA amplification and patch clamping of isolated neurons. These authors concluded that overall the evidence is consistent with the view that there are functional D1and D2receptors in the majority of striatal efferent neurons. From the optimist’s perspective, the material cited previously illustrates numerous levels at which genetic factors could affect DA receptor organization within the basal ganglia. From the pessimist’sperspective, these studies illustrate potential confounds to interpreting any results. The reader is simply reminded to interpret all results cautiously. Vadasz et al. (1992~) lists 15 studies which have examined various aspects of D2receptor binding among inbred strains of mice.2All of the studies used membrane-binding assays and the majority of the studies used young male mice. Nine of the studies made comparisons among multiple inbred strains; however, comparisons of more than 3 strains were made in only four laboratories. Of these only the results of Helmeste and Seeman (1982) and Ciaranello and Boehme and Ciaranello, 1981a,b, 1982; Ciaranello and Boehme, 1981) are directly comparable as they both used young adult male mice from the same source (Jackson Laboratory). Sex was unspecified in the study of Belmaker et al. (1981) and Leprohon-Greenwood and Cinader (1987) used female mice. Normalizing the data to the C strain receptor density as suggested by Vadasz et al. (1992~) and looking only at the 5 strains common to investigations by both laboratories, we find the order of receptor density for Helmeste and Seeman (1982) was: C3H/He, 107%; C, 100%; B6, 75%; CBA, 62%; and D2, 61%. For Ciaranello and Boehme the order was: C, 100%;C3H/He, 90%; B6,76%; CBA, 67%; and D2,63%. The somewhat higher D2 receptor density in the B6 strain compared to that in the D2 strain has been observed in other studies. e.g., Michaluk et al. (1982) and Erwin et al. (1993). Boehme and Ciaranello (1982) examined D2 receptor density in the AKR, C57L, and 5 AKXL RI strains. These authors suggested that the RI data may be bimodally distributed but with only 5 of the 18 available strains examined such conclusions were probably premature (see also Vadasz et ah, 1992~). Leprohon-Greenwood and Cinader ( 1987) examined D2receptor binding among females of five inbred strains (B6, SJL, A, DBA/1, and C3H/ These references are: (1-3) Boehme and Ciaranello (1981a,b, 1982); (4-5) Ciaranello and Boehme (1981, 1982); (6) Belmaker etal. (1981); (7) Severson etal. (1981); (8) Helmeste and Seeman (1982); (9) Bannet et al. (1981); (10) Fink et al. (1982); ( 1 1 ) Michaluk et al. (1982); (12) Leprohon-Greenwood and Cinader (1987); (13) Morgan et al. (1987); (14) Ozsvath and German (1988); and (15) Laszlovsky and Vadasz (1991).

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He). At 7-15 weeks of age, the order of receptor density was B6 < SJL < A = DBA/l = C3H and the range of receptor density values was nearly 100%. Interestingly, these authors found that there were marked declines in receptor density for all strains except B6 such that by 31-45 weeks of age there was no difference among strains. No further changes in receptor density were seen up to 120 weeks. In a subsequent study, these authors found that among three inbred strains (SJL, D2/NNia, B6/NNia) and B6D2 F1 hybrids, animals with a low youthful D2 receptor density, aging had relatively modest effects. In contrast, in the MRL/Mp-‘’ strain, which has a high youthful concentration of receptors, aging caused a marked receptor loss. These authors termed this phenomenon of receptor loss being greatest in the strains with the highest receptor density “economic correction.” Overall, the decrease in D2 receptor density is one of the most consistently reported effects of aging on the basal ganglia; in some but not all studies the losses appear to be progressive with both early and late declines (see Morgan and Finch, 1988; Morelli et al., 1990). Ratty et al. (1990) examined D2receptor density in the TgX15 transgenic formed as a result of insertional mutagenesis. Homozygotes but not heterozygotes express abnormal circling behavior and show a 31% increase in striatal D2 receptor density. Paralleling their D2 receptor studies, Boehme and Ciaranello (1982) also examined 5-HT2receptor density among four inbred strains, B6, D2, AKR, and C57L. The 5-HT2binding was assessed using two different ligands ( [ 3 H ] ~ p i p e r ~and n e [3H]mianserin)which showed good agreement; however, neither method would discriminate among the three subtypes of 5-HT2 receptors (5-HTZA,5-HTZB,and 5-HTZc).Within the striatum the order of receptor binding was AKR > C57L = B6 > D2; the range of difference was 63%. Interestingly, within the frontal cortex, the highest receptor density was found in the D2 strain. Kanes et al. (1993a) were the first to use quantitative receptor autoradiography (QRA) to compare D2receptor density among multiple inbred strains. Brains were cut in 20-p coronal sections from the most rostra1 aspect of the NAc to the most caudal aspect of the retrorubral A8 neurons. Data were obtained for the NAc (6 sections), the dorsomedial caudate-putamen (dCPu) (13 sections), the lateral CPu (1CPu) (14 sections), the SNZc (10 sections), and the VTA (10 sections). For no brain region was there a significant section x strain effect but the strain effect was significant in all sections. Thus, the reported data were collapsed across sections. The data from Table I of this chapter have been recalculated, normalizing all values to that of the C strain (Table V). The QRA data are significantly different from those obtained by membrane-binding assay. Within the striatum (NAc, dCPu, and 1CPu) the strains distributed into two groups: C, AKR, C3H/

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SUMMARY OF SPECIFIC

TABLE V [%]SPIROPERlDOLBINDIKG AMONG

INBRED

MOUSE.%RAINS"

Strain

NAc ? S E

dCPu 2 SE

ICPu 2 SE

SNZc 2 SE

VTA 2 SE

C D2

100 2 6 158 2 16 116 f 9 109 ? 9 107 2 7 148 f 16 112 t 6 142 f 9

100 f 3 119 f 6 106f 3 99 t 6 104f 4 I26 2 7 100 f 3 135 2 4

100 2 3 121 2 6 101 2 6 97 f 4 102 2 4 138 f 8 1002 3 131 f 5

100 f 7 152 t 11 99 f 7 101 2 10 130 t 10 141 f 11 140 2 9 173 f 10

100 f 7 141 t 1 1 82 f 5 100 2 11 93 f 7 109 2 9 123 2 9 148 f 1 1

AKK C3H A B6 CBA LP

"All data have been normalized to that of the C strain. N = 9-12 animals/strain. Adapted from Kanes el al. (1993a)

He, A, and CBA, and D2, B6, and LP. The latter group had a significantly higher receptor density; this difference was greatest in the NAc. Receptor density was significantly higher in the B6 strain compared to the D2 strain only in the 1CPu. Within the SNZc the C, C3H, and AKR strains formed the low receptor density group and the remaining strains formed a high receptor density group. The difference between the C and LP strains was 72%, the largest difference in receptor density noted in any brain region. The correlation between receptor density in the lCPu and the SNZc is not significant ( r = 0.54) although with only eight strains these data should not be overinterpreted. However, such data do suggest differential mechanisms for regulating receptor density in the midbrain and striatum. Within the VTA the low receptor density group was composed of the C, AKR, A, C3H, and B6 strains. Again keeping in mind the caveat noted previously, comparing these data to those from the SNZc may suggest that even within the midbrain receptor density is independently regulated. Intuitively, this observation is congruent with the differing functions of the A9 and A10 DA systems.The reasons for the differences between the membrane-binding assays and QRA are not clear. In some experiments (Helmeste and Seeman, 1982; Boehme and Ciaranello, 1982) butaclamol was used to measure nonspecific binding, but in others (Leprohon-Greenwood and Cinader, 1987; Greenwood and Cinader, 1991; Kanes et al., 1993a) sulpiride was used. Sulpiride has a relatively low affinity for D,, D5,and D4receptors (see Seeman and Van Tol, 1994). To determine if there is a polymorphism either near or a part of Drd2 associated with a difference in D2 receptor density, Kanes et al. (1993b) examined D2 receptor binding in 40 B6D2 F2 hybrids which were also genotyped for three polymorphic microsatellite markers (D9Mzt22, D9Mit4,

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75

D9Mit21) which are near Drd2 on chromosome 9. It was already known that there is a polymorphism in Drd2 between the B6 and D2 strains (Smith et al., 1992). The data for the lCPu and D9Mit21 are shown in Fig. 6. The results illustrate that across the entire rostral-caudal axis, receptor density in the D2 homozygote is significantly higher than that in the B6 homozygote. Essentially identical results were obtained for D9Mit22 and D9Mit4. The data also suggest that there may be some dominance associated with the D2 allele as binding in the heterozygote was nearly identical to that in the D2 homozygote. Finally, the examination of the individual data points for section 17 reveals that the polymorphism near or part of Drd2 is associated with only a small amount of the variance in D2 receptor density. Vadasz et al. (1992~)noted that there had been no multiple-strain comparisons for D1receptors. However, these authors present some preliminary data for five inbred strains (C, C3H, BGBy, CXBI/By, and D2) showing a 22% range in receptor binding data. Kanes et al. (1993a) examined D,

250

a

.-

i

150

.P E

10

330

Section 17 ICPU r = 0.4:

(5

.-0 300-

g

E

In

270-

240 -

I -I

-

I f

210-

I

180-

15

:

20

I-

I-

s:

I

-

-I

I

D

I-

I

76

ROBERT H I T Z E M A "

Pt

al.

receptor binding in parallel with the D4 receptor studies described previously. Receptor binding was examined in the NAc, dCPu, lCPu, and the SNZr. No significant strain x section interaction was detected in any brain region. As with the D2 receptor data, the D1 receptor data have been collapsed across sections and normalized to the C data (Table VI). The range in DI receptor binding is less than that seen for D2receptor binding; the greatest range of difference is seen in the dCPu (37%) (LP vs B6). The correlations between DI and D2 receptor binding in the NAc, dCPu, and lCPu were 0.06, 0.19, and -0.08, respectively. However, given the lack of variance in DI receptor binding, these correlations are not surprising. There are relatively few reports examining D1 or D2 receptor density among inbred rat strains. Striatal D4receptor density is generally reported to be higher in the F344 strain compared to the Buffalo strain (Helmeste, 1983; Cooper et al., 1985; Rosengarten et al., 1986; Kerr et al., 1988; Buzaski et al., 1990). However, the range of difference reported is quite large; Rosengarten et al. (1986) reported a difference of 19% (['H]spiroperidol), while Kerr et al. (1988) reported a difference of >300% (['Hlsulpiride). Luedtke et al. (1992) examined D2 receptor binding among three outbred (Sprague-Dawley, Wistar, and Long-Evans) and five inbred (brown Norway, Buffalo, DA, F344, and LEW) strains of rats using [1251] iodobenzamide as the receptor ligand. Although these authors noted a range of 450580 fmol/mg protein in striatal D2receptor density, there were no statistically significant differences. These authors also examined the differences among the eight strains in the ratio of the long and short splice variants of Dz receptor mRNA. No difference in the long and short isoforms of the mRNA was noted among these strains. Le Fur et al. (1981) found that D2receptor binding, but not the binding of [3H]quiniclidinylbenzilate (QNB) or ['HI LSD, was significantly higher TABLE VI SUMMARY OF SPF.(:IFI(: ['H]SCH23390 BINDING AMONG INRREI) MOUSESTRAINS" Strain

c D2

mn C3H A B6 CBA L.P

NAc 2 SE

dCPu Itl SE

ICPu t SE

SNZc t SE

100 % 3 94 -+ 4 112 t 3 91 f 5 a4 t 2 84 2 2 101 t 4 108 2 6

100 2 4 104 t 2 92 -+ 3 108 2 2 110 t 4 90 +- 2 110 2 3 123 t 3

100 t 2 98 t 2 84 2 72

100 +- 4 83 Itl 5 76 2 5 77 2 4 87 2 4 79 ? 3 104 +- 3 99 f 4

95 2 2 97 t 2 86 t 2 103 t 2 103 +- 2

"All data have been normalized to that of the C strain. N = 9-12 animals/strain. Adapted from Kanes rl nl. (199%)

GENETICS AND THE ORGANIZATION OF THE BASAL GANGLIA

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in SHR rats compared to WKY rats. Lim et al. (1989) examined D2 and D, receptor binding in SHR, WKY, and outbred Sprague-Dawley (SD) rats. In comparison to the WKY and SD strains, the SHR strain had higher D, and D2 receptor densities in the striatum and hypothalamus. Vadasz et al. (1992~)cite only two reports (Rosengarten et al., 1986; Kerr et al., 1988) which compared D, receptor binding among inbred rat strains. Using [3H]SCH 23390, Kerr et al. (1988) found receptor density in the CPu to be modestly higher (16%) in the Buffalo compared to the F344 strain. In contrast, Rosengarten et al. (1986), using [3H]pifluxitol, found receptor density to be somewhat higher (8%)in the F344 strain.

E. NEUROTRANSMITTER TRANSPORTERS Many of the neurotransmitter transporters have been cloned, including transporters for DA, 5-HT, and GABA (Shimada et al., 1991; Kilty et al., 1991; Usdin et al., 1991; Giros et al., 1991; Guastella et al., 1990; Blakely et al., 1991; Mayser et al., 1992). However, there have been relatively few reports examining transporter genetics. Shoemaker et al. ( 1982) examined the whole hain synaptosomal uptake of NE, choline, and GABA in the CXB RI series, the parental strains, and the reciprocal F1 crosses. Although the V,,, values between the parent strains were not greatly different, a significant variation among the RI strains was noted especially for choline uptake where the range was 153%. The data did not distribute bimodally and the authors concluded that the variation in transporter activity was under the control of multiple genes. Moisset et al. (1975) examining NE uptake into cortical synaptosomes prepared from this RI panel and reached a similar conclusion. Durkin et al. (1982) found no difference between the B6 and C strains in basal striatal choline uptake but differences do appear after pharmacological challenges. For example, acute haloperidol treatment increases high-affinity choline uptake in both B6 and C strains but the increase is much greater in the B6 strain (Atweh et al., 1975). Acute ethanol treatment decreases choline uptake in the B6 strain but not in the C strain (Durkin et al., 1982). Ho et al. (1975) found no difference between B6 and D2 mice in whole brain DA uptake. Erwin et al. (1993) found no difference between the B6 and D2 strains in the striatal binding of [3H]CFT, a cocaine analogue, assayed 2 the presence of GBR12909, a DA uptake inhibitor. Similarly, there was no strain x region difference in the binding of [3H]paroxetine, a specific ligand for the 5-HT transporter. Bozy and Ruth (1989) examined the relative ability of cocaine, tropacocaine, and amphetamine to inhibit ['HIDA and [3H]5-HTuptake into whole brain synaptosomes among C3H,

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C, B6, and D2 strains. The authors noted marked strain x drug effects for both DA and 5-HT and concluded that the genetic differences in monoamine inhibition may arise from genetic differences in carrier topology or other site(s) of cocaine interaction. De Fiebre et al. (1989) found no difference between the L S and SS-selected mouse lines in the uptake of DA. George and Ritz (1990) extended this observation when they found no difference between the lines in the binding of [3H]mazindol to the DA transporter. George et al. (1991) also observed that there was no significant difference among the ACI, F344, Lewis, and NBR rat strains in the binding of [3H]mazindol to striatal membrane. Hendley and Fan (1992) examined DA uptake in the SHR, WKY, WKHA, and WKHT rat strains. These authors found to significant difference among strains in striatal uptake. Similarly, Gilad and Gilad (1987) found no difference in DA uptake between the BN and WKY strains.

CHOLINERGIC SYSTEM F. STRIATAL The existence of a DA-ACh interaction within the striatum is now well established (Stoof et al., 1992). Pharmacological studies (Calne, 1978; Doshay and Constable, 1957) initially suggested that a balance between the cholinergic and dopaminergic systems is essential to the control of extrapyramidal motor behaviors. The interaction between these systems in the striatum has been repeatedly confirmed with both functional and anatomical studies. For example, Le Moine et al. (1990) have found, using in situ hybridization techniques, that the majority of cholinergic neurons in the striatum express mRNA for the D2receptor. Both in vivomicrodialysis and in vitro studies have shown that DA agonists inhibit striatal ACh release, while D2 antagonists increase ACh release (Stoof et al., 1992). Given the closeness of the interaction between the striatal dopaminergic and cholinergic systems, the question arises as to whether or not there are shared genetic effects. As will be shown below, a QTL near or a part of Drd2 has a significant effect on the number of cholinergic neurons. From the genetic perspective, variation of cholinergic cell number, function, and associated pharmacology is well described (Albanese et al., 1985; Castellano et al., 1988; Durkin et al., 1977; Ebel et al., 1973; Kempf et al., 1974; Marks et al., 1981, 1989; Schwab et aL, 1988, 1990a,b; Tunnicliff et al., 1973). The data on the behavioral effects of cholinergic agonists and antagonists among inbred mouse strains have shown convincing genetic differences. For example, Marks et al. (1981) examined the effects of oxotremorine, a muscarinic agonist, on open-field behavior among three in-

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bred mouse strains. The order of potency for depression of activity was D2 > B6 > C3H. In a subsequent study, the authors examined the effects of oxotremorine on rotorod performance in the B6, D2, C, and C3H strains. Oxotremorine decreases the time spent on the rotorod. There was no significant difference in EDso among the B6, C, and D2 strains but the EDSO in the C3H strain was two-to three-fold higher. This laboratory has examined the effects of the AChE inhibitor, DFP, on several behavioral indices among the B6, D2, and C3H strains (Smolen et al., 1986). Smolen et al., (1986) found no difference among strains in depression of rotorod performance but found the order of potency for depression of Y-maze activity to be B6 > D2 > C3H. In general, this laboratory noted a similar hierarchy among these strains in their response to nicotine. These authors concluded that the differential cholinergic responsiveness among inbred mouse strains cannot be explained by differences in the number of affinity for muscarinic (or nicotinic) receptors (see below). Castellano et al. (1988) found that both oxotremorine and nicotine are more potent in the D2 strain compared to the B6 strain to depress locomotor activity. Consistent with the idea of greater cholinergic activity in the D2 strain compared to the B6 strain, the D2 strain has been found to be more sensitive to haloperidolinduced catalepsy (Kanes et al., 1993a). Schwab et aZ. (1990a,b) demonstrated that, in comparison to the D2 strain, the B6 strain has a lower number of cholinergic neurons. The deficit in cell number was most pronounced in the striatum (-35%), the nucleus basalis of Meynert (-28%), and the nucleus of the diagonal band of Broca (-32%). These data extend earlier findings showing that the B6 and D2 strains differ in both acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) enzyme activity and regional acetylcholine (ACh) turnover rates (Durkin et al., 1977; Ebel et al., 1973). In contrast to these results, Marks et al. (1981) found no difference among B6, D2, and C3H strains in either ChAT or AChE activity within the “total midbrain,” which contained the striatum. Hashemzadeh-Gargari and Mandel (1989) examined ChAT and AChE activity in the 8 6 and C strains. ChAT activity was significantly higher in the striatum and hippocampus of the C strain (15 and IS%, respectively) but the strains showed no difference in the septum, limbic-temporal cortex, and piriform cortex. A significant difference between the strains in AChE activity was found only in the hippocampus (9% higher in the C strain). Several groups have examined inbred mouse strains for differences in muscarinic receptor density. Aronstam el al. (1979) and Schwab et al. (1992) found that, compared to the D2 strain, the B6 strain has a lower density of muscarinic receptors in the striatum when assayed by the receptor subtype nonspecific ligand [’HIQNB. Marks et al. (1981) found no difference be-

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tween the B6 and D2 strains but did observe that t3H]QNB binding was higher in the striatum of C3H mice. In a subsequent paper and using a somewhat different experimental design, Marks et al. ( 1986) compared striatal ['HIQNB binding among four strains: B6, D2, C, and C3H. These authors found no significant difference in receptor density among the strains although there was a trend to a lower receptor density in the B6 strain. Hashemzadeh-Gargari and Mandel (1989) agreed with Marks et al. (1986) that there is no difference in striatal ['HIQNB binding between the B6 and C strains. Both groups did find a modestly lower binding in the hindbrain/medulla pons of the C strain; this difference reached statistical significance only in the study of Hashemzadeh-Gargari and Mandel (1989). These authors also noted that acute ethanol administration significantly decreases striatal ['H)QNB binding in the B6 strain but not in the C strain.The B6 strain but not the C strain also showed a significant increase in striatal ChAT activity. Baumgold (1987) compared ['HIQNB binding among four mouse strains: B6, C, AKR, and C57BL/10. Data are reported for the midbrain which included the hypothalamus, the thalamus, and the striatum. Receptor density was significantly higher in the B6 strain in comparison to the other three strains. Both Marks et al. (1986) and Schwab et al. (1992) found no difference between the B6 and D2 strains in striatal ['Hlpirenzepine binding although the data of Marks et al. (1986) suggested binding may be somewhat lower in the B6 strain. The affinity of pirenzepine for m, and m4 receptors is 5 and 80 nM, respectively (Waelbroeck et al., 1990). The data reported by Marks et al. (1986) and Schwab et al. (1992) was high-affinity pirenzepine binding and thus, one assumes that the binding was principally to the ml subtype. All five types of muscarinic receptors (ml-5)have been detected in the striaturn (Yasuda et al., 1992) although the m5 receptors can be practically ignored because of their very low density. Using immunoprecipitation techniques, Yasuda et al. (1992) estimated that the striatal ml, m,, m3, and m4receptor densities are 35,12,6, and 45% respectively. Waelbroeck et al. (1990) estimate the densities of the pharmacologically defined striatal MI, M2, MS, and M4 receptors at 27,19,9, and 45%. Both m2and m4mRNAs are expressed in the striatal cholinergic neurons. Given the lower number of cholinergic neurons in the B6 strain compared to the D2 strain (see above), the modest difference in receptor density between strains could be accounted for solely by a difference in m2and/or m4autoreceptor density. Collins and colleagues (Marks et aL, 1989; Miner et al., 1984, 1989) examined the genetic influences on nicotinic receptors in the mouse brain. Nicotinic receptor binding was measured using ['HI nicotine and [aIy5I] bungarotoxin (BTX) which label different populations of receptors. Among 19 inbred strains, there was no significant difference in ['Hlnicotine

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binding in the striatum but significant differences were observed in the midbrain, hindbrain, hypothalamus, and colliculi. In contrast, BTX binding did show significant strain differences within the striatum; the highest binding was found in the D2 strain with the lowest binding in the SWR and BUB/Bn strains. The range of binding values was 75%. Outside of the striatum, BTX binding was generally low in the D2 strain. Recently, we confirmed the differences in the number of striatal cholinergic cells between the B6 and D2 strains and have shown, in addition, that among 10 inbred mouse strains, the range of variation in striatal cholinergic cell number may be as large as 100%depending on the rostralcaudal level examined (Table VII) . For the entire 10-strain panel, the strain X section effect was significant (F= 2.7, df = 45, 235, P < 0.001). The D2, C, and AKR strains formed a group which consistently showed a relatively TABLE VII CHAT CELLNUMBER IN THE STRIATUM OF 10 INBRED MOUSESTRAINS‘ Choline acetyltransferase ( C U T ) cell number/section t SE Strain C

D2 AKR C3H

A B6 P CBA 129 LP

Section 11

Section 14

Section 17

Section 20

Section 22

Section 24

86 t 3 107 2 4 77 C 3 52 t 3 67 t 2 60 t 4 101 t 6 62 2 2 73 t 7 54 t 1

137 t 5 137 t 1 102 C 3 64 2 2 93 t 4 99 t 5 132 t 7 84 C 1 115 -C 7 78 C 3

121 t 6 125 t 3 132 2 5 110 2 3 99 t 9 94 t 3 152 C 9 106 t 4 113 t 2 104 t 6

121 t 4 132 t 7 130 t 3 103 t 3 103 C 5 80 2 4 107 t 5 100 -c 4 94 t 9 103 t 3

93 C 3 106 C 4 108 2 3 84 2 3 105 t 6 69 t 4 85 5 9 81 t 4 75 t 9 74 t 5

102 2 5 85 t 2 90 2 6 62 2 2 69 t 6 66 t 2 63 -C 7 76 -C 8 59 t 4 71 t 1

“Details of the immunocytochemical procedures are found in Hitzemann et al. (1993). Briefly, a Nikon Optiphot microscope with a camera lucida drawing tube was used to obtain a permanent record of cell distribution. The pattern of cell density was traced onto paper and hand counted. Data are reported for six sections. Section 1 1 is immediately rostral to the genu corpus callosum and corresponds to section 157 of the Sidman Atlas. Section 14 is approximately 0.2 mm caudal to section 11 and is characterized by the collapse of the anterior aspects of the anterior commissure into single fiber bundles. At section 17, the nucleus accumbens has disappeared, the medial septal nuclei are well developed, but the anterior commissure has notjoined across the midline. Section 20 is characterized by the most posterior extent of the anterior commissure and the fornix; the third ventricle is clearly evident. The CUT-positive cells of the medial septal area have disappeared and the CUT-positive cells of the substantia innominata are clearly present. At section 22, the dorsal third ventricle and the lateral ventricles have joined and the caudate-putamen is clearly reduced in size. At section 24, the caudate-putamen is further reduced in size, the lateral and third ventricles are again separated, and the fimbria hippocampus is well established. For accurate alignment, a complete striatal section series, stained with cresol violet, was used.

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higher number of cells across all sections. In comparison to the B6 strain, the number of ChAT-positive cells in the D2 strain was 78, 38, 32, 65, 53, and 28% higher in sections 11, 14, 17, 20, 22,and 24, respectively. Forty-seven B6D2 F2hybrids were phenotyped for the number of striatal cholinergic cells and then genotyped for the three polymorphic microsatellites near Drd2. In the rostral, but not in the caudal, aspect of the striatum, significant associations between phenotype and genotype were detected. The data for section 14 and D9Mzt21 are representative of the results obtained (Fig. 7). Consistent with the higher cell number in the D2 strain, the F2 animals homozygous for the D2 allele have a significantly higher number of cholinergic neurons than the animals homozygous for the B6 allele. The heterozygotes were somewhat intermediate. The data also clearly show that most of the variance in the number of cholinergic neurons is not associated with D9Mit21. Assuming for the moment that the QTL detected here is the same as that associated with D2 receptor density, the data provide evidence of a QTL with multiple effects on the organization of the striatum. Lim et al. (1989) examined striatal ['HIQNB binding in the SHR, WKY, and SD rat strains. No difference was detected between the SHR and WKY strains but binding in both of these strains was modestly higher (18%) than that of the SD strain. Gilad and Gilad (1987) compared brown Norway and WKY rats for various indices related to cholinergic function. In the striatum, the WKY strain showed higher ChAT activity (+32%), choline uptake (+65%), and ACh release (+47%).There are no significant difference between strains in [ 3H]QNB binding.

(D2-D2)

h

2

(B6-D2)

9

2 (B6-B6) I

60 80 100 120 140 ChAT Cell Number, section 14, striatum FIG.7. Association of choline acetyltransferase cell number with genotype at D9Mit21. Data are presented for section 14 from the rostral striatum. Experimental details are given in the legend to Table VII.

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The Flinders Sensitive (FSL) and Flinders Resistant (FSR) rat lines were orginally bred for differential responses to the anticholinesterase, DFP (reviewed in Overstreet, 1993). The FSR line is more sensitive to apomorphine-induced stereotypes but less sensitive to raclopride-induced catalepsy (Crocker and Overstreet, 1991); these differences are consistent with increased cholinergic function in the FSL line. There are no significant differences between the lines in ['Hlsulpiride binding as determined by QM. However, the FSL line shows significantlyhigher density of [3H]QNB binding sites in the striatum and hippocampus but not cortex or hypothalamus (Daws et al., 1991; Overstreet et al., 1984).

G. STIUATAL GABASYSTEM (S)

As noted previously, the striatonigral and striatopallidal GABA neurons provide the major output from the striatum. Gabellec et al. (1980) examined GABA levels, glutamate decarboxylase (GAD) activity, and GABA transaminase (GABA-T) activity among the C3H/He and C57BR strains and their F1 hybrids. No difference between strains in any brain area, including the striatum, was found for GAD and GABA-T activity. These data extend the earlier work of Wong et al. (19'74) who observed no difference in whole brain GAD and GABA-T activity among B6, D2, CBA/Ca, CE, LP, and RF strains. Gabellec et al. (1980) did find some regional differences between the C3H and C57BR strains in GABA levels but not in the basal ganglia. Clement et al. (198'7) and Ciesielski et al. (1985) examined regional brain GABA levels and turnover rates in B6 and D2 mice at both 12 and 20 weeks of age. In the striatum and SNZc, as in most brain regions, turnover was substantially higher in the younger animals. Although there was no significant difference between the strains at either age, there was a trend for the D2 strain to a faster turnover in both brain regions. In contrast to the faster turnover, the D2 strain has a lower level of GABA in the striatum (PuglisiAllegra and Mandel, 1980; Simler et al., 1982) examined the effects of isolation-induced aggression on GABA levels in the D2 and B6 strains. A significant increase in aggressive responses was found only in the D2 strain. Both strains showed a decrease of GABA levels in the striatum, septum, olfactory bulb, and posterior colliculus. Only the D2 strain showed an increase of GABA levels in the amygdala. Strain or selected line differences in the striatal binding to benzodiazepine receptors have been detected. For example, Robertson (19'79) found significantly lower (up to 50% lower) [3H]diazepam binding in the whole brain membranes prepared from the C strain compared to the B6, B10, and AKR strains. In a subsequent paper, Robertson noted that ['Hlflunitra-

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zepam binding was somewhat lower in the B6 strain compared to the D2 strain. Shephard et al. (1982) examined the gender and regional differences in [SH]flunitrazepambinding in RHA and FWL rat lines and an unselected control line (RCA).N o difference among the lines was detected in any brain regions including the striatum bul. there was a marked gender effect with the females having a significantly lower receptor binding.

H. ENKEPHALINS, ENDORPHINS, AND OPIATE RECEPTORS Differences in opiod peptides between inbred strains have been reported (see Frischknecht et al., 1988). Within the striatum, met-enkephalin levels are higher in the D2 strain, compared to the B6 strain (Barbaccia et al., 1981); the reverse is true in the amygdala (Kulling et al., 1988). Sanghera et al. (1987) compared C and CBA mouse strains for met-enkephalin content in tissue punches taken from the SNZc, VTA, and CPu. The CBA strain had significantly higher met-enkephalin levels in all three brain regions. Blum et al. (1987) found that alcohol-preferring C58 mice have lower metenkephalin levels in both the CPu and hypothalamus when compared to the alcohol-nonpreferring C3H/CHRGL/2 strain. There were no differences between these strains in other brain areas including the pituitary, amygdala, midbrain, and hippocampus. Baran et al. (1975) measured [3H]nalox~nebinding in whole brain membranes prepared from the CXB RI series, the B6By and CBy parents, and the reciprocal F, hybrids. These authors were the first to note that opiate receptor binding was low in the CXBK strain and high in the CXBH strain. Binding in the remaining RI strains, the parent strains, and the F, hybrids was intermediate. The low binding in the CXBKstrain was consistent with the poor analgesic response in this strain. These data were the first demonstration of genetic variance in opiate receptor binding. Subsequent studies (Jacob et al., 1983; Moskowitz and Goodman, 1985; Reith et al., 1981) showed that the CXBK strain differed primarily in high-affinity p receptor sites in the periaqueductal grey, raphe nuclei, and spinal cord, and not in 6 or K binding sites. Among non-RI inbreds, there is no significant difference in naloxone binding among BGBy, CBy, and C3H strains (Baran et al., 1975; Moskowitz and Goodman, 1985; Reith et al., 1981). Also, there appears to be no difference between the B6 and the C strains. Garcin et al. (1978) were the first to note a significant difference between the B6 and D2 strains in whole brain naloxone binding. Reggiani et al. (1980) proposed that the difference between the B6 and D2 strains in the morphine-induced running fit was associated with difference in either the density or the type of opiate receptor

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85

on DA terminals in the striatum. These authors confirmed that morphine increased DA turnover in the striatum and limbic forebrain of the B6 but not the D2 strain, as evidenced by differences in the accumulation of the DA metabolite, DOPAC. In the striatum, and not the brain stem, forebrain, or neocortex, the B6 strain showed nearly a 100% increase in the binding of the opiate agonists, DAME and Leuenkephalin. Further, &OH DA lesions reduced binding in the B6 strain to the D2 level without affecting binding in the D2 strain. These data suggest that the. B6 strain has a markedly higher density of one or more subtypes of opiate receptors on DA terminals.

I. WEAVER( w v ) MUTANT Mice homozygous for the autosomal recessive gene weaver (wv) show an impaired development of the cerebellum and an ataxia that is evident by Postpatal Days 10-12 (Rackic and Sidman, 1973). Histological examination of the cerebellum reveals a marked loss of granule cells in the cerebellar vermis. The wv gene is located on mouse chromosome 16 (Green, 1981), distal to Sod-1 and approximately 1 cM proximal to Ets-2 (Reeves et al., 1989;Mjaatvedt et al., 1993).The homologous region in the human genome is found on chromosome 21. Gao et al. (1992) has shown that the wv gene encodes an nonautonomous signal for CNS neuronal differentiation. The w mutant is also important to understanding, from the genetic perspective, the functional organization of the basal ganglia. Lane et al. (1977) appears to be the first to note the loss of forebrain DA in the wv/wu mouse. Subsequent studies by numerous groups characterized the nature of the DA deficit (see, e.g., Schmidt et al., 1982;Roffler-Tarlov and Graybiel, 1984; Graybiel et al., 1990; Roffler-Tarlov et al., 1990; Pullara and Marshall, 1989; Simon et al., 1991, 1994; Richter et al., 1992; Simon and Ghetti, 1993; Loughlin et al., 1992; Stotz et al., 1993). The changes found in the DA system may be summarized: (1) there is a specific loss of DA neurons in the dorsal but not the ventral striatum; the loss is more pronounced among the striosomes compared to the matrix compartment; (2) TH-positive neurons are lost in the SNZc and RRF areas but the VTA is relatively spared; (3) prior to the loss of DA, TH activity, or cell death in the striatum, a defect in DA transporter activity can be detected; these data suggest that the DA transporter may be an early target of the wv mutation; (4) amphetamine, but not K', evoked DA release from the residual striatal neurons remaining in the wv/ wv mouse is augmented suggesting that compensatory mechanisms develop to overcome the DA deficits; (5) there is a significant increase of D2 receptor binding in the dorsal but not the ventral striatum when compared to the behaviorally normal heterozygote ( + / w v ) . D1 re-

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el ul.

ceptor binding is decreased throughout the striatum of the w v / w mice; (6);in w v / w but not +/wvrnice, the density ofboth 6 or K opiate receptors is reduced in the CPu and the NAc; the density of p. receptors in both the striosome and matrix compartments of the striatum was unchanged; and (7) compared to +/+ mice, w/wv mice show a 200% increase of 5-HT in the dorsal straitum and a 50% increase of 5-HT in the ventral striatum. Overall, the WL, gene initiates a complex cascade of events which result in the loss of DA neurons and a functional reorganization of the basal ganglia.

V. Conclusions

The studies cited in this chapter provide a sampling of the research which deals either directly or indirectly with genetic influences on the function of the basal ganglia. Numerous citations were not included. For example, there is a much larger genetic literature than cited here on the role of DA in differential ethanol responses among selected lines or inbred strains. Despite such limitations, it is hoped that the reader will be impressed with the genetic diversity of the basal ganglia. Further, it is hoped that this chapter will encourage new investigators to engage in genetic studies. With the advent of new techniques in molecular biology and the availability of dense genetic maps, it is now technically possible to map the QTL associated with any phenotype, providing the phenotype has at least a modest heritability. Clearly, many phenotypes associated with the basal ganglia meet this criteria.

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Schwab, C., Bruckner, G., Biesold, D., Castellano, C., and Oliverio, A. (1988). Trends CZin. Neurophannacol. 3, 251-256. Schwab, C., Bruckner, G., Castellano, C., Oliverio, A,, and Biesold, D. (1990a). Dementia 1, 74-78. Schwab, C., Bruckner, G., Castellano, C., Oliverio, A., and Biesold, D. (1990b). Neurochem. &s. 15, 1127-1133. Schwab, C., Bruckner, G., Roethe, T., Castellano, C., and Oliverio, A. (1992). Neurochem. Res. 17, 1057-1062. Seale, T. W., and Carney, J. M. (1991).J. Addict. Dis. 10, 141-162. Seale, T, W., McLanahan, K,Johnson, P., Carney,J. M., and Rennert, 0.M. (1984a).Pharmacol. Biochem. Behav. 21, 237-244. Seale, T. W., Johnson, P., Carney, J. M., and Rennert, 0. M. (1984b). Phamacol. Biochem. Behav. 20, 567-573. Seale, T. W., Ala, K. A., Cao, W., Parker, K. M., Rennert, 0.M., and Carney, J. M. (1986). P h a m c o l . Biochem. Behav. 25, 1271-1277. Seeman, P., and Van Tol, H. H. M. (1994). Trends Pharmacol. Sci. 15, 264-270. Severson, J. A., Osterburg, H. H., and Finch, C. E. (1981). Neurobiol. Agzng2, 193-197. Shephard, R. A., Nielsen, E. B., and Broadhurst, P. L. (1982). Eur.J Pharmacol. 77, 327-330. Shimada, S., Kitayama, S., Lin, C. L., Patel, A., Nanthakumar, E., Gregor, P., Kuhar, M., and Uhl, G. (1991). Science 254, 576-578. Short, P. H., and Shuster, L. (1976). Psychopharmacology (Berlin) 48, 59-67. Shuster, L., Webster, G. W., Yu, G., and Eleftheriou, B. E. (1975). Psychophamacolugy (Berlin) 42, 249-254. Shuster, L., Yu, G., and Bates, A. (1977). Psychcpharmacolo~(Berlin) 52, 185-190. Simler, S., Puglisi-Allegra, S., and Mandel, P. (1982). Pharmacol. Biochem. Behav. 16, 57-61. Simon, J. R., and Ghetti, B. (1993). Neurochem. Int. 22, (No. 5) 471-477. Simon, J. R., Yu, H., Richter, J. A., Vasko, M. R., and Ghetti, B: (1991). J. Neurochem. 57, 1478- 1482. Simon, J. R., Richter, J. A., and Ghetti, B. (1994). j . Neurochem. 62, 543-548. Skrinskaya, J. A., Nikulina, E. M., and Popova, N. K. (1992). Pharmacol. Biochem. Behav. 42, 261-267. Smith, D. L., Julian, A,, and Erwin, B. G . (1992). Mvuse Genome 90, 439-440. Smolen, A., Smolen, T. N., Oh, E. I., and Collins, A. C. (1986). P h a m c o l . Biochem. Behav. 24, 1077-1082. Sokoioff, P., Giros, b., Martres, M. P., Bouthenet, M. L., and Schwarz, J. C. (1990). Nature (London) 347, 146-151. Sprott, R. L. (1975). Exp. AgingRes. 1, 313-323. Sprott, R. L., and Eleftheriou, B. E. (1974). Gerontologza 20, 155-162. Stoof, J. C., Drukarch, B., De Boer, P., Westerink, B. H., and Groenewegen, H. J. (1992). Neuroscience (Oxford) 47, 755-770. Stotz, E. H., Triarhou, L. C., Ghetti, B., and Simon, J. R. (1993). Brain a s . 606, 267-272. Sunahara, R. K , Niznik, H. B., Weiner, D. M., Stormann, T. M., Brann, M. R., Kennedy, J. I.., Gelernter, J. E., Rozmahel, R., Yang, Y., Israel, Y., Seeman, P., and Odowd, B. F. ( 1990). Nature (London) 347, 80-83. Surmeier, D. J., Reiner,A., Levine, M. S., a n d k i a n o , M. A. (1993). TrendsNeurosci. 16,299-305. Suzuki, T., Otani, R, Koike, Y., and Misawa, M. (1988).Jpn. J. P h a m c o l . 47, 425-431. Suzuki, T., Otani, K., and Misawa, M. (1991).Jpn. J. Phannacol. 57, 455-462. Sved, A. F., Baker, H. A., and Reis, D. J. (1984). Bruin Res. 303, 261-266. Taylor, B. A. (1978). In “Recombinant Inbred Strains: Use in Gene Mapping” (H. C. Morse, ed.), pp. 423-438. Academic Press, New York.

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Tiberi, M., Jarvie, K. R., Silvia, C., Falardeau, P., Gingrich, J.A., Godinot, N., Bertrand, L., Yang-Feng, T. L., Fremau, R. T., and Caron, M. G. (1991). Proc. Natl. Acad. Sn'. U.S.A. 88, 7491-7495. Tolliver, B. K., Belknap, J. C., Woods, W. E., and Carney, J. M. (1994).J. P h a m c o l . Exp. Thm. 270, 1230-1238. Trabucchi, M., Spano, P. F., Racagni, G., and Oliverio, A. (1976). Bruin Res. 114, 536-540. Tunnicliff, G., Wimer, C. C., and Wimer, R. E. (1973). Bruin Res. 61, 428-434. Usdin, T. B., Mezey, E., Chen, C., Brownstein, M..[., and Hoffman, B. J. (1991). Proc. Natl. Acad. Sn'. U.S.A. 88, 11168-11171. Vadasz, C . , Baker, H., Joh, T. H., Lajtha, A., and Reis, D. J. (1982). Brain Res. 234, 1-9. Vadasz, C., Sziraki, I., Murthy, L. R., Vadasz, I., Badalmenti, A. F., Kobor, G., and Lajtha, A. (1987).J. Neurogaet. 4, 241-252. Vadasz, C., Kobor, G., and Lajtha, A. (1992a). Behnv. Brain Res. 48, 29-39. Vadasz, C., Kobor, G., and Lajtha, A. (1992b). Behav. Brain Res. 48, 41-47. Vadasz, C., Laszlovszky, I., De Simone, P. A,, and Fleischer, A. (1992~).J. Neurocha. 59, 793-808. Van Tol, H. H. M., Bunzow, J. R., Guan, H. C., Sunahara, R. K., Seeman, P., Niznik, H. B., and Civelli, 0. (1991). Nature (London) 350, 610-614. Vetulani, J., Sansone, M., and Oliverio, A. (1982). Phamacol. Biocha. Behav. 17, 967-971. Vriend, J., Alexiuk, N. A., Green-Johnson, J., and Ryan, E. (1993).J. Neurochem. 60, 1300-1307. Waelbroeck, M., Tastenoy, M., Camus,J., and Chrisophe,J. (1990). Mol. P h a m c o l . 38,267-273. Wafford, K. A,, Burnett, D. M., Leidenheimer, N. J., Burt, D. R., Wang, J. B., Kofuji, P., Dunwiddie, T. V., Harris, R. A,, and Sikela, J. M. (1991). Neuron 7, 27-33. Wax, T. M. and Goodrick, C. L. (1975). Dew. Psychobiol. 8, 297-303. Wiener, H. L., and Reith, M. E. (1990). Phannucol. Biocha. Behau. 36, 699-701. Witkin, J. M., and Goldberg, S. R. (1990). P h a m c o l . Biocha. Behuv. 37, 339-342. Wong, E., Schousboe, A., Saito, K., Wu, J. Y., and Roberts, E. (1974). Bruin Res. 68, 133-142. Yasuda, R. P., Ciesla, W., Flores, L. R., Wall, S.J., Li, M., Ssatkus, S. A., Weisstein, J. S., Spagnola, B. V., and Wolfe, B. B. (1992). Mol. Phunnacol. 43, 149-157. Yoshimoto, K., and Komura, S. (1989). Alcohol Alcohol. 24, 225-229.

STRUCTURE AND PHARMACOLOGY OF VERTEBRATE GABAA RECEPTOR SUBTYPES

Paul J. Whiting, Ruth M . McKernan, and Keith A. Wafford Neuroscience Research Centre, Merck Sharp & Dohme, Harlow, Essex CM20 2QR, England

I. Introduction to the GABAA Receptor 11. Diversity of the GABA, Receptor Gene Family A. Cloning of Subunit cDNAs B. Additional Variants Created by Alternative Splicing C. Chromosome Assignment of GABAA Receptor Subunit Genes D. Structure of GABAAReceptor Subunit Genes 111. Phosphorylation of GABAA Receptors A. Phosphorylation of PKA B. Phosphorylation by PKC C. Regulation by Other Kinases N. The Composition of GABAA Receptors in Vzuo A. In Sztu Hybridization B. Immunoprecipitation with Subunit-Specific Antisera C. Western Blot Analyses D. Immunolocalization E. Subunit Stoichiometry of GABA, Receptors V. Drug Binding Sites on the GABA, Receptor A. Benzodiazepines B. GABA C. Steroids D. Barbiturates E. Picrotoxin [ t-Butylbicyclophosphorothionate(TBPS)] F. Ethanol G. Loreclezole H. Anesthetics I. Zinc J. Other VI. Conclusion References

1. Introduction to the GABA, Receptor

y-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate central nervous system (CNS). It modulates inhibitory tone throughout the CNS by activating two classes of receptors, GABAA INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 3R

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and GABAB. The latter are, at the molecular level, uncharacterized, although they are known to act via G proteins to activate second messenger systems within the cell. GABABreceptors will not be discussed further. GABA, receptors are ligand-gated ion channels. The binding of the agonist GABA to the multimeric protein which constitutes the receptor complex results in the rapid opening of the integral ion channel through which anions (primarily chloride) flow down their concentration gradient. Electrophysiological experiments suggest that at least two GABA molecules must bind to the receptor for receptor activation, i.e., the receptor contains at least two GABA binding sites (Sakmann et al., 1983). The GABAAreceptor could be considered a pharmacologist’s heaven. As well as the GABA binding site, for which a number of different agonists and antagonists are known, there are several other ligand binding sites. Occupation of these can influence the opening of the channel, either potentiating the effect of GABA or inhibiting it. Barbiturates, neurosteroids, ethanol, and general anesthetics, such as halothane, all act through their independent modulatory sites on the receptor to allosterically enhance the effect of GABA. Loreclezole and ivermectin also appear to allosterically enhance the effect of GABA by binding to distinct sites on the GABAA receptor. Compounds acting at the benzodiazepine (BZ) binding site of the GABAAreceptor can allosterically enhance the effect of GABA (so-called BZ agonists, which are sedative, anticonvulsant, and anxiolytic), inhibit the effect of GABA (so-called BZ inverse agonists, which are proconvulsant), or simply inhibit the effects of the agonists and inverse agonists (so-called BZ antagonists). Finally, picrotoxin acts via a unique site, at or close to the channel, to inhibit the effect of GABA. The pharmacology of recombinant GABA, receptors is discussed in detail under Section V. The purpose of this chapter is not to exhaustively review GABAAreceptors, but to describe how the techniques of molecular neurobiology have, within a relatively short time frame, enabled a revolution in our understanding of the GAF3AA receptor. Our knowledge of the diversity, structure, function, and pharmacology of the GABA, receptor family has increased almost exponentially over the past few years, as we hope will become clear in the subsequent sections of this chapter. II. Diversity of the GABA, Receptor Gene Family

A. CLONING OF SUBUNIT cDNAs Until 1987 evidence for heterogeneity of GABAAreceptors was based primarily on pharmacological and biochemical analyses. Using radiolabeled

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BZ site ligands and performing competition binding assays it was observed that BZ binding sites were not a homogeneous population. Indeed, BZ binding sites could be divided into type I and type 11, the former having higher affinity for certain compounds, such as the triazopyridazine CL 218,872 and some P-carbolines (Squires et aL, 1979). Similarly, by performing photoaffinity labeling experiments with [’HI flunitrazepam and analyzing the labeled protein by SDS-PAGE, several labeled polypeptides could be visualized,one interpretation of which was receptor heterogeneity (Sieghart and Drexler, 1983). One of the key steps toward the molecular cloning of the GABAA receptor was the development by Barnard and co-workers of a protocol for the “large-scale” purification of receptor from bovine brain (Sigel and Barnard, 1984). Microsequencing of tryptic peptides generated sufficient sequence to design oligonucleotide probes for screening of a bovine brain cDNA library. Two cDNA sequences were initially identified, referred to as al and PI (Table I), encoding polypeptides containing open reading frames of 456 and 474 amino acids, respectively (Schofield et al., 1987). Further analysis of cDNAs indicated that a second cDNA sequence had been isolated encoding an open reading frame of 451 amino acids, with 79% identity to a1and only 34% identity to pl; it was thus referred to as ae (Levitan et al., 1989). The cloning and sequencing of these cDNAs was the breakthrough in our understanding of the GABAAreceptor. By screening brain cDNA libraries at low stringency with cDNA probes and degenerate oligonucleotide probes based on sequences conserved between these subunit cDNAs, a family of GABAAreceptor subunit cDNAs was subsequently identified (Table I ) . This work was primarily carried out by Seeburg and colleagues, but many other groups have contributed to the task. The polypeptides are subdivided according to their relative amino acid sequence identities. Thus, within a subgroup, i.e., the CY subunits, there is 70-80% sequence identity, whereas between the subgroups, e.g., CY and /3 subunits, there is only 30-40% sequence identity. In both these comparisons, the regions of highest sequence identity are in the putative transmembrane domains and most of the putative N-terminal extracellular domain, while the regions of the most diversity are the putative signal peptide and large cytoplasmic domain. The dendrogram in Fig. 1 indicates the relative homologies and evolutionary relationships of the subunits of the human GABAAreceptor gene family. The deduced amino acid sequences of GABAA receptor subunit show significant sequence identity (20-30%) with other ligand-gated ion channels [the nicotinic acetylcholine receptor (nAChR), the glycine receptor, and the 5-HT3 receptor] and are thus members of an extended ligand-

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el al.

TABLE I GABA~ RECEPTOR SUBUNITS ~~~

~

Subunit ffl

ff2

Species Chicken Mouse Rat Bovine Human

456

Mouse Rat

45 1

Bovine Human ffS

ff4

ff5

a6

PI

Pz

Mouse Rat Bovine Human

P4

YI

492

Bateson et aL, 1991a Keir et al., 1991; Wang et al., 1992 Seeburg et al., 1990 Malherbe el al., 1990 Schofield et al., 1987 Schofield el al., 1989 Wang et al., 1992 Seeburg et al., 1990; Khrestchatisky et al., 1991 Levitan et al., 1988 Hadingham et al., 1993a Wang et al., 1992 Seeburg et al., 1990; Malherbe et al., 1990 Levitan et al., 1988 Hadingham et id, 1993a Seeburg el al., 1990; Wisden el al., 1991 Ymer et al., 1989a P. J. Whiting, unpublished

Rat Bovine Human

552 550

Rat

464

Human

462

Khrestchatisky et al., 1989; Pritchett and Seeburg, 1990; Malherbe et al., 1990 Wingrove et al., 1991

Mouse Rat Human

443 453 453

Kato, 1990 Luddens et al., 1990 P. J. Whiting, unpublished

Rat Bovine Human

474

Ymer et al., 1989b; Malherbe et al., 1990 Ymer et al., 1989b Schofield el al., 1989

Chicken Rat Bovine Human

PS

Reference

Amino acids

Chicken Rat Human Chicken Chicken Rat Bovine Human

474(P,) 491 ( P d 474 474 474 473 484(Pd 488(P4,) 465

Hatvey et al., 1994 Ymer et al., 198!Jb; Lolait et al., 1989 Ymer et al., 1989b Hadingham et al., 1993b Bateson et al., 1990 Ymer et al., 1989b; Lolait et al., 1989 Wagstaff et aL, 1991 Bateson et al., 1991b Glencourse et al., 1993 Ymer et aL, 1990 Ymer et al., 1990 Ymer et al., 1990 ( continues)

STRUCTURE AND PHARMACOLOGY OF GABA, RECEPTOR SUBlWES TABLE I Subunit

Species

y2

Chicken Mouse Rat

(Continued)

Amino acids

468(y2,) 476(Yz~)

Reference Glencourse et al., 1990 Kofuji et al., 1991 Shivers et aZ., 1989; Malherbe et al., 1990 Shivers et al., 1989; Malherbe et al., 1990 Whiting et al., 1990 Pritchett el al., 1989

Bovine Human Mouse Rat Human

467

Wilson-Shaw et al., 1991 Knoflach el al., 1991; Herb et aL, 1992 Hodingham et al., in press

y4

Chicken

457

Harvey et aL, 1993

6

Mouse Rat Human

449 452

Sommer et al., 1990 Shivers et at., 1989 P. J. Whiting, unpublished

p,

Human

473

Cutting et aZ., 1991

p2

Human

ys

99

Cutting et aL, 1992

a1 a2 a3 a5 a4 a6

Yl P

fl

I

I--

PI P2

P3 I

s Pl I P2

FIG. 1. Dendrogram of deduced-primary amino acid sequences of the human GABA, receptor subunit family. The length of the line separating subunits represents the distance between their sequences.

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PAUL J. WHITING el al.

gated ion channel gene family. GABAAreceptor subunits thus have the same structural motifs found in other members of this gene family: 1. Four hydrophobic regions (TMI -TM4) of membrane-spanning length. 2. A large putative extracellular domain and large putative cytoplasmic domain between TM3 and TM4. 3. A putative signal peptide. 4. Two conserved cysteine residues separated by 13intervening residues (which are believed to be disulfide linked in the nAChR) . 5 . Consensus Nglycosylation sites in the putative N-terminal domain. 6. Consensus phosphorylation sites in the putative large cytoplasmic domain.

VARIANTS CREATED BY ALTERNATIVE SPLICING B. ADDITIONAL Alternative splicing is a mechanism by which additional sequences can be included, o r omitted, from the mature mRNA species. If these sequences are within the coding region of the protein this obviously will result in the inclusion or omission of certain amino acid sequences. It is thus a mechanism for creating additional diversity from a single gene. Within the GABAA receptor gene family, alternative splicing has been identified in four subunits: (Y6, &, &, and y 2 . The splice variant of the (Y6 subunit leads to the omission of 10 amino acids from the N-terminal domain and does not form functional receptors (Korpi et al., 1994). It appears to be present in about 20% of as mRNAs in the mouse cerebellum, arises apparently through incorrect splicing at the 3' splice site within the exon, and has no obvious physiological role. The p2 and y 2 variants arise by inclusion or omission of an additional (Whiting exon, giving an additional 17 (fiZL)(Harvey et aL, 1994) or 8 (yZL) et al., 1990; Kofuji et al., 1991) amino acids within the putative large cytoplasmic domain. While the y2 splice variants are ubiquitous, the P2variants have only been identified in chickens. Interestingly, for both subunits the additional sequence contains a putative protein kinase C phosphorylation site, suggesting a physiologically important role. Further, the yZLsplice variant has been suggested to be necessary for the ethanol potentiation of GABAA receptors (see Sections III,B and V,F). The splice variant of the & subunit arises through an alternative 5' splice donor site, leading to the inclusion (&) of an additional four amino acids in the putative large cytoplasmic domain (Bateson et al., 1991b). Its physiological role is unclear.

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101

C. CHROMOSOME ASSIGNMENTOF GABAARECEPTORSUBUNIT GENES Chromosome assignment of all the genes for all the human CABAA receptor subunits has been performed (Table 11). It is of interest to note the clustering of genes. For instance, a l ,ps, and y2 are clustered and are known to coassemble to form a major receptor population (see Section W ). Similarly, and y2 are clustered, as are a2and yl; these subunit pairs are known to coassemble to form major subunit populations (see Section IV). This clearly suggests some sort of coordinated regulation of gene expression. However, this is obviously somewhat of an oversimplification since, for instance, the y2 subunit is known to coassemble with other a subunits in addition to a1and (Y6. The a5,p3, and y 3 genes are also linked on mouse chromosome 7 (Nakatsu et al., 1993).Mutant mouse lines have been generated which lack all three genes or just the a5and/or y 3 (Nakatsu et al., 1993;Culiat et al., 1994).The mice lacking a5and/or y3genes appear phenotypically normal, while mice lacking all three genes have a lethal cleft palate defect; those mice which do survive show a variable neurological phenotype (tremor, jerky gait). These data suggest that deletion of the p3 gene alone is responsible for the defects. In humans the inherited disorder Angelman syndrome (Angelman et al., 1965),characterized by neurological dysfunctions (mental retardation, seizures, ataxia, absence of speech, jerky movements), has been mapped to the same locus [15qll (Williams et al.,

TABLE I1 CHROMOSOME ASSIGNMENTOF HUMANGABA~ RECEPTORSUBUNIT GENES ~

~

~

GABAAreceptor subunit gene

~~

___

~

_

Human chromosome assignment

~

'Buckle et al. (1989). 2Wilcox et al., unpublished. 'Hicks et al. (1994). 'Wilcox et al. (1992). 5Kn011 et al. (1993). Wagstaff et al. (1991). 'Cutting et al. (1992). *Sommer et al. (1990).

_

_

102

PAUL J. WHITING et at.

1989)] as the a5,p3,and y3 genes (Sinnett at al., 1993). Whether mutation in any of these genes is actually responsible for the disorder is currently unclear.

D. STRUCTURE OF GABAA RECEPTORSUBUNIT GENES Having identified a number of genes which make up the GABAAreceptor family, one obvious question is how is the expression of these genes controlled in what is apparently a very deliberate and precise manner? At the molecular level, one way of getting at this question is to determine the structure of the gene and characterize the elements within the promoter which control transcription. Like other ligand-gated ion channels, GAF5AA receptor subunit genes contain introns. The structures of the mouse 6 (Sommer et aL, 1990) and human p, (Kirkness et al., 1991) subunit genes have been determined. They have nine exons, with the position of the intron/exon boundaries exactly conserved. Thus, the eight introns must have been present in a common ancestral gene before divergence of the receptor subunits. Interestingly, however, when the structures of GABAA receptor and nAChR subunit genes are compared it is clear that the intron/exon boundaries are in different positions. Thus, the intron sequences must have been introduced subsequent to divergence of these genes from a common ancestor. At this time of this writing, promoter regions of the human a1subunit (Kang et al., 1994) and ps subunit (Kirkness and Fraser, 1993) genes and the rat 6 subunit gene (Motejlek et al., 1994) have been identified. It is clear from both sequence analysis and gel mobility shift assays that, like other eukaryotic genes, transcription from these genes will be controlled by numerous protein factors. Such studies are, however, at a relatively early stage.

111. Phosphorylalion of GABA, Receptors

In common with other members of the ligand-gated ion channel family (Huganir and Greengard, 1990), the function of the GABAA receptor is modulated by phosphorylation. Indeed, purified native GBAA receptor protein can be phosphorylated in vitro by cyclic AMPdependent protein kinase A (PKA) and calcium/phospholipiddependent protein kinase C (PKC) (Browning et al., 1990). Analysis of the deduced amino acid se-

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103

quences of GABAAreceptor subunits indicates that the large cytoplasmic loop domains contain consensus sites for phosphorylation by PKA (human as,a4,a67 01,P2, P3,y l , y2, and y ~ )PKC , (all human subunits), and tyrosine kinase (yl, y2, y 3 ) .It is this domain of the nAChR which undergoes phosphorylation (Huganir and Greengard, 1990).

A. PHOSPHORYLATION BY PKA The effects of PKA-mediated phosphorylation on native GABAA receptors have been investigated using PKA activators, such as forskolin [although such data needs to be interpreted with caution, see Ticku and Mehta (1990)], or using purified catalytic subunit of PKA. PKA simulation in rat synaptosomes (Heuschneider and Schwartz, 1989) and cultured chick cortical neurons (Tehrani et al., 1989) and injection of the catalytic subunit of PKA into cultured spinal chord neurons (Porter et al., 1990) all result in a decrease in the GABA-mediated chloride current/flux. The latter study used single-channel analysis to demonstrate that this was primarily a decrease in channel-opening frequency. These data are in accord with the studies of Moss et al. (1992a) who used recombinant GABAA receptors (a1 PI and alPlyZsreceptors) expressed in HEK 293 cells and native GABAA receptors expressed in cultured sympathetic ganglia to demonstrate a PKAmediated decrease in GABA-gated currents. Further, site-directed mutagenesis indicated that this effect was mediated by phosphorylation of Ser409 of the PI subunit. Interestingly, in uitro experiments using purified recombinant polypeptide have demonstrated that P1Ser409can be phosphorylated by both PKA and PKC (Moss et al., 1992b). In contrast to the effects described above, it has been reported that in cerebellar Purkinje cells the activation of PKA results in an upregulation of GABAA receptor function (Sessler et aL, 1989). There is a physiological correlate to this finding; the GABAA-mediatedinhibition of Purkinje cell firing is mediated by the activation of the Purkinje cell PI adrenergic receptors which presumably activate PKA (Parfitt et al., 1990). One could possibly reconcile these two opposite effects of PKA activation on G B A Areceptor function by proposing that Purkinje cells express a different GABAAreceptor subunit from those found in other cell types. Indeed, Purkinje cells are thought to express only the a],P2, P3, and 7 2 subunits (Laurie et al., 1992); not the PI subunit, through which the PKAmediated GABAA receptor inhibition can be mediated (Moss et al., 1992a). Angelotti and co-workers (1993b) found that expression of recombinant alPlyZs (but not alp1)in L929 cells overexpressing PKAresulted in increased GABA-gated chloride currents compared to expression in “wild-type” L929

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PAUL J. WHITING el al.

cells. This increase in GBA-gated currents was prevented by mutation of Ser409 of the PI subunit (cf. Moss et al., 1992a, discussed above). There was no change in the channel properties of the receptors expressed in the cells overexpressing PKA, suggesting an increase in the number of functional receptors through, for example, decreased turnover or increased receptor assembly. PKA-mediated phosphorylation of P,Ser409 may elicit (1) a decrease in receptor function on a short time scale through, e.g., decreasing the frequency of channel opening; and (2) on chronic exposure, increase GABA-gated currents by increasing the number of functional receptors.

B. PHOSPHORYLATION BY PKC The modulation of GABAA receptor function through mediated PKC has been addressed primarily using kinase activators such as phorbol esters. GABAA receptors expressed in Xenopus oocytes as a result of injection of chick brain mRNA (Sigel and Baur, 1988) or a5P2y 2cRNAs are inactivated by application of phorbol esters. Similarly, using a chloride flux assay, phorbol esters inhibited the function of GABAA receptor in mouse brain microsacs (Leidenheimer et aL,1992).Studies using sitedirected mutagenesis have been able to precisely identify the amino acid residues in the receptor subunits which are phosphorylated and correlate this with the functional consequences of phosphorylation of that residue. In uitrolabeling experiments using purified recombinant receptor polypeptides have demonstrated that P1Ser409,y2,Ser327, and y2,Ser343 (the Ser residue in the extra eight amino acids) can be phosphorylated by PKC (Moss et aL, 1992b). In good agreement with this biochemical data, Kellenberger and colleagues (1992) found that mutation of P2Ser4lO (the homologous residues to PISer409) or y2,Ser327 decreased the PKC-mediated inactivation of recombinant alP2y,,receptors expressed in Xenopus oocytes. A more extensive study by Smart and co-workers (Krishek et al., 1994) used sitedirected mutagenesis of receptors expressed in transfected cells to demonstrate biochemically the PKC-mediated phosphorylation of P1Ser409, y2,Ser327, and y2,Ser343. This study also demonstrated that phosphorylation at any one of these residues resulted in a decrease in the GABA-activated current amplitudes; mutation of all three residues provided a receptor which was no longer modulated by phorbol esters. Interestingly, phosphorylation at ~ ~ ~ S e 1 -provided 343 the greatest effect, i.e., the phosphorylation sites are not necessarily equivalent. The previously mentioned studies of Krishek and co-workers clearly demonstrate that the Ser residue in the extra 8 amino acids of yZLis a functional PKC phosphorylation site. This suggests that having two y 2 sub-

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units which differ by the presence or absence of an additional PKC phosphorylation site does indeed have physiological consequences. In this context it is of interest to note that the extra 17 amino acids of the alternatively spliced exon of PeL(which may only be found in the avian genome) also contain a PKC consensus phosphorylation site (Harvey et al., 1994). One property of receptors containing a y2[,subunit is that their GABA-gated currents can be potentiated by ethanol. Indeed, we demonstrated that pL mutation of yzLSer343ablated the ethanol potentiation of a I ~ I yreceptors expressed in oocytes, as did incubation of oocytes expressing wild-type alpIyzLreceptors in the kinase inhibitor H7, suggesting that phosphorylation of yzLSer343is required for ethanol potentiation. The mechanism by which this occurs and the physiological relevence are currently unclear. BY OTHER KINASES C. REGULATION

The y subunits all contain tyrosine kinase consensus phosphorylation sites. Whether these sites can indeed be phosphorylated, and the functional consequences, are currently unknown. A synthetic peptide containing the sequence around y,,Ser343 can be phosphorylated by calcium/calmodulindependent protein kinase I1 as well as by PKC (Machu et al., 1993). Whether this kinase can phosphorylate this site in the assembled receptor is unknown. There may of course be other, as yet unidentified kinases which phosphorylate the GABAA receptor and thereby modulate function. This is suggested by the work of Stelzer et al. (1988), who investigated the “run down” phenomenon observed in electrophysiological experiments, and more recently Gyenes et al. (1994). This run down was prevented by addition of phosphorylation factors (ATP, magnesium) suggesting that, in contrast to the PKA and PKC-mediated events described previously, phosphorylation of the GABAA receptor was actually required for maintainance of receptor function. The emerging data clearly demonstrate that phosphorylation is an important mechanism for regulation of GABAAreceptor function, both in the short term and possibly in the long term. Many issues remain, one of the most obvious being what are the physiological consequences of GABAA receptor phosphorylation? Additionally, how is dephosphorylation of the receptor mediated and what mechanisms regulate dephosphorylation? IV. The Composition of GAB& Receptors in vivo

The pentameric structure of the GABAA receptor is based on analogy with the nicotinic receptor (Unwin, 1989), the physicochemical properties

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of the solubilized receptor (Stephenson, 1988), and electron microscopic studies of purified receptor preparations (Nayeem et ab, 1994).Five subunits are thought to be required to form an intact receptor and in brain these can be selected from a possible repertoire of 13 allowing for a huge multiplicity of GABAAsubtypes. If there were no constraints o n subunit assembly a theoretical maximum of 135(371,293) different receptor subtypes would be possible. However, since at least one cr subunit, one /3 subunit, and one y subunit are required to form a fully functional receptor (Pritchett et al., 1989b), the number of theoretical possibilities is reduced. If the structure of receptors is constrained to contain one of each subunit type this would reduce the theoretical number of possible subtypes to 9126 (6 X 3 X 3 X 13 X 13). Neither of these estimates takes into account restrictions on the positioning of subunits around the channel which may also be an important factor. For example, there is evidence that the benzodiazepine site is formed by both the a and y subunits (see section V,A) and therefore it is likely that these are nearest neighbors in the receptor. Understanding which subunits are involved in forming the many allosteric binding sites on GAJ3AA receptor could also provide contributory information on subunit structure. It is likely that, as for other ligand-gated channels, there is a directed pattern of subunit assembly so that the theoretical number of combinations far exceeds the number of subtypes which are actually formed. The number of subtypes which do indeed occur, and hence, the real extent of GABA, receptor subtype structural and pharmacological diversity, remains unknown. Several experimental approaches are, however, being used to attempt to define which subunits coassemble to form naturally occurring GMAA receptor populations in viva

A. IN SITU HYBRIDIZATION Colocalization of subunits using in situ hybridization has provided evidence of which subunits exist in the same cell type, and hence, might also be assembled in the same receptor molecule. Such an approach is limited by the presence of multiple subunits in one brain region or even one cell type. For example, granule cells of the rat dentate gyrus express 12 different subunits, i.e., all except (YE (Wisden et al., 1992). It would not currently be possible to deduce which subunits coassemble to make a receptor in the cells from this brain region. The extensive mapping studies of Wisden et al. (1992) Laurie et al. (1992), and Persohn et al. (1992) have lent support to the existence of many subtypes included in Table IV.By examining brain structures of cultured neuronal cell preparations which express a restricted

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repertoire of subunits it might be possible to draw some conclusions about the composition of selected GABAA receptors. For immortalized cell lines, examining the subunits expressed seems of limited value. Receptors comprising a&& subunits are expressed in immortalized hypothalamic GT1-7 neurons (Hales et aZ.,1992) and asand fl subunits can be detected in IMR32 neuroblastoma cells (Noble et al., 1993). There is no evidence for the existence of either of these two subunit combinations in viva It should be noted that culturing cells, particularly those which have been differentiated or derived from tumors, may induce the expression of subunit combinations which do not necessarily reflect those present in neurones in viva A wide variety of cell lines have now been screened for GABAA subunit mRNAs using the polymerase chain reaction. More than 10 cell lines have been identified which express mRNA for GABAA receptors but do not have functional GABAAreceptors (Tyndale et al., 1994; Hales and Tyndale, 1994). Primary cultures may more accurately display naturally occurring GABAA receptor subtypes. Cerebellar granule cells have provided a good model in which to study the a&y2 and a1P2y2 subtypes which they express abundantly (Beattie and Siegel, 1993; Bovolin et al., 1992;Mathews et al., 1994; Thompson and Stephenson, 1994). Finally, it is of interest to note that human pancreatic tissue contains just three GABAA subunits a2,&, and y1 (Yang et al., 1994), strongly suggesting that these three subunits form a functional receptor in this organ.

B. IMMUNOPRECIPITATION WITH SUBUNIT-SPECIFIC ANTISERA Solubilized receptors from rat and bovine brain or from affinity-purified receptor preparations have been immunoprecipitated with combinations of antibodies. This method relies heavily on the generation of appropriately selective high-titer, high-affinity antisera, and is only robust for abundant subunits where significant proportions of receptor can be immunoprecipitated. It is deduced that two subunits exist in a receptor when the sum of the proportion of receptors immunoprecipitated by a combination of two antisera is significantly less than the sum of the proportion of receptors immunoprecipitated by those antisera individually.The relative abundance of GABAA receptor subunits has been estimated from the percentages of ['HI muscimol or [3H]benzodiazepine binding sites that are immunoprecipitated from solubilized whole brain or from cerebellum (the most frequently studied tissue by virtue of its well-defined cell morphology and restricted subunit repertoire) as shown in Table 111. The /3 subunits have proven more elusive because they are very well conserved making it difficult to

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PAUL J. WHITING el al. TABLE 111 THEPERCENTAGE OF BINDING SITESIMMUNOPRECIPITATED BY SUBUNITSPECIFIC ANTISERA ~

Subunit

~

% [3H] muscimol

[3H]benzodiazepine

821

81 t 4 Whole brain 40.5 3 28.2 -C 5 13 t 2 24 -C 3 23.9 -t 7 0 0 3-4 4 0 16 -C 2 60 t 3 25 2 6 0 49 + 4 78 2 6 18+3 3.8 0 28 -t 7

>5

722 18 + 3 55 + 3 1957 11.1 + 1 59 t 3 50 + 6 14 C 2 11 2 2 26 + 6 75.9 + 2 53 33 2 4 12.2 t 2 13.8 + 2

0 0 54 t 6 8 2 2 62 t 22 66.8 t 1.2 78.6 rfr 0.7 64 + 4 0 23.2

+

Cerebellum 82 t 88 70 1.4 t 0.7 90 -t 4 7+9 3.6 -C 1.9 8t6 13 + 3 0 0 0 0 57 2 6 50.9 2 0.5 89.0 2 0.1 94 t 7 0 23 C 3 0

Reference Benke et al. ( 1 9 9 1 ~ ) McKernan et al. (1991) McKernan et al. (1991) Marksitzer el al. (1993) Benke et al. (1991b) McKernan et al. (1992) K Quirk et al. (unpublished) Kern and Sieghart (1994) Sieghart et al. (1993) K. Quirk et al. (unpublished) K. Quirk et al. (unpublished) Benke et al. (1994) Benke et al. (1994) Benke et al. (1994) Quirk et al. (1994b) Benke et al. (1990) Quirk et al. (1994b) Quirk et al. (1994b) Togel et al. (1994) Quirk et al. (1994a) Benke et at. (1991a) Duggan and Stephenson (1990)" Luddens et al. (1991) Khan et al. (1994a) Quirk et al. (1994a) Duggan and Stephenson (1990)" Quirk et al. (1994a) Duggan and Stephenson (1990)" Quirk et al. (1994a) Quirk et al. (1994a) Quirk et al. (1994a) Quirk el al. (1994a) Quirk et al. (1994a) Duggan et al. (1992)" Khan et al. (1994a) Fernando et al. (1994) Quirk et al. (1994a) Quirk et al. (1994a) Benke et al. (1991a) Quirk et al. (1994a)

"All studies were carried out using rat or bovine (*) brain. ['Hlbenzodiazepine binding refers to either ['H]R015-1788 or ['Hlflunitrazepam, but not ['H]R015-4513 which labels additional receptors. Studies using affinity purified receptors have been included for ["HI benzodiazepine binding.

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generate antisera which can differentiate between them. A monoclonal antibody, bdl7, which recognizes both p2 and p3 subunits immunoprecipitates 83.5% of [3H]muscimolbinding sites in the cerebellum (Khan et aL, 1994a), confirming that the PI subunit is a minor component in agreement with the relatively lower abundance of mRNA for this species identified by in situ hybridization (Wisden et al., 1992; Laurie et al., 1992; Persohn et al., 1992). In good agreement, Benke et al. (1994) reported that antisera to the PI, p2, and p3 subunits precipitate 18, 55, and 19% of [9H]muscimol binding sites from rat brain.

C. WESTERN BLOTANALYSES Western blot analysis has been carried out following purification of receptors on subunit-specific immunoaffinity resins. This allows the clear identification of two different subunits associated in the same receptor molecule. Using this technique receptors have been identified which contain two types of a subunit (Pollard et aZ., 1993; McKernan et al., 1991; Duggan et al., 1991; Luddens et aZ., 1991; Benke et al., 1991b; Thompson et al., 1994). The a subunit pairs q a 2 ,alas,a2ag,and (Yl(Y6 have been reported. Two copies of the 2 subunit have also been observed to be present in a single receptor (Khan et aL, 199413).There is disagreement over whether the y2 and y3 subunits are present in the same complex. Receptors have been characterized which contain both y 2 and y 3 subunits by Quirk et al. (1994b), whereas Togel et al. (1994) did not identify the 7 2 subunit in receptors purified on a y3-immunoaffinityresin. The yI subunit does not appear to exist in combination with another y subunit or the 6 subunit (Quirk et aL, 1994b). There is some disagreement as to whether the 6 and y2 subunits can exist in the same receptor monomer. One study (Mertens et al., 1993) has identified both subunits in the same receptor molecule immunoprecipitated from rat brain, whereas Quirk et al. (199413) did not find these subunits in combination.

D. IMMUNOLOCALIZATION

As limited number of studies have tried to assess which subunits coexist by direct covisualizationof receptors using immunofluorescence or electron microscopy. Confocal microscopy using antibodies against the a1and a3 subunits has revealed that the majority of 5-HT neurons express as but not a1subunits, whereas the a1 subunit is expressed primarily in GABAergic cells (Gao et al., 1993), including hippocampal interneurones (Gao and

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Fritschy, 1994). Both a1and a3are colocalized in many cells of the raphe nucleus (Gao et al., 1994). This is consistent with other studies described previously in which major populations of GABAAreceptors contain only one a subunit but minor populations can exist which contain two a subunits. Immunocytochemistry using confocal microscopy has colocalized the a1 and p2 subunits to the same neurons in the cortex, hippocampus, and nucleus of the diagonal band of Broca (Benke et al. 1994). Electron microscopy using GABAAreceptor subunit antisera has identified the a1&,9 combination at synapses established by Golgi cell terminals with granule cell dendrites and also at extrasynaptic sites (Somogyi et al., 1989), whereas (26containing receptors are present only at the synaptic sites (Baude et aL, 1992). It is not yet clear whether receptors at the synaptic site contain one receptor population of structure (~,aSp2/~3/~ or two populations, ay1p2/3y2 and ff6&/3?2*

There is now an accumulating body of evidence for the existence of many different GABAAreceptors as outlined in Table IV.This list is by no means comprehensive, comprising only those subtypes for which, to date, there is some reasonable evidence. Other subunit combinations are also likely and as further data becomes available the number of known GABAA receptor subtypeswill increase. A model has been suggested which describes TABLE IV LIKELY SUBUNIT COMBINATIONSOF Receptor

SUBTYPES

Location

Reference

Most abundant subtype present in many brain regions

Benke at al. (1991d; 1994) Laurie et ul. (1992); Widen et at. (1992)

alP"Y2

Cerebellar granule cells Cerebellum

Beattie and Siege1 (1993) Quirk et al. (1994a)

WkYI

Cerebellum, Bergmann Gilia

Laurie et a1 (1992) Quirk et al. (1994a) Yang et al. (1994)

43Y2

Spinal cord, motor neurons, many brain regions

Wisden et al. (1991) Persohn et al. (1992) Benke et al. (1994)

4"Y2

Cerebellum granule layer

Fritschy et al. (1992) Quirk et al. (1994a)

ff4aIP26

Thalamus Hippocampus Cerebellar granule cells Cerebellar granule cells Cerebellar granule cells

Wisden et al. (1992) Wisden et al. (1992) Quirk et al. (1994a) Thompson et at. (1994) Quirk et al. (1994a)

4 2 Y 2

Pancreas

a5PIY2

a6P"Y2 ff6aIP"YZ

4 8

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the subtypes of GABAA receptor present in the cerebellum (Quirk et al., 1994a). The following a and y / 6 combinations are proposed: aeyn (36%), a66 (23%), aly~(28%), a2y1(8%), and a372 (5%).Other groups report a higher proportion of ['HI musimol binding sites to be immunoprecipitated by other a]-subunit antisera (Luddens et al., 1991; Khan et al., 1994a,b) and coexistence of a1and (Y6 in the same receptor complex (Pollard et al., 1993;Mathews et aL, 1994) which would be consistent with some proportion of the (Y672 component also containing an a1subunit. One study using cells transfected with a1and assubunits together has not supported the existence of a1and (Y6 in the same receptor molecule because the characteristics of the receptors formed in doubly transfected cells were not different from those predicted by a mixture of receptors of structure a1p2y2 and a6p2-y~ (Korpi and Luddens, 1993). What is clear is that if a receptor does contain both al and subunits the pharmacology of the benzodiazepine site is dictated by the subunit (Korpi and Luddens, 1993; Thompson and Stephenson et al., 1994; Mathews et al., 1994; Quirk et al., 1994a; Khan et al., 1994a,b). With all the information in the literature derived from immunoprecipitation, in situ hybridization, and Western blot analysis, it is possible to construct a model of the GABAA receptor subtypes present in the cerebellum, where a number of studies have been carried out, and in the whole brain, bringing information together from studies on both rat and bovine brain. A preliminary model of the proportions of major subtypes in the cerebellum and whole brain is shown in Fig. 2. As more information on the structure of individual receptor subtypes becomes available it will be necessary to refine and improve these models. Sufficient data are not available on other brain regions to allow the construction of similar models.

E. SUBUNIT STOICHIOMETRY OF GABAA RECEPTORS While in situ hybridization and immunoprecipitation approaches offer some guide as to which subunits might be combined in a receptor, they do not indicate their relative copy number nor their arrangement. To date this is still uncertain and the number of copies of a, p, and y / S subunits in a single receptor monomer is still a matter of debate. Backus et al. (1993) mutated GABAA receptor subunits to produce changes in the outward rectification of recombinant channels. Using mutated and wild-type a3,&, and y2 subunits they concluded that receptors with three copies of any subunit are not favored, those with two copies each of two subunits fit the model better, and those with two a subunits, one /3 subunits, and two y subunits are the most favorable. There is also evidence from antibody

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FIG.2. A model of proportions of putative GABAAreceptor subtypes in whole rat brain (A) and in rat cerebellum (B).

studies which support a 2 X a, 1 X p,2 X y stoichiometry. Several studies have detected receptors which contain more than one type of a subunit (Pollard et al., 1993; McKernan et al., 1991; Luddens et al., 1991; Khan et al., 1994a) and recently two types of y subunit have been reported to coexist in the same receptor complex (Quirk et al., 1994b). To date there have been no reports of two 0 subunits in a single receptor molecule. It is not known whether all GABA, receptors conform to the same general pattern of assembly, and the role of the 6 subunit remains unclear although from some studies there is evidence that it appears to substitute for the y subunit (Quirk et al., 1994a). Interestingly, if receptors contain 2 X a and 2 X y subunits this would allow for two benzodiazepine sites in a receptor. The only example to date in which a receptor may contain two a subunits which direct different pharmacologies is the ala,J3y2receptors. All receptors appear to have the pharmacology of an a6subunit (see above). Thus, if

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this subunit arrangement does exist, there must therefore be a directed orientation of subunits around the channel with the not the cr1 subunit adjacent to the y2 subunit.

V.

Drug Binding Sibs on he GABAAReceptor

As outlined previously, GABAA receptors can be allosterically regulated by a diverse range of both naturally occurring and synthetic compounds. By using recombinant receptors it has been possible to study the roles played by individual subunits in the actions of many of these compounds and indeed, in some cases, individual amino acids located at the binding sites have been identified.

A. BENZODIAZEPINES The BZ binding site on the GABAA receptor is by far the best pharmacologically characterized primarily because it is the site of action of librium and Valium, two of the most widely prescribed drugs in the Western world. Compounds which act at the BZ site can be divided into several chemical classes: (a) classical BZs, such as flunitrazepam, diazepam (Valium), flumazenil, etc.; (b) P-carbolines, e.g., 6,7-dimethyl-4ethylcarboline-3carboxylate methyl ester (DMCM), and methyl carboline-3carboxylate (pCCM); (c) imidazopyridines, such as zolpidem and alpidem; (d) pyrazoloquinolones, e.g., CGS 8216 and CGS 9896; ( e ) cyclopyrrolones, such as zopiclone, suriclone, and DN-2327; (f) imidazoquinoxalines, U85575, U92330; and (g) triazolopyridazines, for example, CL218,872 and SR951965.All these compounds act by modulating the frequency of channel opening. Their biophysical mechanism of action will not be discussed further here, since it has been described in some detail elsewhere (Macdonald and Olsen, 1994). Prior to the cloning of subunit cDNAs, GABAA/BZreceptors were subdivided into two pharmacological classes, type I and type 11, differentiated by the higher affinity of CL218,872 and zolpidem for type I receptors (Squires et al., 1979). There was also known to be a large population of receptors in the cerebellum which bound the BZ inverse agonist Ro154513 with high affinity, but was insensitive to classical BZ such as diazepam (Sieghart et aL, 1987).The cloning of GABAA receptor subunit families has resolved the molecular basis for these observations, and it is now appreciated that the pharmacology of the BZ site is defined by the type of a and

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y subunit present in the receptor. Photoaffinity labeling experiments using

[3H] flunitrazepam have demonstrated incorporation into both the a and y2 subunits, suggesting that determinants from both a and y subunits make

up the BZ site (Stephenson et aL, 1990). The degree of BZ efficacy as well as affinity is controlled by subunit combination, and a compound may not exert the same degree of efficacy at all receptor subtypes. For example, CL218,872 has a lower affinity at a&y2 receptors compared with that of alPly2.It also has a lower efficacy, potentiating GABA by 30% on a3Ply2 compared with 50% of the response seen with a full agonist at alPly2 (Wafford et al., 1992). Efficacy is a property of a specific compound on a defined particular receptor subtype and cannot be generalized. The role of a, P, and y subunits on the BZ pharmacology of GABAAreceptors has been well characterized and is discussed in detail below. 1. a Subunits

The previously described type I receptors have been shown to consist primarily of receptors containing an a1 subunit (Pritchett et al., 1989a). When alPy2receptors are expressed in transfected cells or Xenopus oocytes they confer high affinity for compounds, such as CL218,872 and zolpidem, both in biochemical and in functional studies, in which zolpidem is a full agonist and CL218,872 a partial agonist (Pritchett et aL, 1989a; Puia et aL, 1991; Hadingham et al., 1993a,b; Wafford et aL, 1992). Type I1 receptors are in fact made up of several other subtype (a@y2,a3Py2,and a5Pm) subunits which have lower affinity for CL218,872 and zolpidem (Pritchett and Seeburg, 1990;Hadingham et aL, 1993a,b).A single amino acid substitution of glycine for glutamate 201 in ag confers high affinity for type I selective compounds, indicating that the region N-terminal to TM1 on the a subunit contributes to the binding site for these particular compounds (Pritchett and Seeburg, 1991). The imidazopyridine zolpidem can be used to distinguish aT and a3containing receptors from those containing a5,having an affinity of 400 nM on a2P3y2and ag/33y2, but >15,000 on a5P3y2(Pritchett and Seeburg, 1990). Conversely, Ro15-4513 exhibits a 10-fold selectivity for a5-containing receptors over all other receptors subtypes (Hadingham et aL, 1993a). To date, no compounds have been described which have differential affinity for a2-and qcontaining receptors. However, there are differences in efficacy. For example, flunitrazepam has a greater efficacy on a3Py2over a2Py2when expressed in HEk293 cells (Puia et al., 1991) or Xenopus oocytes (Wafford et al., 1992). Other a subunits form receptors which have BZ sites with a different pharmacology. a4-and a6-containing receptors have high aMinity for the compounds Ro15-4513 and bretazenil and very low affinity for classical benzodiazepines such as flunitrazepam and diazepam. Since their benzodi-

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azepine site (Luddens et d., 1990) is insensitive to the so-called “classical BZs,” they have been termed “diazepam-insensitive” receptors. They are localized primarily in cerebellar granule cells (a6 receptors) and thalamus (a4receptors) (Malminiemi and Korpi, 1989; Wisden et d., 1992; Laurie et d., 1992). Interestingly, the efficacy of compounds at a&y2 receptors can be quite different from their efficacy at other receptors. For example, Ro15-4513, which binds with high affinity to all &v2 combinations, is a partial inverse agonist on a&y2 but is a partial agonist on a&y2 (K. A. Wafford and P. J. Whiting, unpublished). Mutagenesis studies have allowed the determination of the key residue necessary for high-affinity binding of classical benzodiazepines such as diazepam. The a],a2,as, and a5subunits have a histidine at position 101 (a1 subunit numbering), whereas the a4and (Y6 subunits have an arginine. Substitution of arginine for histidine in confers high-affinity binding of diazepam (Wieland et aL, 1992). Zolpidem affinity was not restored, suggesting other interactions are required for zolpidem binding. Other determinants on the a subunit and/or y subunit, which make up the binding pocket for BZ-like drugs, have yet to be identified. Receptors containing a4 subunits have not been extensively studied. The amino acid sequence of a4is most homologous to and the pharmacological profile of a&, and a&y2 receptors are also very similar (Wisden et al., 1991). Receptor subtypes could thus be categorized into three groups. (1) alcontaining receptors which have the highest aMinity for many BZs; (2) ap,as, and a5-containing receptors which have similar but distinguishable pharmacological properties; and (3) a4and a6-containing receptors which bind many BZs with very low affinity.

2. /3 Subunits It has been suggested that the 0 subunit variant may affect modulation by BZs (Sigel et al., 1990). However, human PI, P2,and Ps subunits have been systematicallycompared in combination with different a subunits and y2 expressed in transfected cells, and no significant differences were found in combinations which differed only with respect to the 0 subunit. Five ligands showed no difference in efficacy to potentiate al&y2, a&y2, and alP3y2 receptors expressed in Xenopus oocytes (Hadingham et aL, 1993b). Other studies have also observed no effect of the p subunit on BZ binding (Pritchett et aL, 1989a; Puia et aL, 1992), and it is concluded that, unlike a and y, this subunit does not contribute to the BZ binding site.

3. y Subunits Since the observation that a y2 subunit is necessary to form a fully functional GABAAreceptor with a high-affinity BZ binding site (Pritchett

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et al., 1989b), two other forms of the y subunit have been reported. These subunits also confer BZ sensitivity to the receptor when combined with an a and /3 subunit, but receptors containing y1or y3 exhibit pharmacological differences from those containing a y2 subunit with respect to both BZ affinity and efficacy (Puia et aL, 1991;Wafford et al., 1993; Herb et al., 1992; Knoflach et aL, 1991; Luddens et al., 1994). Receptors made up of a!l/31yl expressed in 293 cells do not bind [3H]Ro15-1788with high enough affinity to be detected in a radioligand binding assay. They are, however, labeled by [3H]flunitrazepam (Ymer et aL, 1989b). Receptors immunoprecipitated from rat brain with yl-specific antiserum do not bind either ligand with high affinity (Quirk et al., 1994b). In vivo the y1subunit appears to be primarily assembled with the a2subunit rather than a1(see Table N ),and expression of a&yI receptors in Xenopus oocytes revealed a general reduction in both affinity and efficacy for BZ site ligands compared to a&y, (Wafford et al., 1993). The imidazopyridines, such as zolpidem, were markedly reduced in efficacy,whereas CL218 872 was relatively unchanged compared to a2flIy2. Interestingly, BZ site inverse agonists, such as DMCM and Ro15-4513, acted as agonists at 7,containing receptors (Puia et al., 1991; Wafford et al., 1993), and flunitrazepam potentiation was not inhibited by Ro15-1788 or CGS 8216, both BZ antagonists, suggesting low affinity for these compounds. The y3 subunit also confers a unique benzodiazepine profile to the receptor. Although a complete pharmacological profile has not yet been established, a!/3y3receptors maintain a high affinity for Ro15-1788 and Ro154513, but are slightly less sensitive to classical BZs such as flunitrazepam and diazepam (Herb et al., 1992; Knoflach et aL, 1991). Efficacy at receptors containing y3 is, in general, similar to that of y2 (Hadingham et al., in press). The imidazopyridine zolpidem has low affinity for both yT and y3containing receptors (Luddens et al., 1994). Although a benzodiazepine site cannot be formed in the absence of a y subunit, this is not the case with receptors lacking an a subunit, as the By2combination is still sensitive to benzodiazepines (Sigel et aL, 1990; Im et aL, 1993a) suggesting that a /3 or a y can substitute for the a! to form a BZ binding site. Clearly the y subunit variant is a codeterminant of the BZ binding site, and further mutagenesis experiments similar to those performed on a! subunit variants may reveal the amino acids responsible for pharmacological differences generated by y subunits.

4. 6 Subunits The subunit is located in brain regions low in y2 and is present in receptors which are insensitive to classical BZs (Shivers et aL, 1989).To date there have been few reports of studies on recombinant GABAAreceptors

STRUCTURE AND PHARMACOLOGY OF GABA, RECEPTOR SUBTYPES

11 7

containing 6 subunits. Saxena et al. (1994) have reported that a&6 subunits coassemble when expressed in L929 cells, forming a receptor which has a slowed rate of desensitization, is potently blocked by zinc and is diazepam insensitive. As discussed under Section IV there is some controversy over whether &containing GABAAreceptors have a BZ binding site. It is clear that more studies are required to determine the role of the 6 subunit.

5 . p Subunits The p subunits, p1 and p2, are found primarily in the retina and form functional homomeric receptors when expressed in Xenopus oocytes (Cutting et al., 1991, 1992). Although at present classed as receptor subunits they do not appear to coexist with other GABAAsubunits and correspond pharmacologically to the previously described GAB& receptor (Johnston, 1986; Shimada et al., 1992; Qian and Dowling, 1993;Feigenspan et al., 1993; Lukasiewicz et al., 1994). These receptors have not been shown to be sensitive to BZ-site ligands. It is clear that the BZ site is a complex structure, probably formed by contributions from both a and y subunits. We are now beginning to understand the contribution made by different subunits to the benzodiazepine pharmacology. In the future, extensive and detailed mutagenesis studies may allow us to map which amino acids actually contribute to BZ-binding pocket.

B. GABA The molecular pharmacology of the agonist binding site for GABAA receptors has been the subject of a number of recent studies. Expression of any single subunit cDNA results in little or no receptor expression; however, doublet combinations will form channels gated by GABA to varying efficiency (Sigel et al., 1990; Angelotti et al., 1993b; Angelotti and Macdonald, 1993). and a y receptors assemble less efficiently than ap, but the fact that these combinations form GABA-gated channels suggests that construction of a GABA binding site is not dependent on a single type of subunit (Verdoorn et al., 1990). It is also a possibility that incorporation of low levels of an endogenous subunit may enable these doublet channels to be expressed with low efficiency. Several laboratories have published GABA affinities on the subunit combinations they have studied, and although these tend to vary somewhat, depending on which subunits have been expressed together, a general pattern is emerging. The presence of a y subunit confers BZ sensitivity but also appears to slightly reduce the affinity of the receptor for GABA (Sigel et al., 1990; Angelotti and Macdonald, 1993; Horne et al., 1993). A recent study of

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agonists and partial agonists on different receptor combinations demonstrated that affinity for agonists was determined to some extent by individual receptor subunit combinations, but all agonists followed the same pattern of affinity, being most potent on ajpgy2and weakest on a&y2, with GABA showing the largest difference (70-fold between these two combinations) (Ebert et al., 1994). a3-containing receptors also generally had a lower affinity than other a subunit-containing receptors. This low GABA affinity for a3-containing receptors has also been reported by others (Knoflach et al., 1992). Agonist efficacy, however, is clearly determined primarily by a subunits, as the degree of efficacy of the partial agonists, 4,5,6,7tetrahydroisoxazolo [5,4-c]pyridin-3-1 and piperidine-4-sulfonic acid varied according to the a subunit variant but very little between /3 subunits. y1 subunit-containing receptors, however, also had low efficacy for partial agonists compared to those containing y2 and y3 (Ebert et al., 1994). From this study it is clear that, like the BZ site, varying the p subunit has the least effect on GABAAreceptor agonist interactions. Regarding the actual binding site for GABA, several mutagenesis studies have identified amino acids which markedly reduce GABA affinity. Sigel et al. (1992) identified a mutation in the al subunit (a1-Phe64-Leu) which produced a 210fold decrease in affinity for GABA, with comparable reductions for the competitive antagonist SR95531 and bicuculline. Mutation of the same residue in a5also resulted in lower GABA affinity, but equivalent mutations in p2and y2 had much smaller effects. Photoaffinity labeling of the purified bovine receptor using ['HI muscimol, followed by chymotryptic digestion and microsequencing, has also identified this same amino acid residue as carrying the photoaffinity label (Smith and Olsen, 1994), strongly suggesting this to be part of the binding site for GABA or muscimol. Other mutagenesis experiments using the P2 subunit have identified two regions of the N-terminal part of the subunit including four amino acids (Tyr157, Thre160, Thre202, and Tyr205), which when conservatively mutated, resulted in dramatic decreases in GABA affinity (Amin and Weiss, 1993). Again, mutation of corresponding amino acids in either the a,or y2subunits did not have any effect. These mutations did not result in a decrease in maximum current or Hill slope and did not affect the activation by pentobarbital, suggesting an effect on agonist binding rather any subsequent steps in receptor activation. The p1 and p2 subunits are unique in the GABA receptor family in assembling efficiently into a homomeric receptor, and it is unlikely that these subunits assemble together with any of the a,p,y, or S subunits. Consequently, as discussed previously, these receptors are probably best referred to as GAB&, (Johnston, 1986; Cutting et aL, 1991, 1992). These receptors have a different pharmacology to native GABAAreceptors, being

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insensitive to bicuculline, SR95531,barbiturates, and benzodiazepines (Shimada et al., 1992; Polenzani et al., 1991),and have been identified in retinal horizontal cells and bipolar cells (Qian and Dowling, 1993; Feigenspan et al., 1993; Lukasiewicz et al., 1994). The GABA binding site on p1 also has a different structure activity profile, being most sensitive to trans+ aminocrotonic acid (TACA) over GABA and muscimol (Kusama et al., 1993a,b;Woodward et al., 1993). Interestingly, mutagenesis of amino acids which correspond to the critical regions previously described in the p2 subunit also drastically lower GABA affinitywhen mutated in the p, receptor (Amin and Weiss, 1994), thus suggesting that the GABA-binding region is quite well conserved between these two types of receptors.

C . STEROIDS Several pregnane steroids have been shown to interact with a specific site on native (Majewska et al., 1986) and recombinant receptors (Puia et al., 1990).This site has not yet been subject to an extensive study to compare different recombinant receptor combinations; however, some information has been obtained with regard to subunit requirements, and some studies examining brain regional differences in steroid modulation of [ 95S]TBPS binding suggest there may be some subtype selectivity (Sapp et al., 1992). Just as on native GABAAreceptors, 5a-pregnan-3a-ol-2O-one (3a-OHDHP) and 5a-pregnan-3a,21diol-20-one allosterically enhance the response to GABA and at high concentrations directly activate the receptor (Puia et al., 1990; Hadingham et al., 1993b). Single-channel studies have revealed this enhancement to be due to both an increase in the duration of channel opening and an increase in the frequency of openings (Twyman and Macdonald, 1992). Other steroids, such as pregnanolone sulfate and dehydroepiandrosterone (DHEAS), inhibit the response to GABA (Majewska et al., 1988, 1990), and a series of diols have recently been shown to be partial agonists at the steroid site on recombinant GABAA receptor (Belelli et aL, 1994). Puia et al. (1990), demonstrated that the steroids 3aOH-DHP and THDOC potentiated receptors made up of al&y2, or even PI expressed alone in 293 cells. The degree of potentiation was similar for each of these combinations and was much greater than that with BZs, being more comparable to that obtained with barbiturates, although experiments have demonstrated that steroids act at a site distinct to barbiturates (Turner et al., 1989). Unlike the BZ site, steroid potentiation does not depend on the presence of a y subunit and must be present on at least the P subunit (Puia et al., 1990).Shingai et al. (1991),compared potentiation and a& y 2and observed a greater enhanceby 3a-OH-DHP in alply2,a2Ply2,

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ment on alp1y2than on the other combinations suggesting that a subunits may modulate the degree of potentiation. Lan et al. (1991), measured steroid potentiation of [3H]flunitrazepambinding on the same three combinations and observed a greater enhancement on a3P1y2compared to alPly2. A more detailed study comparing all six a subunits is required to reconcile these data. With regard to the P subunit, Hadingham et al. (1993b), have shown no differences in the degree of enhancement with 3a-OH-DHP or 3a,5P-OH-DHP between alPly2,alP2y2,or a1P3y2.There was also no difference in the degree of inhibition with DHEAS. The influence of y subunits on steroid modulation is currently unknown.

D. BARBITURATES Barbiturate drugs have long been known to act via the GABAAreceptor channel, enhancing the action of GABA, and like steroids, directly activating the channel at high concentrations. The in vivo sedative and anesthetic properties of many barbiturates is thought to be due to this action at GABAA receptors. The mechanism of action of pentobarbital has been studied using single-channel patchclamp techniques and has been shown to be due to a prolongation of the channel open time, with no effect on the frequency of channel opening (Jackson et al., 1982; Macdonald et al., 1989). This mechanism leads to a shift to the left of the GABA concentration-response curve and an apparent increase in the maximum response in the presence of pentobarbital (Horne et al., 1993).All GABAAreceptor subunit combinations tested to date have been potentiated by pentobarbital, including doublet and triplet combinations, making it difficult to determine the location of the barbiturate binding site or sites. The degree of potentiation is larger than that of BZs and similar to that elicited by the neuroactive steroids. The /3 subunit does not appear to have any dramatic effect on the potentiation of GABA responses by pentobarbital (Hadingham et al., 199313).The a subunit does not influence the affinity of pentobarbital, but the degree of efficacy does vary, with the smallest effect on a1&y2 and the largest enhancement on a&I2y2 (Thompson et al., 1994). The direct activation of the receptor by pentobarbital is via a different site from that bound by GABA as the response is not altered by mutations which affect the GABA binding site (Amin and Weiss, 1993), and pentobarbital responses are not blocked by the GABA-competitive antagonist SR95531 (Thompson et al., 1994). This agonist site is also affected by the type of a subunit, having low affinity (400 p M ) and low efficacy (50% of maximum GABA current) on a&yZ, a&y2, a&y2, and a5P2y2,but is 10-fold more potent and has a much higher degree of efficacy on a&yn (180% of

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maximum GABA) (Thompson et al., 1994). Further studies using recombinant receptors should reveal more regarding these barbiturate binding sites.

E. PICROTOXIN [ ~BUTYLBICYCLOPHOSPHOROTHIONATE (TBPS)] The plantderived convulsant picrotoxin is a noncompetitive antagonist of the GABAAreceptor. The mechanism of action of picrotoxin is complicated and has been the subject of several detailed electrophysiological studies (Smart and Constanti, 1986; Newland and Cull-Candy, 1992). This compound appears to act at an allosteric site located close to the ion channel pore. The usedependent block is a result of picrotoxin binding preferentially to the agonist bound form of the receptor, stabilizing the desensitized state (Newland and Cull-Candy, 1992). The evidence for an allosteric site located within the lumen of the channel has recently been supported by the identification of an insecticide-resistant form of the Drosophila GABA receptor gene rdl (ffrench-Constant et al., 1991). The two homologues are sensitive or insensitive to the insecticide lindane, which acts by blocking GABA receptor function (ffrench-Constant et al., 1991). The lindane-resistant mutants also have a much reduced sensitivity for picrotoxin. When expressed in Xenopus oocytes the rdl cDNA forms a GABAgated receptor (ffrench-Constant et aL, 1993). The picrotoxin sensitivity is dependent solely on the residue Ala302, which lies in the proposed M2 channel-forming region of the protein (Zhang et aL, 1994). Mutation of this residue to a serine results in a 116-fold reduction in picrotoxin sensitivity and a 970-fold reduction in lindane sensitivity,as well as a small reduction in TBPS sensitivity,which is a cage convulsant also acting at the picrotoxin binding site. Residues in the M2 region of the glycine receptor P subunit have also been implicated in conferring picrotoxin resistance to heteromeric a / P glycine receptors (Pribilla et aL, 1992); hence, it is likely that corresponding amino acids in the mammalian GA13AAreceptor M2 region provide the binding site for picrotoxin. However, as all recombinant forms tested so far are similarly blocked by picrotoxin, the contribution of individual subunits to this site remains to be determined. Another group of compounds also appear to act via the picrotoxin site; several y-butyrolactone analogues have been identified which act as agonists, i.e., enhancing GABA channel activity, or antagonists which reduce the block by picrotoxin but have no intrinsic effect, hence, showing similar properties to the BZ site (Holland et al., 1990).Although little is known about the molecular pharmacology of these compounds, a recent study found that a,P2y2recombinant receptors were sensitive to potentiation by a-tert-butyl-y-butyrolactone,but

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recombinant a’6p2y2receptors were insensitive to this compound (Mathews et aZ., 1994). These results mimicked the developmental switch in granule cell development, where early expression of a1is replaced by c t 6 . Interestingly, picrotoxin sensitivity itself was not changed between alP2y2and a&y2 suggesting that more than one residue must be involved in this compound’s interaction at the picrotoxin site. Further sitedirected mutagenesis experiments should reveal more about how y-butyrolactones interact with this site.

F. ETHANOL Alcohols have been shown to affect a number of different ligand-gated ion channels including the GABAAreceptor (Dietrich et aZ., 1989). Electrophysiological experiments have demonstrated a regional variation in the susceptibility to the potentiating effect of ethanol and this was suggested to be due to GABAAreceptor heterogeneity (Proctor et aZ., 1992). As mentioned under Section I11 potentiation by low concentrations of ethanol appears to be dependent on the presence of the yZLsubunit and indeed requires the PKC phosphorylation site (Wafford et al., 1991; Wafford and Whiting, 1992). It appears that alcohols are able to influence the function of GABAAreceptors directly via this phosphorylation site and at higher concentrations via some other site on the receptor (Mihic et aL, 1994).

G. LORECLEZOLE The anticonvulsant compound loreclezole is effective in a number of behavioral models of epilepsy and may act via a GABAergic mechanism. Loreclezole has recently been shown to strongly potentiate the opening of GABA-gated chloride channels in cultured rat cortical neurons and oocytes expressing human GABAAreceptors (Wafford et d.,1994). Using different combinations of human GABAAreceptor subunits expressed in Xenopus oocytes, the effects of varying a,P, and y subunits were investigated. Unlike BZs, loreclezole-induced potentiation did not require the presence of either an a or a y subunit, but was highly selective for receptors containing the Pn or P3 subunit over those containing the PI subunit. Potentiation was additive with that of BZs, barbiturates, and steroids and was insensitive to the BZ antagonist Rol5-1788 (Wafford et al., 1994). subunit chimeras and sitedirected mutagenesis revealed that a single amino acid in f i n (Asn289) and p3 (Asn290) located at the carboxyl terminal end of the putative channel-forming TM2 region conferred sensitivity to loreclezole (Wingrove et aZ., 1994). The equivalent position in PI was occupied by a

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serine residue, conferring low sensitivity for this compound. The location of this site in the putative pore-forming section of the protein is analogous to the putative picrotoxin binding site; however, picrotoxin does not show any significant subtype selectivity.

H.

ANESTHETICS

A number of general anesthetics are thought to exert their activity through interactions with ligand-gated and voltage-dependent ion channels (Franks and Lieb, 1994). The majority of these have some effect on GABA-mediated synaptic transmission and have been shown to interact with GABAAreceptors (Franks and Lieb, 1994;Jones et al., 1992). Isoflurane, enflurane, and halothane all increase GABA-induced currents in hippocampal neurons (Jones et al., 1992) and Xenopus oocytes injected with mouse cortical mRNA (Lin et al., 1992), resembling the effects of pentobarbital and the neuroactive steroids by prolonging the bursts of channel opening (Yeh et al., 1991). Propofol also has a marked potentiating effect as well as directly activating the GABA-gated chloride channel at higher concentrations (Concas et al., 1991; Hales and Lambert, 1991; Hara et aL, 1993). It is not yet clear whether these anesthetics act via the barbiturate or steroid site or through an independent binding pocket on the receptor. Subunit dependence of anesthetic enhancement has not yet been investigated in detail; however, Lin et al. (1993) have shown that, like pentobarbital, potentiation with enflurane was greater in oocytes expressing aIPIcompared to alP2y2and no difference was seen between yZS-and yZL-containing receptors.

I.

ZINC

The divalent cation zinc has been shown to be an antagonist of GABAA receptors, but displays an unusual property in being less potent at blocking receptors in adult tissue compared to those derived from embryonic cultures (Smart and Constanti, 1990).Zinc blockade appears to be noncompetitive and voltage dependent, without effecting any of the single channel properties of the receptor (Legendre and Westbrook, 1991; Smart, 1992). Zinc reduces the opening frequency of the channel without the flickering block phenomena common to open channel blockers. This indicates that rather than simply plugging the channel, zinc is binding to an allosteric modulatory site close to the channel possibly within the M2 region (Smart, 1992). The developmental profile of zinc blockade has been explained by the finding that incorporation of a y subunit into the receptor complex

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reduces the sensitivity to zinc. Combinations of ap were shown to be highly sensitive to zinc blockade but introducing a y1or y2subunit to the receptor dramatically reduces the effect of zinc (Draguhn et al., 1990; Smart et al., 1991). The y 2 subunit appears to be expressed later in development and so embryonic cultured neurons are more susceptable to zinc antagonism than are adult neurons. A recent study has identified a mutation in the y2 subunit (threonine 142-serine) which increases the efficacy of certain BZs and converts the inverse agonist R015-4513 into an agonist (Mihic et aL, 1994).This mutation also affects the properties of zinc. On wild-type alf12y2, zinc would have little effect; however, when the mutant y2 is expressed with alp2, zinc becomes a positive modulator, potentiating the GABA current. The effects of this mutation are unlikely to be related to changes in the binding site for zinc as it also affects benzodiazepine potentiation, but it is more likely to alter the conformational changes involved when allosteric agents bind to the receptor.

J. OTHER Other compounds have been demonstrated to affect the function of GABAA receptor. However, these have not been investigated in great detail and may be acting via previously identified sites. Lanthanum (La3+)has been shown to potentiate GABA-mediated currents in cells transfected with rat a1f12y2subunit cDNAs (Im et aZ., 1992), with an ECSoof 21 pM and maximum potentiation of 240% compared to an EC50of 200 p M and less than 70% maximum on a& receptors. This effect is mimicked by other lanthanides (Ma et al., 1994) and is opposite to the effect of zinc, which is inhibitory and more effective on receptors lacking the y2 subunit. The potentiation was similar in its subunit dependence to BZs; however, lanthanum potentiation was not blocked by the BZ antagonist Ro15-1’788. Another group of compounds, substituted pyrazinones (U-92813 and U-94863), have also been identified as allosteric modulators of recombinant GABAA receptors (Im et aL, 1993b). This potentiation was, in contrast to BZs, independent of the y2subunit and was not dependent on any particular a subunit. The effects of the compounds were shown not to be through the BZ, barbiturate, or steroid site. These compounds may thus act via a previously unidentified allosteric site on the receptor. Several pesticide compounds have been shown to act at GABAA receptors. The previously mentioned lindane and related compounds endrin and dieldrin are antagonists at the receptor and are thought to act via the picrotoxin site (Tokutomi et al., 1994). Hexachlorocyclohexane has four stereoisomers, of which lindane is the y-isomer. Other isomers also have activity at the GABAAreceptor, especially the 8-isomer, which potentiates

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the GABA response in a manner insensitive to Ro15-1788 and similar to that of barbiturates (Woodward et aL, 1992). It is currently unclear where this compound is acting, but interestingly the p receptor subtype is sensitive to inhibition by y-lindane but not affected by the 8-isomer. Hence, these stereoisomers appear to act at completely different sites on the GABAA receptor. The antihelminthic compound ivermectin is a potent allosteric modulator of GABAA receptors and is independent to other modulatory sites on the receptor (Pong and Wang, 1982; Krusek and Zemkova, 1994). Ivermectin potentiates recombinant human GABAA receptors expressed in Xenopusoocytesand does not require the presence of the y 2subunit; indeed, ivermectin affected GABA responses to the PI subunit expressed alone (Arena et aZ., 1993; K. A. Wafford, 1994, unpublished observations). The molecular pharmacology of this site thus remains to be investigated in detail. The nonsteroidal anti-inflammatory drugs, felbinac, fenbufen, or biphenyl acetic acid, have been shown to be proconvulsant when administered together with antibacterial quinolones (norfloxacin and ciprofloxacin) . While having weak effects alone, these compounds have recently been shown to act in combination at the GABAAreceptor to produce a potent antagonist effect (Haliwell et aZ., 1991; Akaike et aZ., 1991; Squires and Saederup, 1993). The mechanism of this interaction has not yet been determined and these compounds have not yet been studied o n recombinant GABAAreceptors. Penicillin also acts as a noncompetitive antagonist at recombinant GAElAA receptors and, unlike picrotoxin, shows no agonist dependence, suggesting a different mechanism of action (Horne et aZ., 1992). The anxiolytic/anticonvulsant compounds chlormethiazole and trichloroethanol, the metabolite of chloral hydrate, have also been shown to interact with GABAA receptors in a manner similar to that of barbiturates potentiating GABA responses and have been shown to have a direct effect at high concentrations (Moody and Skolnick, 1989; Hales and Lambert, 1992b; Peoples and Weight, 1994). It is not currently known whether these compounds act at the same site as barbiturates or at one of those previously described. Clearly a number of neuroactive compounds interact directly with the GABA/BZ receptor complex, and differences in their effects may be due to selectivity for receptors containing different subunits.

VI. Conclusion

Our knowledge of the GABAAreceptor family has clearly grown exponential since 1987when the first subunit cDNAs were identified. As is always

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the case, we are generating more questions than answers. Why are there so many receptor subtypes?What are the functions of these subtypes?What controls their expression, assembly, and sorting? How is their function regulated, and why? What is the structure of the receptor at the molecular level, and how is it activated/deactivated/modulated? Hopefully, the coming years will reveal the answers to some of these questions.

References

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Belelli, D., Lambert, J. J., Peters, J. A,, Lan, N. C., and Gee, K. W. (1994). Modulation of human (al@lyZL) recombinant GABA&receptors by pregnanediols. Can. J. Physiol. Phannacol. 72, P13.3.18. Benke, D., Mertens, S., Trezciak, A,, Gillesen, D., and Mohler, H. (1990). Identification of the y2 subunit protein in native GABA receptors in brain. Eur.J. Phannacol. 189,337-340. Benke, D., Mertens, S., Trzeciak, A., Gillesen, D., and Mohler, H. (1991a). Identification and immunohistochemical mapping of GABAAreceptor subtypes containing the 6 subunit in rat brain. FEBS Lett. 283, 145-149. Benke, D., Cicin-Sain, S., Mertens, S., and Mohler, H. (1991b). Immunochemical identification of the a 1 and a 3 subunits of the GABAAreceptor in rat brain.J Recqt. Res. 11, 1-4. Benke, D., Mertens, S., and Mohler, H. ( 1 9 9 1 ~ )Ubiquitous . presence of GABAA receptors containing the a 1 subunit in rat brain demonstrated by immunoprecipitation and immunohistochemistry. Mol. Neurophannucol. 1, 103-1 10. Benke, D., Mertens, S., Trzeciak, A,, Gillesen, D., and Mohler, H. (1991d). GABA, receptors display association of y2 subunit with a 1 and p2/3 subunits.J Biol. C h a . 266,4478-4483. Benke, D., Fritschy, J.-M., Trzeciak, A,, Bannwarth, W., and Mohler, H. (1994). Distribution, prevalence, and drug binding profile of y-aminobutyric acid type A receptor subtypes differing in the &subunit variant. J Bzol. C h a . 269, 2700-27107. Bovolin, P., Santi, M. R., Puia, G., Costa, E., and Grayson, D. (1992). Expression patterns of y-aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella. Proc. Natl. Acad. Sn'. U.S.A. 89, 9344-9348. Browning, M. D., Bureau, M., Dudek, E. M., and Olsen, R. W. (1990). Protein kinase C and CAMP dependent protein kinase phosphorylate the P-subunit of the purified GABAA receptor. Proc. Natl. Acad. Sd. U.S.A. 87, 1315-1318. Buckle, V. J., Fujita, N., Ryder-Cook, A. S., Derry, J. M. J., Barnard, P. J., Lebo, R. V., Schofield, P. R., Seeburg, P. H., Bateson, A. N., Darlison, M. G., and Barnard, E. A. (1989). Chromosomal localization of GABAA receptor subunit genes: Relationship to human genetic disease. Neuron 3, 647-654. Concas, A,, Santoro, G., Serra, M., Sanna, E., and Biggio, G. (1991). Neurochemical action of the general anesthetic propofol on the chloride ion channel coupled with GABAA receptors. Bruin Res. 542, 225-232. Culiat, C. T., Stubbs, L. J., Montgomery, C. S., Russell, L. B., and Rinchik, E. M. (1994). Phenotypic consequences of deletion of the y3, a5, o r P3 subunit of the type A yaminobutyric acid receptor of mice. Proc. Natl. Acad. Sn'. U.S.A. 91, 2815-2818. Cutting, C . R., Lu, L., O'Hara, B. F., Kasch, L. M., Montrose-Mzadeh, C., Donovan, D. M., Shimada, S., Antonarakis, S. E., Guggino, W. B., Uhl, G. R., and Kazazian, H. H., Jr.. (1991). Cloning of the gamma-aminobutyric acid rho1 cDNA A GABA receptor subunit highly expressed in retina. Proc. Natl. Acad. Sd. U.S.A. 88, 2673-2677. Cutting, G. R., Curristin, S., Zoghbi, H. Y., O'Hara, H. Y., Seldin, M. F., and Uhl, G. R. (1992). Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit p2 and colocalization of the genes encoding p2 (GABARR1) and pl (GABARRl) to human chromosome 6q14q21 and mouse chromosome-4. Genomics 12, 801-806. Deitrich, R. A,, Dunwiddie, T. V., Harris, R. A,, and Erwin, V. G. (1989). Mechanism of action of ethanol: Initial central nervous system actions. Phannacol. Reu. 41, 489-537. Draguhn, A,, Verdoorn, T. A., Ewert, M., Seeburg, P. H., and Sakmann, B. (1990). Function and molecular distinction between recombinant rat GABAA receptor subtypes in Zn". Neuron 5, 781-788. Duggan, M. J., and Stephenson, F. A. (1990). Biochemical evidence for the existence of 7aminobutyrateA receptor isooligomers. J. Biol. C h a . 265, 3831-3835.

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Pritchett, D. B., Sontheimer, H., Shivers, B. H., Ymer, S., Kettenmann, H., Schofield, P. H., and Seeburg, P. H. (1989b). Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature (London) 338, 582-585. Pritchett, D. B., and Seeburg, P. H. (1990). Gamma aminobutyric acidAreceptor a5 subunit creates novels type I1 benzodiazepine receptor pharmaco1ogy.J. Neurochem. 54,1802-1804. Pritchett, D. B., and Seeburg, P. H. (1991).y-aminobutyric acid type A receptor point mutation increases the afFinity of compounds for the benzodiazepine site. h c . Natl. Acad. Sci. U.S.A. 88, 1421-1425. Proctor, W. R., Allan, A. M., and Dunwiddie, T. V. (1992). Brain regiondependent sensitivity of GABA, receptor-mediated responses to modulation by ethanol. Alcohol. Clin. Exp. Res. 16, 480-489. Puia, G., Santi M., Vicini, S., Pritchett, D. B., Purdy, R. H., Paul, S. M., Seeburg, P. H., and Costa, E. (1990). Neurosteroids act on recombinant GAB& receptors. Neuron 4,759-765. Puia, G . , Vicini, S., Seeburg, P. H., and Costa, E. (1991). Influence of recombinant yaminobutyric acidA receptor subunit compositions on the action of allosteric modulator of y-aminobutyric acidgated chloride currents. Mol. Phannacol. 39, 691-696. Puia, G., Ducic, I., Vicini, S., and Costa, E. (1992). Differences in the negative allosteric modulation of y-aminobutyric acid receptors elicited by khlorodiazepam and by a Bcarboline-3carboxylate ester: A study with natural and reconstituted receptors. Proc. NatZ. Acad. Sn'. U.S.A. 89, 3620-3624. Qian, H., and Dowling.J. E. (1993).Novel GABA responses from roddriven retinal horizontal cells. Nature (London) 361, 162-164. Quirk, K, Gillard, N. P., Ragan, C. I., Whiting, P. J., and McKernan, R. M. (1994a). Model of subunit composition of y-aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their Q and y / 6 subunits. J. Biol. C h a . 269, 16020-16028. Quirk, K , Gillard, N. P., Ragan, C. I., Whiting, P. J., and McKernan, R M. (1994b). yaminobutyric acid type A receptors in rat brain <:ancontain both y2 and y3 subunits, but y l does not exist in combination with another y subunit. Mol. Pharmucol. 45,1061-1070. Sakmann, B., Hamill, 0.P., and Bormann, J. (1983).Patch-clamp measurements of elementary chloride currents activated by the putative inhibitory transmitters GABA and glycine in mammalian spinal neurons. J. Neural Trunsm. SupPl. 18, 83-95. Sapp, D. W., Witte, U., Turner, D. M., Longoni, B., Kokko, N., and Olsen, R. W. (1992).Regional variation in steroid anaesthetic modulation of ['HITBPS binding to y-aminobutyric acid receptors in rat brain. J. Phannacol. Exp. Thm 262, 801-808. Saxena, N., and Macdonald, R. L. (1994). Assembly of GABA-A receptor subunits: role of the 6 subunit. J. Neurosn'. 14, 7077-7086. Schofield, P. R., Darlison, M. G., Fujita, N., Burt, I). R., Stephenson, F. A., Rodriguez, H., Rhee, L. M., Ramachandran, J., Reale, V., Glencourse, T. A., Seeburg, P. H., and Barnard, E. A. (1987). Sequence and functional expression of the GABA, receptor shows a ligand gated ion channel family. Nature (London) 328, 221-227. Schofield, P. R., Pritchett, D. B., Sontheimer, H., Kettenmann, H., and Seeburg, P. H. (1989). Sequence and expression of human GABA, receptor a1 and Bl subunits. FEBS Lett. 244, 361-364. Seeburg, P. H., Wisden, W., Verdoorn, T. A., Pritchett, D. B., Werner, P., Herb, A,, Luddens, H., Sprengel, R., and Sakmann, B. (1990). The GABA, receptor family: Molecular and functional diversity. Cold Spnng Harbor Symp. @ant. Biol. 55, 29-40. Sessler, F. M., Mouradian, R. D., Cheng, J.-T., Yeh, H. H., Lui, W., and Waterhouse, B. D. (1989).Noradrenergic potentiation of cerebellar purkinjee cell responses to GABA Evidence for mediation through the &adrenoceptor-coupled cyclic AMP system. Brain R ~ s499, . 27-38.

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Shimada, S., Cutting, G., and Uhl, G. R. (1992). y-aminobutyric acid A or C receptor? yaminobutyric acid p l receptor RNA induces bicuculline, barbiturate and benzodiazepine insensitive y-aminobutyric acid responses in Xmopus oocytes. Mol. Pharmucol41,683-687. Shingai, R., Sutherland, M. L., and Barnard, E. A. (1991). Effects of subunit types of the cloned GABAA receptor on the response to a neurosteroid. Eur.J. Pharmucol. 206, 77-80. Shivers, B. D., Killisch, I., Sprengel, R., Sontheimer, H., Kohler, M., Schofield, P. R., and Seeburg, P. H. (1989). Two novel GABA, receptor subunits exist in distinct neuronal populations. Neuron 3, 327-337. Sieghart, W., and Drexler, G. (1983). Irreversible binding of ['Hlflunitrazepam to different proteins in various brain regions. J . Neurochem. 41, 47-55. Sieghart, W., Eichinger, A., Richards,J. G., and Mohler, H. (1987). Photoaflinity labelling of benzodiazepine receptorproteinswith partial inverse agonist [3H]Ro154513: A biochemical and autoradiographic study.J. Neurochem. 48, 46-52. Sieghart, W., Item, C.,Buchstaller,A.,Fuchs,R, Hoger, H., and Adamiker, D. (1993). Evidence for the existence of differential Oglycosylated a5subunits of the y-aminobutyric acidA receptor in the rat brain. J. Neurochem. 60, 93-98. Sigel, E., and Barnard, E. A. (1984).A y-aminobutyricacid/benzodiazepine receptor complex from bovine cerebral cortex. Improved purification with preservation of regulatory sites and their interactions. J. Biol Chem. 259, 7219-7233. Sigel, E., and Baur, R. (1988).Activation of protein kinase C differentiallymodulates neuronal Na+,Ca", and y-aminobutyratetype Achannels. Proc. Nutl. Acad. Sci. U.S.A.85,6192-6196. Sigel, E., Baur, R., Trube, G., Mohler, H., and Malherbe, P. (1990). The effect of subunit composition of rat brain GABA, receptors on channel funciton. Neuron 5, 703-711. Sigel, E., Baur, R., Kellengerger, S., and Malherbe, P. (1992). Point mutations affecting antagonist and agonist dependent gating of GABA, receptor channels. EMBO J. 11, 2017-2023. Sinnett, D., Wagstaff, J., Glatt, K., Woolf, E., Kirkness, E. J., and Lalande, M. (1993). High resolution mapping of the y-aminobutyric acid receptor f33 and a 5 gene cluster on chromosome 15ql1-15q13, and localization of breakpoints in two Angelman Syndrome patients. Am. J. Hum. Gaet. 52, 1216-1229. Smart, T. G. (1992). A novel modulatory binding site for zinc on the GABA, receptor complex in cultured rat neurones. J. Physiol. (London) 447, 587-625. Smart, T. G., and Constanti, A. (1986). Studies on the mechanism of action of picrotoxinin and other convulsants at the crustacean muscle GABAA receptor. Proc. R SOC.London B 277, 191-216. Smart, T. G., and Constanti, A. (1990). Studies on the mechanism of action of picrotoxinin and other convulsants at the crustacean muscle GABA receptor. Br.1. Phannucol. 99, 643-654. Smart, T. G., Moss, S.J., Xie, X., and Huganir, R. L. (1991).GABA, receptors are differentially sensitive to zincdependence on subunit composition. Br. J. Phannacol. 103, 1837-1839. Smith, G. B., and Olsen, R. W. (1994). Identification of a [SH]muscimolphotoaiiinity substrate in the bovine y-aminobutyric acidA recptor a subunit. J. Biol. Chem. 269, 20380-20387. Sommer, B., Poustka, A,, Spurr, N. R, and Seeburg, P. H. (1990). The murine GABAA receptor &subunit gene: Structure and assignment to human chromosome 1. DNA Cell Biol. 9, 561-568. Somogyi, P., Takagi, H., Richards, J. G., and Mohler, H. (1989). Subcellular localization of benzodiazepine/GABAAreceptors in the cerebellum of rat, cat and monkey using monoclonal antibodies. J. Neurosci. 9, 2197-2209. Squires, R. F., and Saederup, E. (1993). Indomethacin/Ibuprofen-like anti-inflammatory agents selectively potentiate the y-aminobutyric acid-antagonistic effects of several

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NEUROTRANSMlllER TRANSPORTERS: MOLECULAR BIOLOGY, FUNCTION, AND REGULATION

Beth Borowsky* and Beth J. Hoffman Building 36, Room 3A17, Unit on Molecular Pharmacology, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892-4090

I. Introduction A. Historical Perspectives B. Interest in Transporters 11. Molecular Biology of Transporters A. Nat/C1--Dependent Neurotransmitter Transporters B. Glutamate Transporters C. H'-Dependent Vesicular Transporters 111. Shared Structural Features A. Nat/C1--Dependent Transporters B. Glutamate Transporters C. Vesicular Transporters DI. Regulation of Transporters V. Ionic Requirements A. Nat/C1--Dependent Transporters B. Glutamate Transporters C. Vesicular Transporters VI. Transmitter Emux VII. Transporter Localization A. Distribution B. Glial vs Neuronal C. Subcellular Localization VIII. Transporter Heterogeneity IX. Future Directions References

1. Inhodudon

Synaptic neurotransmission is dependent on four processes (Fig. 1). First, the neuron must synthesize and store the neurotransmitter. Next, an action potential stimulates the release of the neurotransmitter from the presynaptic terminal. The released neurotransmitter then enters the synap-

* Current address: Department of Molecular Biology, Synaptic Pharmaceuticals, 215 College Road, Paramus, New Jersey 07652-1431. INTERNATIONAL REVlEW OF NEUROBIOLOGY, VOL. 98

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Copyright 0 1995 by Academic Press, Inc. All righm of reproduction in any form reserved.

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ammo ncia Precursor

FIG.1. Schematic model of a nerve synapse illustrating components of synaptic neurotrane mission. ( 1 3 Neurotransmitters are synthesized and then packaged in presynaptic vesicles by vesicular transporters. (3) Depolarization-induced release of neurotransmitters from presynaptic vesicles into the synaptic cleft. (4) Neurotransmitters interact with pre- and postsynaptic receptors as well as glial receptors. ( 5 ) Released neurotransmitters are transported back into presynaptic terminals and surrounding glia via membrane-bound transport proteins. NTT, neurotransmitter transporter; R, receptor; VT vesicular transporter.

tic cleft and interacts with both post- and presynaptic receptors. Finally, the neurotransmitter must be removed for the synapse. The predominant mechanism for the removal of released neurotransmitters is by active transport into presynaptic neurons or surrounding glia via membrane-bound transport proteins. Pharmacologic studies have suggested that distinct transporters exist for each of the monoaminergic and amino acid neurotransmitters. The monoaminergic transporters have been of particular interest because they are sites of action for most clinically acting antidepressants as well as for psychostimulants. Acetylcholine is the only major classical neurotransmitter without a transporter. The two major functions of these plasma membrane-bound transporters, inactivation and recycling of released neurotransmitter, are

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accomplished by different mechanisms for acetycholine. Released acetylcholine is enzymatically degraded, and choline, a precursor for the synthesis of acetylcholine, is taken back into the terminal by a specific transporter. The recent cloning of many of these transport proteins has greatly expedited research on individual transporters as well as on the mechanism of transport. This chapter integrates recent molecular biological advances with earlier biochemical and functional studies on transporters to describe what is known, and what remains a mystery, about neurotransmitter transporters.

A. HISTORICAL PERSPECTIVES Uptake of neurotransmitters was described initially for epinephrine (EPI) and norepinephrine (NE) (see Iversen, 1975, for review; Axelrod, 1971). Burn (1932) was the first to suggest that catecholamines might be sequestered by tissue storage sites. Subsequently, the in uiuo administration of large doses of NE and EPI was shown to increase the catecholamine content of many peripheral tissues (Raab and Gigee, 1955; Stromblad and Nickerson, 1961). The importance of uptake in the distribution of circulating catecholamines was demonstrated by Axelrod et al. (1959) and Whitby et al. (1961). Following the intravenous administration of small doses of [3H]NE or ['HIEPI in mice and cats, these investigators found that much of the dose was rapidly removed from the circulation and stored unmetabolized in various peripheral tissues. The observation that ['HI NE uptake was greatest in tissues with dense sympathetic innervation suggested that this neurotransmitter was being sequestered by sympathetic neurons. Further support for this concept came from a series of studies demonstrating that NE uptake was greatly diminished after destruction of sympathetic innervation. For example, superior cervical ganglionectomy greatly diminished the amount of NE accumulated by tissues innervated by this ganglion (Stromblad and Nickerson, 1961; Hertting et al., 1961; Fischer et al., 1965). In addition, destruction of catecholaminergic neurons with Ghydroxydopamine significantly decreased the amount of [3H]NEuptake in both peripheral tissues (Thoenen and Tranzer, 1968; Malmfors and Sachs, 1968) and the brain (Uretsky and Iversen, 1970). Autoradiographic studies, using both light and electron microscopy, provided the first direct evidence that sequestered catecholamines were localized in adrenergic nerve terminals (Wolfe et al., 1962; Marks et al., 1962; Wolfe and Potter, 1963). Using fluorescence histochemistry, Hamberger et al. (1964) demonstrated that NE uptake occurs not only in nerve terminals but also in cell bodies and axons. While NE transport was the first to be characterized, similar systems have since been well documented for other neurotransmitters. Thus, uptake

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systems for dopamine, serotonin, GABA, glutamate, and glycine have been well established. As discussed under Section I, B, molecular cloning has recently revealed additional transporters not previously characterized.

B. INTERESTIN TRANSPORTERS Interest in transport and transporters has focused on a wide spectrum of topics ranging from basic neurochemistry to drug development. The more basic research includes the mechanism of action of transporters as well as their roles in neurotransmission and transmitter recycling. In addition, transporters have been useful tools for identifying and characterizing neurotransmitter systems. For example, the recent cloning of high-affinity transporters for proline and taurine support synaptic roles for these putative neurotransmitters (Fremeau et aZ., 1992; Smith et aL, 1992b; Uchida et aZ., 1992; Liu et aL, 1992a). Transport proteins and transporter mRNAs have also proven to be useful for mapping the anatomic distribution of neurotransmitter systems, some of which lack other specific markers. The use of uptake inhibitors has also helped to determine the physiological roles of neurotransmitters. For example, while a response to a direct-acting agonist suggests that a given receptor is coupled to a given response, it does not necessarily indicate that the receptor is normally innervated by the appropriate neurotransmitter. On the other hand, since uptake inhibitors basically amplify ongoing synaptic activity, responses to uptake inhibitors might reflect more physiologically relevant roles of neurotransmitters. Aconsiderable amount of research has focused on the role of transporters as the site of action of both antidepressants and psychomotor stimulants. Since imipramine was first observed to alleviate symptoms of depression, numerous monoamine uptake inhibitors have been targeted as antidepressants (for review, see Hollister, 1986).The therapeutic effects of the tricyclic antidepressants have long been attributed to their ability to block the uptake of norepinephrine and serotonin. In addition, more selectiveuptake inhibitors are also effective antidepressants. The serotonin uptake inhibitors fluoxetine, paroxetine, citalopram, zimelidine, and setraline, the norepinephrine uptake inhibitors desipramine and protriptyline, as well as the dopamine uptake inhibitor bupropion are all effective in treating depression. The psychomotor stimulant cocaine has been shown in nitro to inhibit the uptake of dopamine, serotonin, and norepinephrine (Ritz et aZ., 1987; Ross and Renyi, 1967, 1969). However, most of cocaine’s effects, such as its rewarding properties, its psychomotor stimulant, reinforcing, and stereotypic effects, are believed to be related to its inhibition of dopamine uptake (Heikkila et al., 1979; Reith et aZ., 1986; Ritz et al., 1987). There is

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considerable interest in understanding the role of the dopamine transporter in the reinforcing effects of cocaine and in developing potential treatments for cocaine abuse (see Kuhar et aL, 1991 for review).

II. Mdecukw Biology of Transporters

Just as advances in molecular biology greatly enhanced our understanding of neurotransmitter receptors in the 1980s,similar advances are expanding out knowledge of transporters in this decade. Since 1990, a growing number of transporters have been cloned and identified. On the basis of sequence homology, ion dependence, and predicted topology, these transporters have been classified into several families (Fig. 2). The first family of transporters functions at the plasma membrane, is Na+ and C1dependent, and has 12 potential transmembrane domains. Members of this family include transporters for dopamine, norepinephrine, serotonin, GABA, glycine, proline, taurine, and betaine. A second family consists of Na+-dependent transporters with 8-10 potential transmembrane domains which function at the plasma membrane. This family includes three transporters for glutamate and a neutral amino acid transporter. The third family consists of vesicular transporters which package neurotransmitters into synaptic or neuroendocrine vesicles by transporting neurotransmitters from the cytoplasm into the vesicle lumen. Examples of this family include the two vesicular monoamine transporters and the vesicular acetylcholine transporter.

A. N~+/CI--DEPENDENT NEUROTRANSMITTER TRANSPORTERS The rat GABA (GAT-1; Guastella et aL, 1990) and human norepinephrine transporters (Pacholczyk et aL, 1991) were the first to be cloned. Purification of the rat GABA transporter protein (Radian et aL, 1986) allowed Guastella and colleagues (1990) to degrade and sequence fragments of this protein. An oligonucleotide probe, designed from one of those fragments, was then used to isolate a rat GABA transporter cDNA from a rat brain cDNA library. Expression in Xenopus oocytes demonstrated that this transporter has a high affinity for GABA, is Na+and C1- dependent, and has a pharmacologic profile similar to that of neuronal GABA transporters. The GAT-1 cDNA encodes a protein of 599 amino acids with a predicted molecular weight of 67 kDa. Hydropathy analysis of the deduced protein

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.J.

HOFFMAh

FIG.2. Classification of the three families of neurotransmitter transporters.The proposed topology of each family is illustrated showing the putative number of transmembranedomains, relative loop sizes, and potential glycosylation sitcs. For glutamate ( B ) , both the 8- and 10transmembranc domain models have been postulated (see tcxt).

shows 11-13 highly hydrophobic segments which may represent membranespanning domains. The human norepinephrine transporter was isolated by Pacholczyk and colleagues (I99 1) using an expression cloning strategy. COSl cells transfected with pools of clones from human SK-N-SH cells were incubated with the norepinephrine analogue miodobenzylguanidine ( [lZ5I]mIBG) . DNA was rescued from positive COSl cells following autoradiography. A single positive clone was isolated, sequenced, and further characterized in HeLa cells. The human norepinephrine transporter rDNA predicts a protein of

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FIG 2. (Continued)

617 amino acids, with 12 or 13 potential membrane-spanning domains. Amino acid comparison between the norepinephrine and GAl3A transporters reveals 46% identity. Allowing for conservative amino acid substitutions, the similarity rises t o 68%. This similarity suggested the existence of a novel family of neurotransmitter transporters and enabled investigators to readily search for other members. Two basic strategies, both relying on conserved sequences, have been used to clone other members of this family. One strategy has been to use degenerate primers to amplify portions of novel transporters from cDNA librarirs using the polyinerasc chain reaction. These amplified frag-

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r

G1*-1.

d Bcp-1 r GAT-? r GAT-3

h TAUT r TAW

r CrcoT r OAT 1

UT

7

puKI(sAmG

9

n

1

h 5HTT r 5HTT r DAT b DAT h NET h DAT r ProT r GlyT-2 r GlyT-la d BGT-1 r GAT-2 r GAT-3 h TAUT r TAUT r CreaT r GAT-1

186 186

166 166 162 166 143 299 133

143 139 152 148 148 158 151

h 5HTT r 5HTT r DAT b DAT h NET h DAT r ProT r GlyT-2 r GlyT-la d BGT-1 r GAT-2 r GAT-3 h TAUT r TAUT r CreaT r GAT-1

229 229 2 13 211 212 214 191 314 193 189 184 199 196 196 211 188

FIG.3. (Continued)

3 OI 3 01.

h 5HTT r 5HTT

r DAT b DAT

281

11 NET

23t 285

28e

11 11x1'

266

r Pro'l' r GlyT-2 r GlyT-la d BGT-1 r GAT-2 r GAT-3 h TAUT I

447 26t

2 52 2 57 272

269 259 284 2 63

TXlT

r CrcdT

r GAT-l

377 379 3 53 361 361 3 64

h SHTT r 5HTT r DAT b 1)AT

t i UET

h

DAT

341 522 341 337 332 347 344

r ProT r GlyT-2 r GlyT-la d BGT-1 r GAT-2 r GAT-3 h TAUT r TAUT r CreaT

344 359 333

r GAT-1

FIG 3. (Continued)

h r r b h h

5HTT

452 454 437 435 435 438 415 596 415 411

5HTr

DAT DAT NET DAT r ProT r GlyT-2 r GlyT-la d BGT-1

r GAT-2 r GAT-3

406

421 418 418 433 412

h TAUT r TAUT r CreaT r GAT-1

h 5HTT r 5HTT r DAT

524 526 508 506

b DAT h NET h DAT r ProT r GlyT-2 r GlyT-la d BGT-1 r GAT-2 r GAT-3 h TAUT r TAUT r CreaT r GAT-1

506 509 487

668 488 486

481 496 493

493 508 485

FIG.3. (Crmtinued)

h r r b h h r

r r d r

r h r r r

5HTT 5HTT DAT DAT NET DAT ProT GlyT-2 GlyT-la BGT-1 GAT-2 GAT-3 TAUT TAUT CreaT GAT-1

h 5HTT r 5HTT

r DAT b DAT h NET h DAT

r r r d r r h r r

ProT GlyT-2 GlyT-la BGT-1 GAT-2 GAT-3 TAUT TAUT CreaT r GAT-1

598 599 582 580

580 583 561 742 562 561 556 571 567 568

*

* TPTEIPCGDIRLNAV TEIPCGDIFMNAV HQLVDRGEVRQFTLRHWLLL TEHGRVDSGEVRQFTLHHWLW NEHHLHLVAQRDIRQFQLQHLAI KDRELVDRGEVRQFTLRHV SLEENRTGMYVATLAGSQSPKPLMVHMRKYGGITSFENTAIEWREIAEEEEESMM PDWGPFLAQHRGERYKNMIDPLGTSSLGLKLPVKDLELGTQC RDWGPALLEHRTGRYAPTTTPSPEDGFFJQPLHPDKAQIPIVGSNGSSRLQDSRI PSLPQPKQHLYLYLDGGTSQDCGPSPTKEGLIVGEKETHL DLPQKSQPELTSPATPMTSLLRLTELESNC LKMRGKLGASPRMVTVNDCEAKVKGDGTISAITMETHF WAVEREGATPYNSRTVMNGALVKPTHIIVETMM AVEREGATPFHSRATLMNGALMKPSHVIVETMM WGLHHLEYRAQDADVRGLTTTPVSESSKWWESVM

EDIVRPENGPEQPQAGSSASKEAYI

FIG.3. (Continued)

583 559

628 629 619 617 600

620 637 799 633 575 602 621 619 621 635 599

NEUROTRANSMITTER TRANSPORTERS

151

ments, or unique portions of them, can then be used as probes to isolate full-length cDNAs. The second strategy has been to use degenerate oligonucleotide primers to screen cDNA libraries at low stringency (Usdin et al., 1991; Borowsky et al., 1993). Using these methods, as well as expression cloning strategies (Hoffman et al., 1991; Uchida et al., 1992), transporter cDNAs have been isolated for dopamine (Shimada et al., 1991; Kilty et al., 1991; Usdin et aL, 1991; Giros et aZ., 1991, 1992), serotonin (Hoffman et al., 1991;Blakely et aZ., 1991; Ramamoorthy et al., 1993a;Corey et al., 1994b), GABA (Clark et aL, 1992; Borden et al., 1992; Liu et al., 1993a), glycine (Smith et aL, 1992a;Guastella et al., 1992; Liu et al., 1992b, 1993b; Borowsky et al., 1993; Kim et aL, 1994), proline (Fremeau et al., 1992), taurine (Smith et al., 199213;Uchida et aZ.,1992;Liu et aZ.,1992a),betaine/GABA (Yamauchi et aL, 1992), and creatine (Guimbal and Kilimann, 1993). The predicted amino acid sequences for these transporters are shown in Fig. 3, while descriptions of each transporter are listed in Table I. Each of these cDNAs encodes a Na+- and C1--dependent transporter with a length of approximately 600 amino acids. Each has 12 hydrophobic segments likely to be transmembrane domains, with both the amino and carboxy termini predicted to be intracellular (Fig. ZA, top). As for many membrane proteins, the amino terminus is predicted to extend into the cytoplasm due to the absence of a “signal peptide.” Members of this family also have a large second extracellular loop with consensus sites for N-linked glycosylation. In addition, there are consensus sites for phosphorylation in intracellular domains. The overall amino acid identity among members of this family is about 20%, but approaches 40% if conservative amino acid substitutions are considered. Based on amino acid sequence, this family can be divided into

FIG.3. Comparison of predicted amino acid sequences for cloned members of the Na+/ C1-dependent transporter family. Shaded residues indicate amino acids that are conserved in at least half of the members. Asterisks below the sequences indicate amino acids that are conserved only among the amino acid transporter subfamily. Solid lines above the sequences indicate putative transmembrane domains. Sequences are shown for the human (h) (Ramamoorthy et al., 1993a) and rat (r) (Hoffman et al., 1991; Blakely et al., 1991) serotonin transporters (5-H’IT); rat (Kilty et al., 1991; Giros et al., 1991; Shimada et al., 1991), bovine (b) (Usdin et al., 1991), and human (Giros et al., 1992) dopamine transporters (DAT); human norepinephrine transporter (NET; Pacholczyk et al., 1991); rat proline transporter (ProT; Fremeau et al., 1992); rat glycine transporters [GLYT-2 (Liu et al., 1993h) and GLW-la (Guastella et al., 1992, Borowsky et al., 1993)l; dog (d) hetaine/GABA transporter (BGT-1; Yamauchi et al., 1992); rat GABA transporters [GAT-1 (Guastella et al., 1990), GAT-2 (Borden et al., 1992), and GAT-3 (Clark et al., 1992; Borden et al., 1992)l; rat (Smith et al., 1992b) and human (S. M. Jhiang, GenBank Accession No. 218956) taurine transporters (TauT); and rat creatine transporter (CreaT; Mayser et al., 1992).

TABLE I Na'/Cl--DEPENDENT FAMILY OF TRANSPORTERS

Transporter DAT

5-HR

Species

Substrate

& (pm)

No. amino acid

mRNA size (kB)

cow

Dopamine

31.5

693

3

Rat

Dopamine

0.3-1.2

619

3.6, 3.7

Inhibitors ( K J " Mazindol, GBR12909 < cocaine < amphetamine < nomifensine Amfolenic acid, GBR12909, mazindol < nomifensine < cocaine < amphetamine Amfolenic acid, GBR 12909, mazindol, nomifensine < cocaine < amphetamine

Human

Dopamine

1.2

620

-

Rat

Serotonin

0.5

630

3.1, 3.7

Paroxetine, chlomipramine < imipramine, fluoxetine, MDMA < cocaine < amphetamine

Human

Serotonin

0.5

630

3, 4.9, 6.8

Paroxetine, chlomipramine < imipramine, fluoxetine, MDMA < cocaine < amphetamine

Distribution

Reference (original name)

Brain

Usdin el al. (1991)

Brain

Kitty et al. (1991); Giros et al. (1991); Shimada et al. (1991);

Brain

Giros et al. (1992)

Brain, adrenals, gut. stomach, kidney, uterus, spleen Brain, adrenals, gut, stomach, kidney, uterus, spleen, placenta

Hoffman el al. (1991); Blakely et al. (1990) (SERT)

Ramamoorthy et al. (1993a)

r

u1

Drosophila Serotonin

0.6

581

3.3

Mazindol, nomifensine < cocaine < fluoxetine < chlomipramine, amphetamine

Newous system

Corey et al. (1994b)

NET

Human

Norepinephrine

0.5

617

5.8

Mazindol < desipramine, nomifensine, nortripytline < amphetamine, imipramine

Brain, adrenal medulla

Pacholczyk et al. (1991)

GAT-1

Rat

GABA

7.3

599

4.2

Nipecotic acid < AHCH, DABA

Brain

Guastella et al. (1990)

GAT-2

Rat

GABA

8

602

2.4

GABA

18

602

2.5

Brain, retina, liver kidney Brain, retina, liver, kidney

Borden et al. (1992)

Mouse

PAlanine < DABA < guavacine PGPA < P alanine < DABA, hypotaurine < guavacine

Rat

GABA

2.3, 12

627

4.7

Brain

Clark et al. (1992) (GAT-B); Borden et al., (1992)

Mouse

GABA

0.8

627

5

@-Alanine < Pproline, nipecotic acid, guavacine, DABA < THPO < ACHC K P A , DABA < hypotaurine < j3alanine < guavacine

Brain

Liu et al., (1993a) (GAT-4)

Dog

Betaine

614

2.4, 3

Quinidine < phloretin, palanine < nipecotic acid, DABA

Kidney

Yamauchi et al. (1992)

01

GAT-3

BGT-1

GABA

398 93

Liu et al. (1993a) (GAT-3)

(continued)

TABLE I- Continued

Transporter

Species

Substrate

& (pm)

No. amino acid

mRNA size (kB)

Inhibitors

(y)"

Distribution

Reference (original name)

633

3.7

Sarcosine < glycine methyl ester, glycine ethyl ester

25

633

3.2

Sarcosine

100

638

3.8

Brain

Glycine

72

638

3.6

Sarcosine < glycine methyl ester, glycine ethyl ester Sarcosine

Glycine

90

692

3.6

Sarcosine

Brain, kidney

Kim et al. (1994)

Rat

Glycine

17

799

8

glycine methyl ester

Brain: spinal cord

Liu et al. (1993b)

PROT

Rat

Proline

637

4

Pipecotic acid < sarcosine, 3.4 dehydroproline, norleucine, phenylalanine < histine, cysteine

Brain

Fremeau et al. (1992)

TAUT

Rat

Taurine

621

6.2

Hypotaurine, palanine, GES < GABA

Brain, kidney, heart, spleen, ileal mucosa, lung, epididymis

Smith et al. (1992b)

GLYT-la

Rat

Glycine

Mouse

Glycine

GLYT-Ib

Rat

Glycine

Human GLIT-lc

Human

GLYT-2

33,94

9.7

43

Brain, spinal cord, stomach spleen, liver lung, thymus, uterus Brain, spinal cord

Brain

Guastella et al. (1992) (GLyT1); Borowsky et al. (1993) (GLW2)

Liu et al. (1992b) (GLW Smith et af. (1992a) (GLYTI) Kim d al. (1994)

Creatine

Canine

Taurine

Mouse

Taurine

Rabbit

Creatine

12

4.5

35

655

7

Hypotaurine, palaine, GES < GABA

590

7.5

Hypotaurine, palanine, GES < GABA

635

4.5. 3.2

GP, GB < GAA, AGP

Brain, kidney, heart, spleen, ileal mu cosa, lung, epididymis Brain, kidney, heart, spleen, ileal mucosa, lung, epididymis

Uchida el al. (1992)

Lung, kidney, skeletal muscle, epididymis, heart, brain

Guimbal and Kilimann (1993)

Liu et al. (1992a)

MDMA, 3,4methylenedioxy-methamphetamine(ecstasy); ACHC, cis-Saminocyclohexane carboxylic acid; DABA, 2,Qdiaminobutyrc acid; W P A , P-guanidinopropionic acid; THPO, 4,5,6,7-tetrahydroisoxazolo [4,5c]pyridine-3-01; GES, Pguanidinoethanesulfonic acid; GP, 3gaunidinopropionate; GB, 4-guanidinobutyrate; GAA, guanidinoacetic acid; AGP, 2-aminc~3guanidinopropionate.

NEUROTRANSMITTER TRANSPORTERS

157

three subfamilies: monoamine, amino acid, and atypical neurotransmitter transporters. Interestingly, there is a higher degree of similaritywithin each subfamily than between subfamilies. For example, while many amino acid residues are conserved by members of both the amino acid and monoamine subfamilies, certain residues are conserved only within one subfamily (Figs. 3 and 4). Members of the atypical Na+/Cl--dependent transporter family have about 30%identity with members of the other subfamilies and about 60% identity with other members within the subfamily. Like the monoamine and amino acid transporters, these transporters have 12 predicted transmembrane domains and a large second extracellular domain with consensus sites for N-linked glycosylation (Fig. 2A, bottom). However, these transporters also have an extensive fourth extracellular loop with consensus sites for N-linked glycosylation and longer amino and carboxy termini. Members of this family include V7-3-2, V17-2-1, V17-3-2 (Uhl et aL, 1992), NTT4 (Liu et aL, 1993c), and XT1 (El Mestikawy et aL, 1994). The substrates for these orphan clones have not yet been determined.

B. GLUTAMATE TRANSPORTERS While cloning strategies based on sequence homology of the GABA and norepinephrine transporters resulted in the isolation and identification of many transporters, these strategies were unsuccessful in cloning a transporter for the excitatory amino acid neurotransmitter glutamate. It is now clear that these strategies were ineffective because the glutamate transporters define a new family of neurotransmitter transporters with no significant homology to the Na+/Cl--dependent family of transporters (Kanai et al., 1993, 1994; Kanner, 1993). Three distinct, but structurally similar, cDNAs

FIG.4. Sequence homology and predicted membrane topology of Na+/Cl--dependent transporters. Depicted here is the deduced amino acid sequence and predicted membrane topology of the rat 5-HT transporter (Hoffman et al., 1991; Blakely et al., 1991). Amino acid residues that are shaded are conserved among all the monoamine members of the Na'/CI-dependent family: human norepinephrine (Pacholczyk et aL, 1991), rat dopamine (Kilty et al., 1991; Giros et at., 1991; Shimada et al., 1991),cow dopamine (Usdin et ai, 1991), human dopamine (Giros et al., 1992), human 5-HT (Ramamoorthy et al., 1993a), and rat 5-HT transporters. Amino acid residues in black are conserved among all members of the Na'/CI--dependent family. Potential N-linked glycosylation sites are indicated. The triangle indicates a potential phosphorylation site that is conserved among all members of the Na'/Cl-dependent family, while circles indicate potential phosphorylation sites conserved among the monoamine subfamily members.

158

BETH BOROWSKY AND BETH J. HOFFMAN

encoding glutamate transporters have recently been isolated using methods not dependent on sequence homology (Fig. 5 , Table 11). Kanai and Hediger (1992) used a Xenopus oocyte expression system to clone a glutamate transporter (EAACl) cDNA from rabbit small intestine. Pines and colleagues (1992) used an antibody against a purified glutamate transporter to isolate a glutamate transporter (GLT-1) cDNA from a rat brain cDNA library. Finally, Storck et al. (1992) used an oligonucleotide probe, based on the sequence of a protein that copurified with a ceramide galactosyl transferase, to isolate a third glutamate transporter (GLAST) cDNA. Recently, three human glutamate transporters have also been cloned (Arriza et aL, 1994). EXAT1 has 96% identity with the rat GLASTl sequence, EAAC2 has 95% identity with the rat GLT-1 sequence, while W T 3 has a 92% identity with the rabbit EAACl sequence (Arizza et al., 1994). The three glutamate transporters are structurally very similar, with 51-55% amino acid identity (Fig. 5 ) . In contrast, they show no sequence similarity to members of the Na'/Cl--dependent family. The number of membrane-spanning domains originally predicted were 6 for GLAST (Storck et al., 1992), 8 for GLT-1 (Pines et al., 1992), and 10 for EAACl (Kanai and Hediger, 1992). Hydropathy analysis for each clearly predicts 6 transmembrane domains on the amino terminal half of the protein. However, there is some controversy as to the number of transmembrane domains at the carboxy terminus. The proposed 8- and 10-transmembrane domain models are shown in Fig. 2B, while a 9-transmembrane domain model is shown in Fig. 5. Additional structural information will be necessary before a more definitive topological model can be established. A recently cloned neutral amino acid transporter (Arriza et al., 1993; Shafqat et al., 1993) is also structurally related to the glutamate transporters (34-39% amino acid identity). However, while the glutamate transporters are dependent on both Na' and K', the neutral amino acid transporter appears to be dependent only on Na+. Interestingly, members of this family have significant sequence homology to the proton-coupled glutamate transporters of Eschenchia coli and other bacteria (glt-P; Tolner et al., 1992), as well as to the dicarboxylate transporter of Rhizobium meliloti (dct-A, Jiang et al., 1989).

C. H+-DEPENDENT VESICULAR TRANSPORTERS The transporters discussed previously are all located on the plasma membrane and function to transport released neurotransmitters from the synapse into the cytoplasm of neurons and glia. In contrast, the role of the vesicular transporter is to repackage this cytoplasmic pool of neurotransmit-

h ASCTl h EAATl r GLASTl h EAAT2 r GLTl h EAAT3 rb EAACl

333

h ASCTl h EAATl r GLASTl h EAAT2 r GLTl h EAAT3 rb EAACl

408 428 428 427

h h

483 503 503

353 353 352 351 322 321

426

397 396

ASCTl EAATl r GLASTl h EAAT2 r GLTl h EAAT3 rb EAACl

h

ASCTl EAATl r GLASTl h EAAT2 r GLTl h EAAT3 rb EAACl h

502 501 472 471

KGEQELAEWVEAIPNCKSEEETSPLVTHQNPAGPVASAPELESKESVL PYQLIAQDNETEKPI-DSETKM PYQLIAQDNEPEKPV ECKVTLAANGXSADCSVEEEPWKREK

IDECKVTLAANGKSADCSVEEEPWKREK

DSDTKKSYINGG

FIG.5. (Continued)

532 542 543 574 573 525 524

NEUROTRANSMITTER TRANSPORTERS

161

ters into presynaptic vesicles (for review, see Edwards, 1992). During synaptic transmission, these vesicles fuse with the plasma membrane and release their contents into the synapse. Two similar, but distinct, monoamine vesicular transporters have been cloned (Fig. 6, Table 111).An adrenal chromaffin granule amine transporter (CGAT) cDNA was cloned from a PC12 cDNA library by examining the ability of mammalian cells, transfected with groups of cDNAs, to survive in the presence of the toxin MPPt (Liu et aL, 1 9 9 2 ~ )A . second vesicular amine transporter was isolated from a rat basophilic leukemia cell cDNA library using an expression cloning strategy (MAT; Erickson et aL, 1992) and from a rat brain stem cDNA library by low stringency screening with CGAT (SVAT; Liu et aZ.,1 9 9 2 ~ )The . adrenal and brain vesicular transporters have been renamed VMATl and VMAT2, respectively. The two vesicular amine transporters are proton dependent, transport serotonin, dopamine, norepinephrine, epinephrine, and histamine, and are inhibited by reserpine and tetrabenazine. The two transporters show different ranks orders of potency for monoamines: serotonin > epinephrine > dopamine > norepinephrine > histamine forVMAT1, and serotonin > dopamine > norepinephrine > epinephrine > histamine for VMAT2 (Liu et al., 1992c; Erickson et aL, 1992). In addition, VMAT2 has a 10-fold higher affinity for histamine than does VMATl (Peter et al., 1994). From initial studies, VMATl was found only in chromaffm granules, while VMAT2 was found in monoaminergic neurons in the CNS as well as in peripheral tissues. In further studies, both VMATl and VMAT2 have been identified in the adrenal medulla. VMATP is present in both chromaffin cells and sympathetic ganglion cells, while VMATl is present only in chromaffin cells (Hoffman and Mezey, 1993;Mahata et aL,1993).The cDNA for each transporter predicts a protein with 12 transmembrane domains and a large first loop with potential N-linked glycosylation sites facing the vesicle lumen. The transporters are 62% identical to each other and also share limited, but significant, homology to bacterial drug-resistance genes (for review, see Schuldiner, 1994). Interestingly, a recently cloned bovine chro-

FIG.5. Comparison of predicted amino acid sequences for cloned members of the glutamate transporter family. Shaded residues indicate amino acids that are conserved in at least half of the members. Solid lines above the sequences indicate putative transmembranedomains [using the nine-transmembrane model from Arriza et al., (1994)l. Sequences are shown for human (h) ASCTl (Arriza et al., 1993), human EAATl (Arriza et al., 1994; Shashidharan and Plaitakis, 1993; Kawakami et al., 1994), rat (r) GLASTl (Storck et al., 1992), human EAATP (Arriza et al., 1994; Shashidharan et al., 1994), rat GLTl (Pines et al., 1992; Kanner, 1993), human EAAT3 (Arriza et al., 1994; P. Shashidharan, GenBank Accession No. U08989), and rabbit (rb) EAACl (Kanai and Hediger, 1992).

TABLE I1 GLUTAMATE FAMILY OF TRANSPORTERS

K (gm)

No. amino acids

mRNA size

(y)"

Reference (original name)

Transporter

Species

Substrate

EEACl

Rabbit

Glutamate

12

524

3.5, 2.5

THA < AAD < DHK

Brain, kidney, small intestine, liver, heart,

Kanai and Hediger (1992)

GLAST

Rat

Glutamate

77, 11

543

4.5

THA

Brain

Storck et al. (1992)

2

573 532

11 2.2-5.1

THA < AAD, DHK

Brain

Pines et al. (1992)

-

Brain, heart, placenta, liver, kidney, skeletal muscle, pancreas, lung

Arriza et al. (1993); Shafquat et al. (1993) (SAlT)

Inhibitors

(kB)

Distribution ~

~

~~

CLT-1

Rat

Glutamate

ASCTl

Human

Alanine, 71 88 Serine, Cysteine 29

FAATl

Human

Glutamate

48

542

4.2

THA < PDC < SOS < DHK. KA

Brain, heart, muscle, placenta lung

Aniza et al. (1994)

FAAT2

Human

Glutamate

97

574

10

PDC < THA < DHK < KA <

Brain, placenta

Aniza et al. (1994)

Human

Glutamate

62

525

3.8

THA < PDC < SOS < DHK. KA

Kidney, brain lung, Aniza et al. (1994) liver, heart placenta, muscle

i

m

N

FAAT3

sos

THA, m-threo phydroxyaspartate; AAD,La-aminoadipate; DHK, dihydrokainate; SOS, L-serine, Osulfate; KA, kainic acid.

FIG.6. Sequence homology and predicted membrane topology of vesicular monoamine transporters. Depicted here is the deduced amino acid sequence and predicted membrane topology of the rat vesicular monoamine transporter (VMAT2; Erickson et al., 1992; Liu et aZ., 1992~).Potential N-linked glycosylation sites are shown facing the vesicle lumen. Potential protein kinase C sites are indicated by circles and potential protein kinase A sites are indicated by triangles. Amino acid residues in black and protein kinase sites darkly shaded are conserved among the rat, human (Surratt et aZ., 1993; Peter et aZ., 1993; Erickson and Eiden, 1993), and cow (Krejci et aZ., 1993; Howell d aL, 1994) VMAT2, while lightly shaded residues and protein kinase sites are conserved between the rat VMATl (Liu et aZ., 1992c) and VMAT2.

TABLE 111 VESICULAR FAMILY OF TRANSPORTERS

Transporter

Species

Substrate

& (pm)

No. amino acids

mRNA size (kB) Inhibitors

(y)

Distribution

Reference (original name)

VMATl

Rat

Serotonin epinephrine, dopamine, norepinephrine

521

3

Reserpine < tetrabenazine < ketanserin

Adrenal gland

Liu et al. (1992~) (CGAT)

W T 2

Rat

Serotonin, dopamine, norepinephrine, epinephrine, histamine Serotonin, dopamine, norepinephrine, epinephrine, histamine

515

4, 2.2, 2.9

Reserpine < tetrabenazine < ketanserin

Brain, stomach

Erickson ef al. (1992) (MAT); Liu ef al. (1992~) (SVAT)

514

Reserpine < tetrabenazine < ketanserin

Brain, stomach

Surratt et al. (1993) (VAT2); Peter d al. (1993) (SVAT); Erickson and Eiden

Reserpine < tetrabenazine < ketanserin

Brain adrenal gland

Krejci et al., (1993) Howell ef al., (1994)

Human

(1993) (VMAT1)

WG17

VAChT

cow

Serotonin, dopamine, norepinephrine, epinephrine, histamine

518

C. eleganj

Acetylcholine

527

2

Nervous system

Alfonso et al.

Torpedo

Acetylcholine

51 1

3

Electric lobe

(1993) Varoqui et al. (1994)

Rat

Acetvlcholine

531

3

Human

Acetylcholine

533

Vesicamol

Brain, spinal cord

Erickson et al. ( 1994) Erickson et al. ( 1994)

NEUROTRANSMITTER TRANSPORTERS

165

m a n granule vesicular transporter (bVMAT2; Krejci et al., 1993; Howell et aZ., 1994) is more similar to the rat and human VMAT2 (Surratt et aL, 1993; Peter et aL, 1993; Erickson and Eiden, 1993) than to the rat VMATl transporter. A putative vesicular acetylcholine transporter gene, unc-17, was cloned from Cmnorhabditis ekgans (Alfonso et al., 1993). The protein encoded by this gene has 12 potential transmembrane domains and is 37% identical to the rat VMATl and 39% identical to the rat VMAT2 (Fig. 7, Table 111). A homologue of unc-17was cloned from the Torpedoelectric lobe (Varoqui et aL,1994).Vesamicol (-) tran~-2-(4phenylpiperidino)cyclohexanol, which binds to cholinergic vesicles and blocks acetylcholine storage, was shown to bind to membranes from cells transfected with either unc-17 or the Torpedo homologue (Varoqui et al., 1994). Rat and human homologues of this vesicular acetylcholine transporter have also been isolated (VAChT; Erickson et aL, 1994). Interestingly, the entire cDNA sequences for the human VAChT and unc-I7 are contained within the first introns of the human and C. ekgans choline acetyltransferase genes, respectively (Erickson et al., 1994;Alfonso et al., 1994).This represents a unique and evolutionarily conserved “ACh locus,” which may allow coordinated transcription of two essential cholinergic genes. These vesicular transporters thus define another family of neurotransmitter transporters (Fig. 2C). The similarity of this family to bacterialresistance genes, which remove toxic substances from the cytoplasm, as well as the ability of members of this family to function as antiporters has led Schuldiner (1994) to suggest referring to these transporters as Toxin Extruding ANtiporters, or TEXANS. 111. Shared Structural Features

The actual process by which these families of transporters bind substrates and ions and transport them across a membrane is not yet fully understood. However, predictions about the structure and function of these transporters can be made by examining common structural motifs within families, the results of mutagenesis and deletion studies, and by analogy to other systems. As the first and largest of the families cloned, more information has been amassed about the Na+-/C1--dependent family than the glutamate and vesicular families. A. N~+/CI--DEPENDENT TRANSPORTERS The most conserved regions in this family are in the 12 hydrophobic stretches (20-24 amino acids) predicted to be transmembrane domains,

FIG.7. Sequence homology and predicted membrane topology among H+-dependentvesicular transporters. Depicted here is the deduced amino acid sequence and predicted membrane topology of the rat vesicular monoamine transporter (VMAT2; Erickson et aL, 1992; Liu d al., 1992~). Potential N-linked glycosylation sites are shown facing the vesicle lumen. Potential protein kinase C sites are indicated by circles and potential protein kinase A sites are indicated by triangles. Amino acid residues that are shaded are conserved among all members of the vesicular family: human (Surratt et al., 1993; Peter et al., 1993; Erickson and Eiden, 1993) and cow (Krejci et al., 1993; Howell et al., 1994) VMATP, rat VMATl (Liu d al., 1992c), and the vesicular acetylcholine transporters from C. &guns (Alfonso et nl., 1993), ToTpedu (Varoqui et al., 1994), and rat and human (Erickson et al., 1994).

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suggesting a role for these regions in transport. Between these transmembrane domains are five intracellular and six extracellular loops. With the exception of the second extracellular loop (50-75 amino acids) and the fourth extracellular loop in the atypical subfamily (90 amino acids), these extracellular loops are fairly small (6-25 amino acids). Transmembrane domains 1, 2, and 4-8 are the most highly conserved within the Na+/CI-dependent family, suggesting that these regions may be involved in general transport functions. In contrast, residues conserved only within subfamilies might be involved in substrate and inhibitor recognition. The currently proposed model is that a fraction of the transmembrane domains form a pore to which the substrate and ions bind extracellularly. This binding results in a conformational change which exposes the substrate and ions to the cytoplasm. The transmembrane domains most likely to be associated with the pore are ones that are amphipathic cy-helices, with hydrophobic residues facing the membrane and hydrophilic residues facing the pore. Based on an Eisenberg analysis, Brownstein and Hoffman (1993) found that at least three transmembrane domains, 1, 6, and 7, are likely to be amphipathic. The ligand-binding domains of neurotransmitter receptors may provide clues to the binding of these substrates to their transporters. For example, two serine residues in the fifth transmembrane domain of adrenergic recep tors have been proposed to act as a hydrogen bond donor in the binding of the hydroxyl group of the catechol ring (Strader et aZ., 1989).In addition, a conserved aspartate residue in adrenergic receptors has been implicated in the binding of ligands with protonated amine groups (Horstman et al., 1990; Strader et aL, 1987). By analogy, polar amino acid residues within transmembrane domains may be important for substrate binding in transporters. In support of this, an aspartate residue (Asp79 in the first transmembrane domain of the dopamine transporter) has been demonstrated to be necessary for function of the dopamine transporter (Kitayama et aZ., 1992). These investigators demonstrated that replacement of Asp79 with alanine, glycine, or glutamate reduced both [SH]DAuptake and the binding affinity of the cocaine analogue C R . This aspartate is conserved in the monoamine subfamily but substituted by glycine in the amino acid subfamily, suggesting that this residue may be important in neurotransmitter recognition by monoamine transporters. These investigators further demonstrated that replacement of two serine residues (Ser356 and Ser359 in the seventh transmembrane domain) by alanine or glycine reduced ['HI DA uptake with less effect on C R binding. These serine residues are conserved throughout the family, and thus may play an important mechanistic role

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in transport of both monoamines and amino acid neurotransmitters. Chimeric transporters have also been used to identify regions of transporters involved in substrate and inhibitor recognition (see Future Directions). Kanner and colleagues used several approaches to identify regions of the GABA transporter critical for function. Measuring transport activity following protease treatment originally suggested that deletion of 1520 kDa (out of 80 kDa) left a functional transporter that was truncated at both the amino and the carboxy termini (Mabjeesh and Kanner, 1992). Further work, using a series of deletion mutants, demonstrated that transporters could only be truncated to within a few amino acids of the first and last transmembrane domains without impairing function (Bendahan and Kanner, 1993).The discrepancy between the proteolysis and mutagenesis data may be explained by the atypical mobility of hydrophobic proteins (Takagi, 1991). Since GABA is a zwitterionic molecule and its cosubstrates Na+ and C1are charged, it is likely that charged amino acid residues in the membranespanning domains are important in their transport. Pantanowitz et al. (1993) used site-directed mutagenesis to examine the role of the five charged amino acids in the transmembrane region (Arg69, Lys231, Arg257, Glu467, Lys497). Only one of these, Arg69, was found to be absolutely essential for function. Since other charged residues did not restore function, it is unlikely that it is simply the positive charge which is critical. This Arg lies in the highly conserved region between the first and second transmembrane domains and may be important in the binding of C1- ions. It is reasonable to assume that Na+ions would bind to negatively charged residues in the membrane. However, Glu467, the only negatively charged amino acid residue in the membrane, was shown not to be essential for function (Pantanowitz et al., 1993). Other residues which could bind Na+ include those capable of electrostatic interactions with positive charges. Aromatic amino acids, such as tryptophan, have been shown to be important in the structure and function of acetylcholinesterase (Sussman et al., 1991) and barnase (Loewenthal et aL, 1991). Site-directed mutagenesis of the 10 tryptophan residues predicted to be in transmembrane domains of the GABA transporter revealed three residues critical for function (KleinbergerDoron and Kanner, 1994). Replacement of tryptophan in position 68,222, or 230 with serine or leucine severely impaired GABA transport. Replacement of Trp68 or 230 with the aromatic amino acids phenylalanine or tyrosine did not effect transport, while replacement of Trp222 with these amino acids impaired GABA transport. The lack of activity following replacement of Trp230 with serine was attributed to inefficient targeting to the plasma membrane. Interestingly, Trp68 is located in the same region

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as Arg69 and Asp79, which have also been shown to be required for function (Pantanowitz et aL, 1993; Kitayama et aL,1992). Trp222 is conserved within the amino acid subfamily but not in the monoamine subfamily, suggesting that it may be involved in neurotransmitter binding. Transmembrane domains rich in prolines may also play important roles in transport. Prolines, normally absent in transmembrane domains in nontransport proteins, are located in both hydrophobic and hydrophilic domains in transport proteins (Brand1 and Deber, 1986). Structural changes following isomerization of X-proline peptide bonds could contribute to conformational changes necessary for transport (Brand1and Deber, 1986). Prolines also introduce bends in a-helices which might contribute to the formation of pockets for binding substrates and ions. In the Na+/Cl-dependent family, 5 of the 12 transmembrane domains contain conserved proline residues (1, 2, 5, 11, and 12). The large putative extracellular loop between transmembrane domains 3 and 4 shows little homology among members of the family. However, all members contain several consensus sites for N-linked glycosylation in this loop. Regional variations in the glycosylation pattern for the dopamine transporter may account for the differences in the apparent molecular weight of this transporter in the nucleus accumbens and striatum (Lew et aZ., 1991). Zalecka and Erecinska (1987) demonstrated a 40% decrease in the V,, for dopamine transport following treatment of synaptosomes with neuraminidase, suggesting that glycosylation may regulate transport function. Further, mutagenesis studies have demonstrated that elimination of glycosylation impairs or prevents substrate transport and radioligand binding (Keynan et aZ., 1992). While this study suggests that glycosylation is required for a functional transporter, it must also be considered that these mutants might not target to the plasma membrane efficiently. Using both tunicamycin treatment and mutagenesis studies, Tate and Blakely (1994) demonstrated that glycosylation of the serotonin transporter is required for optimal stability of the transporter in the membrane, but not for serotonin transport or ligand binding. Cysteine residues are often critical for appropriate folding and secondary structure. Two conserved cysteine residues in the &-adrenergic receptor have been shown to be important in ligand binding, perhaps through the formation of a disulfide bond (Dixon et aZ., 1987). Interestingly, while the large extracellular loop of the Na+/Cl--dependent transporters shows little conservation, two cysteines, nine amino acids apart, are present in all members of this family. By analogy, tertiary structure in this extracellular loop, resulting from disulfide bonds, may be important for transporter function, as has been demonstrated by Meiergerd and Schenk (1994).

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B. GLUTAMATE TRANSPORTERS The three cloned eukaryotic glutamate transporters are between 51 and 55% identical, while the neutral amino acid transporter is somewhat less homologous. Although there is some controversyas to the number of transmembrane domains in these transporters, hydropathy plots suggest similar tertiary structures. A reasonable model predicts 8,9, or 10 transmembrane domains (see Figs. 2B and 6). There are four highly conserved stretches in this family, predominantly in hydrophobic regions. In the 10-transmembrane domain model, these conserved regions include transmembrane domains 2 and 7-10, as well as a portion of the large second extracellular loop. Regions of the transporter important in the binding and transport of substrates and ions have not yet been determined. One potentially important region is a serine motif in the seventh transmembrane domain (using the 10 TMD model; Fig. 2B).As mentioned previously, serine residues have been implicated in the binding of ligands to adrenergic receptors (Strader et al., 1989) and in the transport of dopamine across the plasma membrane (Kitayama et al., 1992). Other potentially important residues include Glu383 (in EAAC1; Kanai and Hediger, 1992), the only polar amino acid in a highly conserved hydrophobic stretch, and conserved charged amino acids Lys269 and His296 (in EAACl) in transmembrane domains 5 and 6, respectively. Members of this family have two consensus sites for N-linked glycosylation in similar positions in the large second extracellular loop. Deglycosylation studies suggest that GLT-1 and GLAST are glycosylated at one or two sites (Storck et al., 1992; Danbolt et aL, 1992). Glycosylationof EAACl, which has an additional consensus site for N-linked glycosylation in the first extracellular loop, has not yet been studied. The significance of glycosylation in the function of the glutamate transporters has not been determined.

C. VESICULAR TRANSPORTERS There are a number of conserved charged and polar amino acid residues within the transmembrane domains of the vesicular transporters (see Fig. 7). These residues may be important in the binding and translocation of substrate and H' (Erickson et al., 1992). In particular, there are four conserved aspartate residues which may be important for interacting with amino groups on substrates. IV. Ragdotion of Transporters

Since reuptake into presynaptic nerve terminals and surrounding glia is the predominant mechanism for terminating the action of released neu-

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rotransmitters, changes in the activity of transporters should have a significant impact on the amount and duration of neurotransmitters present in the synapse. These changes, in turn, would alter the response of both preand postsynaptic receptors to released neurotransmitters. Changes in the level of transport could be due to either changes in the affinity of the transporter for substrates or changes in the maximal velocity of the transporter. The latter could result from either changes in the rate at which the transporter cycles from occupied to unoccupied, or changes in the number of active transporters at the cell surface. Alterations in Na+-dependent transport have been identified in a number of neurological and psychiatric disorders (Coppen et al., 1978; Tuomisto et al., 1979; Meltzer et al., 1981; Kaplan and Mann, 1982; Palmer et al., 1986; Cross et al., 1986; Haberland and Hetey, 1987; Markianos and Sfagos, 1988; Arora and Meltzer, 1989; Malmgren et al., 1989; Rothstein et al., 1992). For example, changes in serotonin transport have been implicated in several diseases. Although one study found no changes in imipramine binding in frontal cortex from postmortem suicide victims (Arora and Meltzer, 1989), others have reported a decrease in the B,,,, for imipramine binding in postmortem brains from depressed patients (for review, see Langer and Schoemaker, 1988). Studies on platelets from cirrhotic and hypertensive patients demonstrated a decrease in serotonin uptake without a change in imipramine binding (Ahtee et al., 1974, 1981; Bhargava et al., 1979; Kamal et al., 1984). Similarly, an increase in K, for platelet serotonin transport has been demonstrated in patients with multiple sclerosis (Markianos and Sfagos, 1988). However, caution should be exercised in interpreting these data since imipramine is not specific for serotonin, and platelets may not be an adequate model for serotonin neurons. A study examining postmortem tissue from schizophrenic patients found an increase in both the K,,, and the V,, for dopamine and norepinephrine transport in the nucleus accumbens and caudate nucleus (Haberland and Hetey; 1987). In addition, several groups have found that the density of glutamate transporters is decreased in patients with amyotrophic lateral sclerosis, Huntington’s disease, and Alzheimer’s disease (Rothstein et al., 1992; Palmer et aL, 1986; Cross et al., 1986). Whiel these studies suggest that altered transport activity is associated with a number of diseases, the mechanisms by which these transporters become dysfunctional have not yet been determined. In addition to disease states, there is also growing evidence that normal transporter function is subject to physiologic regulation. Such regulation could occur at the level of transcription or translation or by posttranslational modifications. Much of the focus thus far has been on the role of second-messenger systems. Potential pathways through which secondmessenger systems might regulate transport function acutely are illustrated

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A,

~u b e a t e

I<+

K+

NO

cGMP

B.

substrate

NO cGMP FIG.8. Schematic models illustrating potential mechanisms for phosphorylation-mediated regulation of neurotransmitter transport. (A) Direct effect of phosphorylation. Activation of protein kinases following an increase in intracellular second-messenger concentration (CAMP, IPS,DAG, NO, cGMP, or Ca2+)can alter transport activity directly by phosphorylation of the transporter. (B) Indirect effect of phosphorylation. Activation of protein kinases following an increase in intracellular second-messenger concentration can regulate transport activity indirectly by altering ion gradients through phosphorylation of ion channels or Na'/K+ ATPases. (C) Redistribution of transporter molecules between active and inactive pools. This schematic depicts the possible life cycle of transport proteins. Activation of membrane-bound receptors through signal transduction pathways (1; for example, see 8A and 8B) leads to phosphorylation of one or more target proteins. These phosphoproteins signal (2) the movement of transportercontaining vesicles (3) to the plasma membrane. The fusion of these vesicles with the plasma membrane results in the redistribution of transporters (4), thus leading to an increase in neurotransmitter transport through an increase in the number of transport proteins ( 5 ) . Receptor signaling may lead to phosphorylation of other target proteins (1) which, in turn, signal retrieval of transporters from the plasma membrane by invagination

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C.

FIG.8. (Continued)

in Fig. 8. Many of these pathways converge by activating protein kinases. Regulation of transport could occur directly by phosphorylation of the transport protein (Fig. 8A), or indirectly by phosphorylation of other proteins, such as Na+/K+-ATPase,phosphatases, or proteins affecting ion gradients (Fig. 8B). Alternatively,as illustrated in Fig. 8C, phosphorylation could affect the distribution of transporters in active and inactive pools, as has been demonstrated for the insulin-responsive glucose transporter (Birnbaum, 1989). Cholera toxin, which increases CAMPlevels, increases the V,, and decreases the Kd for serotonin uptake in the human placental choriocarcinoma cell line JAR (Cool et al., 1991). This effect is mimicked by isobutylmethylxanthine, dibutyryl CAMP,and forskolin and blocked by the protein

FIG.8. (Continued) of membranes (6)followed by fission to form vesicles (7). This results in a decrease in neurotransmitter transport by removing transport proteins from the plasma membrane. These vesicles (7) then fuse with larger endosomes (8). Transporters destined to be recycled segregate and are pinched off to form new vesicles (9). These concepts are proposed based on the traflicking of glucose transporters (Lienhard et al., 1992). A, Na+/Kt ATPase; C, ion channel; DAG, diacyl glycerol; E, effector; G, guanine nucleotide binding protein; IPS, 1,4,5-inositol triphosphate; L, ligand; R, receptor; T, transporter.

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kinase inhibitor N ( 2-aminoethyl)-5-isoquinolinesulfonamide. The stimulatory effect of cholera toxin is only evident after 16-24 h, suggesting that this effect occurs at the transcriptional level. In contrast to the stimulatory effect of cholera toxin in JAR cells, cholera toxin rapidly decreases serotonin uptake in rat adrenal pheochromocytoma cells (PC12 cells) suggesting that regulation may be cell specific (King et aL, 1992). In contrast to the stimulatory effects of CAMP, activation of protein kinase C (PKC) inhibits serotonin transporter function. Phorbol-12myrisate-13-acetate (PMA), an activator of PKC, decreases the V,, without af€ecting the K , of serotonin transport in perfused rabbit lung and bovine pulmonary artery endothelial cells (Myers and Pitt, 1988;Myers et al., 1989). In human platelets, the PKC activator 4~-12-tetradecanoyl-phorbol-l3acetate (p-TPA) decreased the V,, of serotonin transport without affecting the number of serotonin transporters at the platelet surface (Anderson and Horne, 1992). Inhibition of serotonin uptake by PMA is reversed by the PKC inhibitor, staurosporine, and is accompanied by a translocation of PKC activity from the cytosol to the membrane in bovine pulmonary artery endothelial cells (Myers et al., 1989). The translocation of activated PKC to the membrane may affect serotonin uptake directly by phosphorylating the transporter or indirectly by phosphorylating other membrane proteins such as the Naf/Kf-ATPase. The calmodulin antagonist CGS 9343BA decreases serotonin transport in two human placental choriocarcinoma cell lines,JARand BeWo (Jayanthi et aL, 1994). This decrease is associated with a decrease in the maximal velocity and affinities for Naf and C1- and an increase in the apparent affinity for serotonin. CGS 9343BA has no effect on alanine uptake through the Naf-dependent ASC transport system and stimulated leucine uptake through the Naf-independent amino acid transport system, eliminating changes in ion gradients as the cause for altered serotonin transport. Further, neither serotonin transporter mRNA content nor binding of the cocaine analogue RTI-55 are altered by the calmodulin antagonist. These findings suggest that the human serotonin transporter is regulated by calmodulindependent post-translational modification. Although several studies have demonstrated regulatory roles for secondmessenger systems, the physiological mechanisms which trigger the regulation of transporters have not been defined. Taking advantage of the presence of both the serotonin transporter and the G protein-coupled As adenosine receptor in rat basophilic leukemia cells (RBL 2H3), Miller and Hoffman (1994) investigated the ability of the serotonin transporter to be modulated by receptor activation. Treatment of RBL 2H3 cells with the As adenosine receptor agonist 5'-hrethylcarboxamidoadenosine (NECA) increases serotonin uptake, with an increase in both the V,, and the K,.

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The effect is mimicked by the cGMP analogue 8-bromocGMP, and NECA increases cGMP levels. The adenosine receptor antagonist xanthine amine congener blocks both the increased serotonin uptake and the elevation in cGMP. The nitric oxide donors Snitroso-Nacetylpenicillamine(SNAP) and hydroxylamine increase serotonin uptake, while the nitric oxide synthase inhibitors M-nitro-L-arginine methyl ester hydrochloride and NGmonomethyl-L-arginine acetate (NMMA) block the effect of NECA on u p take. In contrast to the effect of NECA, stimulation of PKC with the phorbol ester PMA decreases serotonin uptake in RBL 2H3 cells by decreasing the V,,,. In rat brain synaptosomes, the effects of SNAP, 8-bromocGMP, and PMA are similar to those seen in RBL 2H3 cells (K. J. Miller and B. J. Hoffman, unpublished observations, 1994). These results suggest that the rapid increase in serotonin transport following stimulation of As adenosine receptors is mediated by an increase in cGMP through the generation of the gaseous second-messenger nitric oxide. Interestingly, since it freely diffuses across membranes, nitric oxide could regulate presynaptic transport through either presynaptic or postsynaptic receptors. Treatment of RBL 2H3 cells with PMA, SNAP, or &bromo cGMP did not affect alanine transport through the Na'dependent ASC system, and had opposite effects on leucine uptake through the L-amino acid system, suggesting that the effects of these agents on serotonin transport are not due to changes in ion gradients. Further, the changes in V,, elicited by PMA and NECA were not accompanied by changes in the number of transporters at the cell surface, as determined by [3H]paroxetine binding. Thus, the effects of NECA and PMA on serotonin uptake most likely result from posttranslational modification of the transporter. In contrast to the enhanced uptake of serotonin following treatment with SNAP (Miller and Hoffman, 1994), Pogun and colleagues (1994) report that this N O donor decreases [3H]dopamine, ['HI serotonin, and ['Hlglutamate uptake without affecting ['HI norepinephrine uptake. These differences may be explained by the fact that in the former case, the NOproducing compounds were washed off before uptake experiments were performed, while in the latter case, these compounds were still present during uptake experiments. It has been proposed that the presence of NO+ could lead to nitrosylation of the transporters, perhaps destroying or creating disulfide bridges, which would change the secondary structure of the transporter and cause a decrease in uptake (K. J. Miller and B. J. Hoffman, unpublished observations, 1994). Thus, changes in uptake in the presence of NO-generating compounds might reflect nitrosylation of the transporter rather than an effect mediated through cGMP. Since the end result of most second-messenger systems is the activation of protein kinases, regulation of transporters likely involvesphosphorylation

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of the transporter or other membrane proteins. Interestingly, the phosphatase inhibitor okadaic acid enhances the 8-bromo-cGMP-inducedelevation of serotonin uptake in RBL 2H3 cells, suggesting that the serotonin transporter is dynamically regulated by both protein kinases and phosphatases (K. J. Miller and B. J. Hoffman, unpublished observations, 1994). Transport of the amino acid neurotransmitters GABA and glutamate is also subject to regulation by second messengers. Activation of PKC with 12-Qtetradecanoylphorbol 1%acetate (TPA) stimulates glutamate uptake in primary cultures of glia from rat brain cortex (Casado et al., 1991). In contrast, activation of PKC by Ph4A inhibits GABA transport in these glial cells (Gomeza et al., 1991). Neither glutamate nor GABA uptake is altered by PKC activation in neurons from primary cultures. The effects of TPA and PMA are mimicked by the diacylglycerol analogue oleoylacetylglycerol and the diacylglycerol kinase inhibitor R59022 and suppressed by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and H7, respectively. The effect of PMA on GABA uptake is also mimicked by exogenously added phospholipase C. In addition, the ionophore amiloride does not stimulate glutamate flux suggesting that the effect of TPA is not mediated by activation of Na+-H+ exchange. The disparate effects of PKC activation on glia and neurons may reflect either the presence of different isoforms of PKC in these cells or a smaller pool of PKC available for translocation in neurons, as has been suggested (Mudd and Raizada, 1990). By incubating a purified glutamate transporter preparation with PKC and [y-32P]ATP,Casado et al. (1993) provided direct evidence for PKCmediated phosphorylation of the glutamate transporter. Phosphoamino acid analysis indicates that phosphorylation occurs predominantly on serine residues. Treatment of C6 glioma cells with TPA elevates glutamate transport and the level of phosphorylation with similar kinetics. TPA also elevates glutamate transport in HeLa cells transiently transfected with rat GLT-1 glutamate transporter. Mutation of Serll3, the only serine residue in GLT1 that forms part of a consensus site for PKC phosphorylation, abolishes the effect of PMA on glutamate uptake in transfected HeLa cells. This study thus provides the first direct evidence that phosphorylation of a single residue on a transporter can alter its activity. In contrast to the inhibitory effect of PKC activation on GABA transport in primary glia and the lack of effect of PKC activation on primary neurons (Gomeza et al., 1991), activation of PKC increases the activity of GAT-1 expressed in Xenopus oocytes (Corey et al., 1994a). Treatment with the PKC activators PMA, 1-oleoyl-2acetyl-sn-glycerol,N-heptyl-5-chloro-1naphthalenesulfonamide, or indolactam increases GABA uptake by raising the V,,,, with no effect on the K,. The PKC inhibitor bisindolylmaleimide decreases basal- and PMA-stimulated GABA uptake. Treatment with cyclo-

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hexamide does not inhibit PMA-induced stimulation, suggesting that this effect is not dependent on protein synthesis. Further, GABA uptake is also enhanced following treatment with the protein phosphatase inhibitor okadaic acid, as well as the protein phosphatase 2B inhibitor cyclosporin A. Removal of all three intracellular consensus PKC sites by sitedirected mutagenesis does not affect the modulation of transport by either PMA or bisindolylmaleimide, suggesting that the PKC effect on transport activity may be indirect. The glucose transporter is indirectly modulated by insulin primarily by translocating the transporter from subcellular compartments to the plasma membrane (Wardzala et aZ., 1978; Blok et al., 1988;Calderhead et aZ., 1990). To determine if a similar regulatory process occurs for the GAT-1 transporter, Corey et aZ., 1994a) measured uptake in subcellular fractions of the oocyte. Untreated, all detectable GAT-1 protein is found in the trans-Golgi. However, treatment with PMA results in a translocation from the trans-Golgi to the plasma membrane. Thus, PKC-mediated regulation of GAT-1 in Xenqbus oocytes occurs predominantly through redistribution of transporters to and from intracellular storage compartments and the plasma membrane. There is also evidence that GABA transport is regulated by protein kinase A (PKA). Treatment of striatal synaptosomes with the phosphatase inhibitor okadaic acid decreases GABA uptake (Tian et al., 1994). GABA uptake is similarly inhibited by forskolin and 8-bromoadenosine-3,5-cyclic monophosphate. In contrast, none of these treatments alter dopamine uptake into striatal synaptosomes. These findings suggest that the GABA transporter in the striatum is regulated by an adenylyl cyclasedependent system. While it appears clear that phosphorylation of the glutamate transporter alters its activity, the physiological role for such regulation has yet to be defined. However, the well-accepted toxicity of excessive extracellular glutamate makes it tempting to speculate about possible mechanisms. High levels of glutamate in the synapse might activate a low-affinity glutamate receptor located either presynaptically or on glia. This in turn could stimulate the phosphatidylinositol cascade leading to an increase in PKC activity. The more active phosphorylated transporter would lead to a decrease in extracellular glutamate. In a similar manner, dephosphorylation of the transporter, when glutaminergic stimulation is desirable, would lead to a decrease in transport. In response to ischemic insult, extracellular glutamate concentrations increase in rat hippocampal slices (Lu et aZ., 1993).This increase is reduced by daurisoline, an N-type Ca*+channel blocker, and H-7, a PKC inhibitor, and is enhanced by the PKC activator phorbol-l2,13-dibutyrate.Levels of diacylglycerol and PKC are elevated in synaptosomes exposed to ischemic

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insult. The putative blocker of the non-NMDA type excitatory amino acid receptor 6,7-dinitroquinoxaline-2,3dione, but not the NMDA receptor anacid, reduced ischemia-induced tagonist ~,~-2-amino-5-phosphonovaleric release of glutamate from hippocampal slices and inhibited PKC activity in synaptosomes. Interestingly, the inhibitory effect of PKC activation on glutamate transport demonstrated in synaptosomal and slice preparations (Lu et al., 1993) is the opposite of the stimulatory effect of PKC demonstrated in primary glial cells. This may reflect a difference in the effect of PKC activation in neurons and glia. Obviously, ischemic insult involves a cascade of complex effects. While it is likely that glutamate transporters play an important role in this cascade, the mechanism and effect on transport remain to be determined. Under conditions such as cerebral ischemia, hypoxia, or hypoglycemia, nonesterified fatty acids accumulate rapidly in the brain (Gardier et aL, 1981; Broniszewska-Ardeltand Dabrowiecki, 1976). Unsaturated fatty acids inhibit the Naf-dependent transport of glutamate, aspartate, GABA, and glycine (Rhoads et al., 1982; Chan et al., 1983; Troeger et aL, 1984;Peterson and Raghupathy, 1985; Barbour et aL, 1989; Zafra et al., 1990). Transport of leucine through the Naf-independent L system is only slightly affected by unsaturated fatty acids (Zafra et aL, 1990). In addition, this effect is produced almost exclusively by cis- and not trans-unsaturated fatty acids and is reversed by bovine serum albumin, a protein that binds fatty acids. Unsaturated fatty acids may modify transport function by altering the lipid environment surrounding the transporter protein (Zafra et aL, 1990). Long-term potentiation (LTP) is triggered by the postsynaptic entry of CaZfthrough the NMDA receptor channel, while presynaptic mechanisms, such as activation of PKC, may aid in maintaining LTP (Bliss and collingridge, 1993; Madison et d., 1991; Linden and Routtenberg, 1989). Arachidonic acid and nitric oxide have been proposed as potential membrane-permeant retrograde messengers for carrying information from postsynaptic to presynaptic cells (Dumuis et al., 1988; Williams et aL, 1989; Schuman and Madison, 1991; O'Dell et aZ., 1991). Arachidonic acid had a prolonged inhibitory effect on high-affinity glutamate uptake in salamander retinal glia (Barbour et al., 1989). Further, both arachidonic acid and reactive oxygen species inhibited glutamate uptake in astrocyte cultures (Volterra et al., 1994). Thus, alterations in the concentration of glutamate in the synapse, by regulation of glutamate transport, may be involved in LTP. Cholera toxin stimulation of serotonin uptake in JAR cells is blocked by cyclohexamide, an inhibitor of protein synthesis, and actinomycin D, an inhibitor of mRNA synthesis, and is accompanied by an increase in human placental serotonin transporter mRNA and an increase in the maxi ma1 binding capacity of the cocaine analogue 2/3-~arbomethoxy-3/3-(4['~~I]-

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iodophenyl) tropane (Ramamoorthy et al., 1993b). These findings suggest that elevations in intacellular CAMP produce an increase in the density of serotonin transporters at the cell surface as the result of increased steadystate levels of transporter mRNA. Dopamine transporters may be regulated in a similar manner since treatment of a rat hypothalamic cell line with dibutyryl CAMP or forskolin enhances dopamine transport (Kadowaki et al., 1990). Studies on the regulation of transport have not been limited to direct effects of second messengers. Long-term administration (21 days) of selective and nonselective inhibitors of serotonin uptake decreases steady-state levels of serotonin transporter mRNA in the rat midbrain raphe complex due to either changes in the level of transcription or changes in mRNA stability (Lesch et al., 1993). Thus, regulation of the serotonin transporter at the level of gene expression may contribute to the therapeutic effects of antidepressants which inhibit uptake. Pifieyro and colleagues (1994) studied in vivo reuptake of serotonin by measuring the time to recovery of firing activity in hippocampal pyramidal neurons following application of serotonin. The prolonged recovery period in response to acute paroxetine is diminished in rats previously subjected to a 21-day paroxetine treatment. This decreased uptake is associated with a decrease in the B,,,, but not the & for paroxetine binding, suggesting a decrease in the number of transporters as a result of a decrease in transporter mRNA. In goldfish retina, serotonin transport is higher in animals subjected to darkness than in light-adapted controls (Lima and Schmeer, 1994).This increased uptake probably reflects a dark-associated increase in melatonin synthesis, for which serotonin is a precursor. The regulation of transport by hypertonicity is another interesting example of physiologic regulation. Accumulation of both betaine and taurine in Madin-Darby canine kidney (MDCK) cells is enhanced in cells cultured in hypertonic medium (Nakanishi et al., 1988; Uchida et al., 1991). The increased activity is associated with an increase in the level of mRNA for the betaine and taurine transporters (Yamauchi et al., 1992; Uchida et al., 1992). There is also evidence that transporters on glial cells may be regulated by monoamine receptors. In primary astroglial cultures from newborn rat cerebral cortex, stimulation of a,-adrenergic receptors increases the V,, for glutamate uptake without affecting uptake of GABA or taurine (Hansson and Ronnback, 1991). Stimulation of P-adrenergic receptors decreases glutamate uptake while stimulating uptake of GABA and taurine. In addition, stimulation of 5HT2 receptors increases taurine uptake without affecting glutamate or GABA uptake. Transport of the antiepileptic drug valproic acid ( W A ) into primary astroglial cultures is also affected by receptor

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stimulation. The a,agonist phenylephrine and the glutamate agonists glutamate, quisqualate, and kainate increase the V,, and K, for VPA transport (Nilsson et aZ., 1992). In contrast, the a2agonist clonidine decreases the V,, and K,,,for VPA transport. While the mechanism by which glial receptors influence transporters is unknown, one possibility is a second messengermediated phosphorylation of the transporter. Alternatively, receptors and transporters may interact at the membrane level, either directly or through G proteins.

V. Ionic Requirements

A. N a ' / C i - - D ~TRANSPORTERS ~ ~ ~ ~ ~ ~ ~ As their name suggests, members of this family are dependent on extracellular Na+and C1-. Studies demonstrating the ionic dependence of neurotransmitter transport have been extensively reviewed by Kanner and Schuldiner (1987) and Rudnick and Clark (1993). The energy necessary for the active transport of substrates, often against their concentration gradients, is derived from energy stored in ion gradients generated by the Na+/K'ATPase. In the proposed alternate access pore model for transporters (see below for discussion), substrates, along with Na+ and C1-, bind to the extracellular side of the transporter (Fig. 9A). This binding then causes a conformational change which exposes these binding sites to the intracellular side. The substrates are then released inside the cell. Lastly, the unoccupied transporter is recycled so that the binding sites once again face the extracellular side. In the case of serotonin transport, this last, and probably rate-limiting step is facilitated by the binding and transport of K' or H+ from the intracellular to the extracellular side. The apparent stoichiometry varies between members of this transporter family. For norepinephrine, the stoichiometry appears to be 1 Na+: 1 C1- : 1 NE' (Ramamoorthy et al., 1992;Friedrich and Bonisch, 1986). Ratios of 2 Na' : 1 C1- : 1 neurotransmitter are proposed for dopamine (McElvain and Schenk, 1992), GABA (Radian and Kanner, 1983;Keynan and Kanner, 1988; Mager et al., 1993; Clark and Amara, 1994), and glycine (Zafra and GimCnez, 1989). For serotonin, the apparent stoichiometry is 1 Na+ : 1 C1- : 1 K' : 1 5-HT' (for review, see Rudnick and Clark, 1993; Mager et al., 1994). Many questions about the transport process, including the roles of ions, turnover rates, voltage dependence, and the nature of individual steps, can be addressed by electrophysiological analysis. Within the Na'/

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FIG.9. Proposed stoichiometry for transport of substrates and ions by (A) the Na+/Cl-dependent transporters, (B) the glutamate transporters, and (C) the vesicular transporters.

C1--dependent family, such studies have focused largely on GABA and serotonin (for review, see Lester et al., 1994). At physiological pH, GABA exists primarily in the zwitterionic form. Given the proposed stoichiometry, this predicts that the transport of GABA is electrogenic. Voltage-clamp studies on horizontal cells of skate retina (Malchow and Ripps, 1990), crayfish stretch receptor neurons (Kaila et al., 1992), and Xenopus oocytes expressing the GAT-1 GABA transporter (Kavanaugh et al., 1992; Mager et al., 1993) support the electrogenicity of GABA transport. The fact that GABA transport is electrogenic and voltage dependent makes this transporter a good candidate for electrophysiological studies. Several interesting findings have come from two recent studies examining the electrophysiological properties of the GAT-1 GABA transporter expressed in Xenopus oocytes (Kavanaugh et al., 1992; Mager et al., 1993). Among the findings are that GABA transport increases with hyperpolariza-

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tion and with increasing extracellular concentrations of GABA, Na', or C1-. At low extracellular concentrations of GABA, GABA becomes rate limiting. At these concentrations, GAT-1 is less sensitive to voltage, suggesting that the interaction of GABA and the transporter is voltage independent. On the other hand, when the extracellular concentration of GABA is high, Na' becomes rate limiting in a voltagedependent manner. The mechanism of C1- binding is less well understood, but there is evidence that C1- binding increases the affinity of Na+ for the transporter. The GABA uptake inhibitor nipecotic acid, which is a substrate for the transporter, causes a dose-dependent inward current, with a maximal current approximately 86% of that for GABA (Kavanaugh et al., 1992). The GABA uptake inhibitor SKF89976A blocks GABA-induced currents and charge movements (Mager et d., 1993). Since the structurally related inhibitor NO-328 binds only in the presence of Na' (Braestrup et al., 1990), it has been proposed that these inhibitors work by stabilizing a state in which Nat is bound to the outside of the transporter, as has been suggested for the effects of phlorizin on the glucose transporter (Parent et al., 1992a,b).The time to complete a single transport cycle was shown to be 160 ms at -80 mV, 96 mM Na', and 22°C. Extrapolation to physiological conditions, 140 mM Na' at 37"C, predicts a cycle time of approximately 10 ms, which is near the characteristic decay time of GABAAsynaptic currents (Mager et al., 1993). In addition to transport-associated ion flux, a transient current was measured which likely reflects a capacitative charge movement across the membrane within the transporter protein. By measuring ['Hlserotonin uptake and [Iz51] RTI-55 binding in Xenopus oocytes expressing the serotonin transporter, Mager et al. (1994) demonstrated that serotonin transport occurs with a turnover rate of approximately 0.5 molecules of serotonin per second per molecule of transporter. Howbinding reflects both membrane-bound and ever, because the [ 1251)RTI-55 intracellular serotonin transporters, these authors suggest that a more reasonable estimate is l per second. This turnover rate is in agreement with previous estimates in native tissues (Talvenheimo et al., 1979; Rudnick and Nelson, 1978). Given the positive charge of serotonin, the stoichiometry predicts that the transport of serotonin is electroneutral. This is supported by several studies demonstrating that serotonin transport is not affected by changes in membrane potential (for review, see Rudnick and Clark, 1993),while one study demonstrates that serotonin transport is dependent on membrane potential (Kanner and Bendahan, 1985). A recent detailed analysis of the electrophysiological characteristics of the rat serotonin transporter expressed in XenOpus oocytes reveals that serotonin transport is not electroneutral and actually involves several conducting states (Mager et aL, 1994).

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The first is an inward, steady-state current associated with serotonin transport. This voltagedependent current is induced by external serotonin, dependent on external Nat, and inhibited by serotonin uptake inhibitors. The second conducting state is a transient inward current produced in response to voltage jumps to negative potential. This current occurs only in the absence of serotonin and is suppressed by serotonin uptake inhibitors. While this current can be carried by Nat or Lit, Kt cannot substitute. This transient current is proposed to represent a voltagedependent opening of the transporter in the absence of serotonin. Lastly, a small steadystate leakage current is revealed in the absence of serotonin. This current is suppressed by uptake inhibitors and can be carried by Na’, Li+, or K+. The presence of three conductance states for serotonin transport has led these authors to propose that the serotonin transporter may share some permeation properties with ion channels. The currently accepted alternative access pore model for transport pedicts that the transporter has a central aqueous pore with binding sites for ions and substrates. “Gates” on the extracellular and intracellular surfaces of the transporter alternatively open to expose either one or the other side to these binding sites. However, in order for currents to be associated with transport both gates must be open simultaneously. Thus, it has been proposed that the gates open simultaneously during the transport cycle and during extreme negative potential. In addition, the leakage current may reflect the gates being slightly open at rest. These findings suggest that ion-coupled transporters, in which the flux of one ion is tightly coupled to the flux of the substrate or other ions, and ion channels, in which the flux of each ion is relatively self-determined, may share similar structural and functional properties. However, it remains to be determined if the conductance states demonstrated in oocytes occur in native tissue.

B. GLUTAMATE TRANSPORTERS The ion coupling system for members of the glutamate transporter family is quite distinct from that of the Nat/C1--dependent family (Fig. 9B). High-finity glutamate transport in retinal glial cells is coupled to the cotransport of two Na+ ions and the countertransport of one Kt ion and one OH- ion (Kanner and Schuldiner, 1987; Nicholls and Attwell, 1990; Bouvier et al., 1992). Arriza and colleagues (1994) demonstrated that the transport of glutamate is electrogenic. The exact stoichiometry for the three recently cloned glutamate transporters has not yet been determined.

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C . VESICULAR TRANSPORTERS In contrast to the plasma membrane transporters, which rely on the Na+ gradient generated by the Na+/K+-ATPaseto drive the cotransport of ions and substrates, vesicular amine transport depends on the electrochemical gradient generated by the vacuolar Hf-ATPase (for review, see Kanner and Schuldiner, 1987). The transport of a cytoplasmic amine into the vesicle lumen is coupled with the transport of a proton out of the vesicle (Fig. 9C). Dependence on both ATP and the transmembrane electrochemical proton gradient has been demonstrated in chromaffin granules (Kanner and Schuldiner, 1987) and permeabilized CV-1 cells expressing the VMAT2 vesicular transporter (Erickson et aL, 1992).

VI. Tmnsmimr Efflux

In addition to shuttling neurotransmitters from the extracellular space into nerve terminals and glia, there is growing evidence that transporters also function to release neurotransmitters. In contrast to the classical release of neurotransmitters by fusion of presynaptic vesicles with the plasma membrane, release through transporters is not dependent on Ca". While there is evidence that release of neurotransmitters from transporters may occur in both physiological and pathological states, questions remain concerning the functional significance of this release (for review, see Bernath, 1992; Attwell et aL, 1993). There is strong evidence that GABA is released by reverse transport. Depolarization-induced release of GABA from retinal horizontal cells, neurons, and type-2 astrocytes is dependent on extracellular Na+,independent of extracellular Ca2+,and is inhibited by the GABA uptake blocker nipecotic acid (Schwartz, 1982; Yazulla and Kleinschmidt, 1983; Ayoub and Lam, 1984; Pin and Bockaert, 1989; Gallo et aZ., 1991). GABA is released more effectively by application of glutamate, which raises the intracellular concentration of Na+, than by Kt-induced depolarization. In addition, voltageclamp studies demonstrate that depolarization of a retinal horizontal cell in Ca*+-freemedium evokes release of GABA from the apposed bipolar cell (Schwartz, 1987).Taken together, these findings have led to the suggestion that depolarization of a retinal horizontal cell dendrite by glutamatergic input from photoreceptors induces GABA release both by making the membrane potential more positive and by increasing the intracellular concentration of Na+ (Attwell et aL, 1993). Recent work by Dong and colleagues (1994) demonstrates the coexistence of GABA receptors and GABA trans-

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porters on catfish cone horizontal cells. Thus, eMux of GABA through the transporter could form a feedback loop to GABA receptors on the same or neighboring horizontal cells. In astrocytes, the rise in intracellular Na' through non-NMDA glutamate channels is predicted to be a stronger stimulus for GABA release than is the associated depolarization (Attwell et aL, 1993).The release of GABA following glutamatergic stimulation may serve a protective function by quieting the activity of glutamate neurons. Both Ca2+-dependentand Ca2+-independentrelease of glutamate have been demonstrated following K+-induced depolarization (for review, see Nicholls and Attwell, 1990;Bernath, 1992).Release of glutamate by reverse transport may be particularly important during ischemia and anoxia for several reasons. First, vesicular release of glutamate is inhibited by anoxia (Gribkoff and Bauman, 1992;Sanchez-Prieto and Gonzalez, 1988).In addition, after a few minutes of anoxia or ischemia, extracellular K+rises,depolarizing the cells, while extracellular Na+ decreases (Siesjb, 1990).These ionic and voltage gradients produced during ischemia or anoxia promote the release of glutamate through the transporter. Efflux of glycine has been demonstrated in synaptic plasma membrane vesicles from both rat brain (Arag6n and GimCnez, 1986) and C6 glioma cells (Zafra and GimCnez, 1988). In both cases, efflux is dependent on Na+ and C1-, suggesting that it may occur through the plasma membrane transporter. In addition, non-NMDA glutamate agonists stimulate the release of glycine from astrocytes (Levi and Patrizio, 1992).Because glycine is a coagonist at the NMDA receptor channel complex, it is tempting to speculate that glycine eMux from glia may help regulate the excitability of glutamate neurons. Transporter-mediated eMux has also been demonstrated for the monoamine neurotransmitters. Amphetamine treatment releases monoamines by two mechanisms. First, the inward transport of amphetamine through the monoamine plasma membrane transporters is coupled to the outward transport of monoamines (Azzaro et aL,1974;Fischer and Cho, 1979).As a weak base, amphetamine also dissipates the pH gradient across vesicles, resulting in an increase in cytoplasmic monoamines and promoting reversed transport (Sulzer and Rayport, 1990;Sulzer et d., 1993). 3,4Methylenedioxymethamphetamine (MDMA), Fhloroamphetamine (PCA), and fenfluramine are amphetamine derivatives that preferentially release serotonin. As with amphetamine, eMux of serotonin is due to both an exchange at the plasma membrane transporter and by an effect on vesicles (Fuller et aL, 1975;Johnson et aL, 1986).Interestingly, MDMA and fenfluramine inhibit vesicular uptake of serotonin by dissipating the pH gradient and by directly blocking vesicular transport, while PCA only dissipates the pH gradient (Schuldiner et aL, 1993).

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By transfecting a human dopamine transporter cDNA into COS cells, Eshleman and colleagues (1994) studied transporter-mediated dopamine release in a cell lacking vesicular storage and release mechanisms. Kt-induced depolarization or low extracellular Nat stimulates CaZt-independent release of dopamine in transfected cells. This release is inhibited by several dopamine uptake blockers, such as mazindol, GBRl2909, bupropion, nomifensine, and benztropine. Other drugs, such as methamphetamine, amphetamine, and ethanol, enhance release. Cocaine, phencyclidine, and WIN35428 have no effect on release. Since all drugs with abuse potential either enhance or have no effect on release, it may be possible to identify structural features of the transporter that interact with abused and nonabused drugs. Norepinephrine has also been demonstrated to be released by reverse transport. Inhibition of the Nat pump by oubain results in an increase in intracellular Na+ and depolarization and is accompanied by a desipraminesensitive efflux of norepinephrine (Trendelenburg, 1991). Outward transport of norepinephrine may also be important in certain pathological states. Glucose deprivation leads to an efflux of norepinephrine, presumably due to lack of ATP which is required for both the Nat pump and vesicular transport (Russ et al., 1991). The lack of ATP would result in efflux of monoamines from vesicles. With an impaired Na' gradient, and an increase in cytoplasmic neurotransmitter, the cells are primed for reverse transport. Hypoxia also leads to an efflux of norepinephrine (Trendelenburg, 1991). Interestingly, when hypoxia is coupled with glucose deprivation, a greatly potentiated norepinephrine efflux is observed (Russ et al., 1991; Schomig et al., 1987). VII. Transplter Localization

While recent molecular advances have greatly expanded our knowledge about transporters, there are still many aspects of transporters which have yet to be fully explored. One such topic concerns the localization of transporters. In addition to determining which regions of the brain and which peripheral tissues express each transporter, it will also be important to determine which types of cells express each transporter and where on these cells the transporters are found. A. DISTRIBUTION For some transporters, such as serotonin and dopamine, the distribution of transporter mRNA closely parallels the known distribution of cell bodies

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for the given neurotransmitter (Hoffman et al., 1991; Usdin et al., 1991; Lorang et al., 1994). For other transporters, mapping the anatomic distribution by in situ hybridization histochemistry has produced some unexpected results, suggesting novel roles for certain neurotransmitters. For example, the study of glycine neurotransmission has been limited by the lack of good markers for glycine cells as well as the lack of selective pharmacologic tools. In situ hybridization histochemistry along with immunohistochemistry surprisingly revealed that GLYT-la mRNA is localized in macrophages in a number of peripheral tissues, including thymus, spleen, and lung (Borowsky et al., 1993). While a specific function for glycine in macrophages has not yet been defined, the presence of a high-affinity transporter for glycine in these cells suggests that glycine may be important in cellular communication or that high extracellular levels of glycine may be toxic to neighboring cells. Within the CNS, glycine is considered primarily as an inhibitory neurotransmitter in the brain stem and spinal cord. The widespread distribution of GLYT-la and GLW-lb mRNA, demonstrated by in situ hybridization, supports the emerging status of glycine as a supraspinal neurotransmitter (Borowsky et al., 1993).

B. GLIALvs NEURONAL Another question which has not been completely addressed is whether the cloned transporters are localized in neurons or glia. Uptake into glia is thought to be particularly important for the amino acid neurotransmitters GABA, glycine, and glutamate. In addition, serotonin and catecholamine uptake have been demonstrated in primary astrocytes (Kimelberg, 1986). Since multiple transporter subtypes have been identified for each of these neurotransmitters, it will be necessary to determine if a given subtype is restricted to either glia or neurons, or whether a subtype can be localized in both cell types. For example, while GABA uptake occurs in both neurons and glia, it is not clear which of the cloned GABA transporters is localized in glia. Using an antibody to the purified GAT-1 transporter, Radian and colleagues (1990) demonstrated that GAT-1 is localized in both neurons and glia. In situ hybridization histochemistry revealed that GAT-1 mRNA is localized primarily in neurons, but is found in Bergmann glia in the cerebellum (Rattray and Priestley, 1993). While the pharmacologic profile of GAT-3 is similar to that reported for GABA transport in glia, in situ hybridization revealed that GAT-3 mRNA is localized primarily in neurons (Clark et al., 1992).The cellular distribution of GAT-2 has yet to be reported. Further localization studies will be necessary to determine which, if any, of the cloned GABA transporters are localized predominantly in glia. mRNA

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for the GLYT-lb glycine transporter is found only in white matter, suggesting a glial localization (Borowsky et al., 1993). GLYT-la mRNA is found only in gray matter, but is also present in C6 glioma cells, suggesting that it may be localized in both neurons and glia (Borowskyet al., 1993;Guastella et al., 1992). Using both PCR and in situ hybridization, both of these glycine transporters have been identified in rat bipotential oligodendrocyte type-2 astroctyte progenitor cells, as well as the differentiated oligodendrocytes and type-2 astrocytes derived from these cells (Borowsky and Hoffman, 1994). The cellular distribution of GLYT-2 has not yet been reported. Of the three cloned glutamate transporters, immunohistochemical analysis demonstrates that at least one, GLT-1, is localized only in astrocytes (Danbolt et al., 1992). In situ hybridization suggests that GLAST mRNA may be present in Bergmann glia in the cerebellum, while the cellular localization of GLAST mRNA in other parts of the brain has not yet been determined (Storck et al., 1992).Lastly,while the distribution of EEACl mRNA is similar to that of glutamate cell bodies, double-labeling studies will be necessary to demonstrate a neuronal localization (Kanai and Hediger, 1992).

C. SUBCELLULAR LOCALIZATION In addition to determining which types of cells express each transporter, it is also important to determine the subcellular distribution of these membrane proteins. This type of information could help to better define the boundaries of the synapse and the potential range of a released neurotrans mitter. Based on electrochemical evidence, Garris and colleagues (1994) suggested that dopamine transporters may be localized outside the synaptic density. Immunohistochemical localization of GAT-1 shows this transporter to be present in axons but not cell bodies of GABAergic cells (Radian et al., 1990). Electron microscopy studies show GAT-1 immunoreactivity is also present in glial processes but not in glial cell bodies (Radian et al., 1990).In fully polarized hippocampal neurons, GAT-1 is restricted to z o n a l surfaces (Pietrini et al., 1994). Conversely, the betaine transporter (BGT1) is targeted to basolateral surfaces under hyperosmotic conditions in the polarized epithelial cell line, MDCK (Yamauchi et al., 1991). Interestingly, after transfection into MDCK cells, GAT-1 is localized primarily to the apical surface, while BGT-1 remains localized to the basolateral surface (Pietrini et al., 1994).It remains to be determined what factors regulate the differential sorting of these transporters and whether different subtypes are targeted to axons, dendrites, or cell bodies. Future immunohistochemical studies will be necessary to determine the ultrastructural localization of other transporters.

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VIII. Transporter Heterogeneity

To date, a single transporter has been identified for each of the monoamine neurotransmitters. On the other hand, multiple subtypes of transporters have been identified for the amino acid neurotransmitters GABA, glycine, and glutamate. In some instances there appear to be functional differences among the subtypes, while in other cases such distinctions are less clear. For glutamate transporters, differences in distribution and affinity for glutamate have prompted speculation about distinct functions for the three subtypes (for review, see Kanai et aL, 1993). The localization of GLT1 in astrocytes throughout the brain (Danbolt et aL, 1992) and the very high affinity for glutamate (& = 2 p M ; Pines et aL, 1992) make it a good candidate for maintaining the low, 1 p M , extracellular concentrations of glutamate. GLAST has a lower affinity for glutamate (X, = 11 or 77 p M ) and is distributed throughout the cerebrum as well as in Bergmann glia in the cerebellum (Storck et al., 1992; Kanai et al., 1993). Thus, GLAST may act as a reserve transporter for keeping glutamate from excitotoxic levels (Kanai et aL, 1993). Lastly, the parallel distributions of EEACl and glutamate neurons, along with the similarity in the affinities of EEACl transporters (X, = 12 ph4; Kanai and Hediger, 1992) and AMPA receptors (EC,, = 19 pM; Patneau and Mayer, 1990), for glutamate make EAACl an ideal candidate for a synaptic transporter. Two of the glycine transporters, differing only at the amino termini, are alternatively spliced or alternatively promoted from a single gene in a tissue-specific manner (Borowsky et aL, 1993). GLYT-lb mRNA is found in all fiber tracts in the brain, while the distribution of GLYT-la mRNA closely parallels the distribution of mRNA for both strychnine-sensitiveand strychnine-insensitive glycine receptors in the brain. Further, GLW-la is found in a number of peripheral tissues, while GLYT-lb is restricted to the CNS. Despite their distinct distributions, the two transporters show similar affinities for glycine and similar pharmacologic profiles. Evidence that GLkT-la and GLYT-lb may be regulated by distinct promoters suggests that the relative amounts of each transporter may vary during development and under various conditions (Borowskyand Hoffman, 1993).It is proposed that GLYT-lb, being present in white matter, would maintain a consistent presence to keep extracellular glycine low, while the abundance of GLYTl a would vary with the amount of glycinergic activity at both the inhibitory glycine receptor and the glycine binding site on the NMDA receptor. The third glycine transporter, GLW-2, is restricted to the brain stem and spinal cord, has a somewhat higher affinity for glycine, and, unlike GLYT-la and GLW-lb, is not inhibited by sarcosine (Liu et aZ., 1993b). Thus, this

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transporter is likely associated only with the activity of glycine at the inhibitory glycine receptor and not at the NMDA receptor complex. The functional significance for the multiplicity of GABA transporters is less clear than that for glutamate and glycine. In terms of distribution, GAT-1 and GAT-3 are restricted to the brain, while GAT-2 is also found in peripheral tissues (Borden et al., 1992). While there are differences in the distribution of GAT-1 and GAT-3, there are overlaps as well (Clark et al., 1992). GAT-2 and GAT-3 are more sensitive than GAT-1 to the glial-specific drug, P-alanine, but less sensitive to other presumed glial-specific agents such as guvacine, nipecotic acid, and hydroxynipecotic acid (Borden et al., 1992). Further, the potency of L-DABAis higher at GAT-1 and GAT-2 than at GAT-3 (Borden et al., 1992). Thus, pharmacologic distinctions between glial and neuronal GABA transporters may be more complicated than previously thought, and the cloned transporters cannot readily be categorized into glial or neuronal divisions. One additional distinction among the GABA transporters that may account for some functional differences is their differential dependence on extracellular C1-. There is increasing dependence on C1- for GAT-2, GAT-3, and GAT-1, respectively (Borden et al., 1992). Future studies on the transporters for GABA, glycine, and glutamate should help to clarify the necessity for multiple subtypes. The vesicular monoamine transporters, VMATl and VMAT2, are highly conserved and pharmacologically very similar. Among the potential reasons for having two transporters are differences in affinity for substrates, as shown for histamine (Peter et aL,1994), and differential expression in distinct cell types, perhaps being associated with specific monoamine neurons. In support of this, VMAT2, but not VMATl, has been identified in histaminergic neurons (Gonzalez et al., 1994;B.J. Hoffman, unpublished observations, 1994). Lastly, different VMATs may be targeted to different populations of vesicles or granules (for example, see Liu et al., 1994).

IX. Future Directions

In the near future, it is likely that more transporters will be cloned and identified. For example, plasma membrane transporters for choline and adenosine and vesicular transporters for GABA/glycine, glutamate, and ATP. In addition, identification of the substrates for the orphan transporters as well as other potential transporters may define novel neurotransmitter systems. Future studies on the function of the three families of transporters should identify the transmembrane domains involved in forming the pore and determine what roles the other transmembrane domains may play.

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The molecular cloning of transport proteins should greatly aid in the identification of the causes of various disease states as well as the design of novel therapeutics. Disease states which could potentially be treated by drugs interacting with transport proteins include schizophrenia, Parkinson’s disease, Huntington’s chorea, Alzheimer’s disease, amyotrophic lateral sclerosis,and epilepsy. In addition, understanding how antidepressants interact with the monoamine transporters, during both acute and chronic treatment, could lead to improved treatment regimens for this disease. Similarly, a better understanding of how drugs of abuse, such as cocaine and amphetamine, interact with the dopamine transporter could lead to the development of drugs which block the effects of these agents without themselves blocking uptake. Giros and colleagues (1994) addressed this issue by constructing a series of chimeric proteins containing portions of the dopamine (DAT) and norepinephrine (NET) transporters. Exchanging either the amino termini or the regions between the first intracellular loop and the fifth transmembrane domain of these transporters had no effect on either substrate or inhibitor affinities. In contrast, replacing the last four transmembrane domains and the carboxy terminus of the DAT with that of the NET increased the affinity of the DAT for DA and NE toward values expected for the NET. This chimera also loses stereoselectivity for the two isomers of amphetamine, suggesting that the carboxy terminal confers the high substrate affinity for the NET, but does not dictate specificity for uptake blockers. Replacing transmembrane domains 5-8 of the NET with those of the DAT produced a 1000-fold increase in K, for tricyclic antidepressants without affecting substrate specificity. Thus, the affinity of the NET for desipramine and nortriptyline shifted from nanomolar (NET-like) to micromolar (DATlike). Interestingly, putting transmembrane domain 5-8 of the NET into the DAT resulted in a loss of affinity for cocaine and nomifensine without affecting the affinity for substrates such as amphetamine. These findings suggest that it may be possible to design drugs which could block the effects of cocaine without blocking uptake. In a similar study, Buck and Amara (1994) used chimeric dopaminenorepinephrine transporters to analyze the structural requirements for catecholamine and MPP+ selectivity. Transmembrane domains 2 and 3 of the dopamine transporter were found to be important in selectively increasing the rate of dopamine uptake compared to that of norepinephrine. Further, these studies suggested that transmembrane domains 4-8 of the dopamine transporter might affect the relative rate with which MPP’ is accumulated. Following the observation that tricyclic antidepressants were more potent at the human serotonin transporter, while d-amphetamine was more potent at the rat serotonin transporter, Barker and colleagues (1994) ana-

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lyzed cross-species chimeras of the serotonin transporter. These investigators isolated a region distal to amino acid 532 that imparts species preferences for these drugs and suggest that a region near transmembrane domain 12 may be involved in both substrate and antagonist recognition. Determining the chromosomal localizations of the cloned neurotransmitter transporters is an important first step toward discovering possible links between transporter dysfunction and neurological diseases. For several transporters, this has already been done. The human dopamine transporter has been assigned to the distal part of chromosome 5 at 5p15.3 (Giros et aL, 1992). Although not confirmed in different sets of families, a possible linkage of chromosome 5 in familial schizophrenia has been reported (Sherrington et al., 1988; Kennedy et aL, 1988).The GLYT-1 gene has been localized to the human chromosome 1, at 1~31-32,and to the mouse chromosome 4 (Kim et al., 1994). Interestingly, the mouse GLYT-1 gene is near the locus for the mouse neurological disorder clasper. The human serotonin transporter has been localized to chromosome 17 at 17qll.l-17q12 (Ramamoorthy et aL, 1993a). In addition, the human neutral amino acid transporter has been assigned to chromosome 2 (Shafqat et aZ., 1993). The atypical orphan transporter, XTl, is localized on the mouse chromosome 3 near the locus for the mouse neurologic mutation spastic (El Mestikawy et al., 1994). Lastly, the human vesicular monoamine transporters, VMATl and VMAT2, have been assigned to chromosomes 8p21.3 and 10q25, respectively (Peter et aL, 1993). Chromosomal mapping of the remainder of transporters may reveal potential links between transporter dysfunction and disease.

Acknowledgments

We thank Dr. Keith Miller, Ms. Lydia Gould, and Dr. Rusty Johnson for critically reviewing the manuscript.

References

Ahtee, I.., Pentikainen, L., Pentikainen, P. J., and Paasonen, M. K. (1974). Experientia 30, 1328-1329. Ahtee, I>.,Briley, M., Raisman, R., LeBrec, D., and Langer, S. Z. (1981). LijeSci. 29,2323-2329. Alfonso, A,, Grundahl, K, Duerr, J. S., Han, H.-P.,and Rand,J. B. (1993). Science261,617-619. Alfonso, A., Grundahl, K., McManus, J. R., Asbury, J. M., and Rand, J. B. (1994).J. Mol. Bid. 241, 627-630.

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Shafqat, S., Tamarappoo, B. K., Kilberf, M. S., Puranam, R. S., McNamara, J. O., GaudanoFerraz, A., and Fremeau, R. T., Jr. (1993).J. Biol. Chem. 268, 15351-15355. Shashidharan, P., and Plaitakis, A. (1993).Biochim. Biophys. Acta 1216, 161-164. Shashidharan, P., Wittenberg, I., and Plaitakis, A. (1994).Biochim. Biophys. Acta 1191,393-396. Sherrington, R., Brynjolfsson, J., Petursson, H., Potter, M., Dudleston, K., Barraclough, B., Wasmuth, J., Dobbs, M., and Gurling, H. (1988).Nature (London) 336, 164-167. Shimada, S., Kitayama, S., Lin, C.-L., Patel, A,, Nanthakumar, E., Gregor, P., Kuhar, M., and Uhl, G. (1991).Science 254, 567-578. Siesj6, B. K. (1990).News Physiol. Sci. 5, 120-125. Smith, K. E., Borden, L. A., Hartig, P. R., Branchek, T., and Weinshank, R. L. (1992a).Neuron 8,927-935. Smith, K. E., Borden, L. A,, Wang, C.-H. D., Hartig, P. R., Branchek, T. A., and Weinshank, R. I. (199213).Mol. Pharmacol. 42, 563-569. Storck, T., Schulte, S., Hofmann, K., and Stoffel, W. (1992).Proc. Natl. Acad. Sci. U.S.A. 89, 10955-10959. Strader, C. D., Sigal, I. S., Register, R. B., Candelore, M. R., Rands, R., and Dixon, R. A. F. (1987).Proc. Natl. Acac. Sci. U.S.A. 84, 4384-4388. Strader, C. D., Sigal, I. S., and Dixon, A. F. R. (1989).Am.]. Respir. Cell Mol. Biol. 1, 81-86. Stromblad, B. C. R., and Nickerson, M. (1961).J.Phannacol. Exp. Ther. 134, 154-159. Sulzer, D., and Rayport, S. (1990).Neuron 5, 797-808. Sulzer, D., Maidment, N. T., and Rayport, S. (1993).J.Neurochem. 60,527-535. Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991).Science 253, 872-879. Surratt, C. K., Persico, A. M., Yang, Z., Edgar, S. R., Bird, G. S., Hawkins, A. L., Griffin, C. A,, Li, X., Jabs, E. W., and Uhl, G. R. (1993).Proc. Natl. Acac. Sci. U S A . 89,9730-9733. Takagi, T.(1991).Adv. Ekctrophm. 4, 391-406. Talvenheimo, J., Nelson, P. J., and Rudnick, G. (1979).J. Biol. Chem. 254, 4631-4635. Tate, C. G., and Blakely, R. D. (1994).J. Biol. Chem. 269, 26303-26310. Thoenen, H., and Tranzer, J. P. (1968).Naunyn-Schmiedebergs Arch. Phannakol. Exp. Pathol. 261, 271-288. Tian,Y., Kapatos, G., Granneman,J. G., andBannon, M.J. (1994).Neurosci. Lett. 173,143-146. Tolner, B., Poolman, B., Wallace, B., and Konings, W. N. (1992).J.Bactaiol. 174, 2391-2393. Trendelenburg, U. (1991).Trends Pharmacol. Sci. 12, 334-337. Troeger, M. B., Rajalowska, U., and Erecinska, M. (1984).J.Neurocha. 42, 1735-1742. Tuomisto, J., Tukiainen, E., and Ahlfors, U. G. (1979).Psychopharmacology (Berlin) 65,141-147. Uchida, S., Nakanishi, T., Kwon, H. M., Preston, A. S., and Handler, J. S. (1991).J. Clin. Invest. 88, 656-662. Uchida, S., Kwon, H. M., Yamauchi, A,, Preston, A. S., Marumo, F., and Handler, J. S. (1992). Proc. Natl. Acac. Sci. U.S.A. 89, 8230-8234. Uhl, G. R., Kitayama, S., Gregor, P., Nanthakumar, E., Persico, A,, and Shimada, S. (1992). Mol. Brain. Res. 16, 353-359. Uretsky, N.J., and Iversen, L. L. (1970).J. Neurochem. 17, 269-278. Usdin, T. B., Mezey, E., Chen, C., Brownstein, M. J., and Hoffman, B. J. (1991).Proc. Natl. Acad. Sci. U.S.A. 88, 11168-11171. Varoqui, H., Diebler, M.-F., Meunier, F.-B., Rand, J. B., Usdin, T. B., Bonner, T. I., Eiden, L. E., and Erickson, J. D. (1994).FEES Lett. 342, 97-102. Volterra, A,, Trotti, D., and Racagni, G. (1994).Mol. Pharmacol. 46, 986-992. Wardzala, L. J., Cushman, S. W., and Salans, L. B. (1978).J. Biol. Chem. 253, 8002-8005. Whitby, L. G., Axelrod, J., and Weil-Malherbe, H. (lUSl).J.Pharmacol. Exp. T k . 132, 193-201.

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Meyer B. Jackson Department of Physiology and Center for Neuroscience, University of Wisconsin, Madison, Wisconsin 53706-15 3 2

I. Introduction 11. Motor Nerve Terminals

A. K' Channels B. Cap' Channels C. Presynaptic Transmitters D. Channel Locations 111. Squid Synapse A. Ca2+Channels B. K+ Channels C. Channel Locations D. Presynaptic Transmitters IV. Posterior Pituitary A. Ca2' Channels B. K' Channels C. Na+ Channels D. Neurotransmitters V. Ciliary Ganglion A. Cap+Channels B. Other Channels C. Neurotransmitters VI. Goldfish Retina Bipolar Cells A. CaZ+Channels B. Neurotransmitters VlI. Honorable Mention MII. Summary and Conclusions References

I. Introduction

An action potential in a cell body is often regarded as a decisive event that guarantees the transmission of electrically encoded information to the next battery of neurons in a synaptic relay. However, when an action potential reaches the nerve terminal, the amount of neurotransmitter secretion depends on much more than a simple condition of excitation in a binary sense. The all-or-noneproperty of the presynaptic action potential does not INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 38

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carryover to simple all-or-none behavior for secretion of neurotransmitter. Information undergoes a great deal of processing within nerve terminals, as complex patterns of activity and various presynaptic neurotransmitters work together to specify the precise secretory output evoked by a presynaptic action potential. The ion channels of the nerve terminal membrane play a major role in this form of information processing. The most obvious role for ion channels in nerve terminals is to permit the entry of calcium to trigger secretion. Many additional functions for presynaptic ion channels can easily be envisaged. Ion channels determine the extent of impulse propagation within a nerve terminal and whether excitation reaches crucial secretory regions. Furthermore, the shape of an action potential, and thus its efficacy in inducing calcium entry, ultimately depends on the properties of many types of channels. As a result, a group of specialized proteins in the nerve terminal membrane provide foci for the regulation of secretion. Much has been learned in recent years about how ion channels in nerve terminals contribute to the regulation of neurosecretion. This chapter reviews work on the excitability of nerve terminals and discusses how these membrane properties contribute to function. Despite the obvious importance of the nerve terminal membrane to synaptic function, progress in understanding presynaptic excitability has until recently been very slow. The key problem is the small size of most nerve terminals. In contrast to nerve cell bodies, most of which are sufficiently large for direct recording, the small size of most nerve terminals severely limits electrophysiological examination. Investigation of ion channels in nerve terminals has depended on the development of suitable preparations and techniques for electrical study. Tissues with large accessible nerve terminals have been hard to find. But the importance of presynap tic mechanisms has drawn a number of investigators to this technical challenge, resulting in the development of various preparations especially for electrophysiological study of nerve terminals. This chapter reviews work in which direct electrophysiological measurements have been made from nerve terminals. Presynaptic membrane biophysics has become a highly specialized field with progress depending on the advantages and disadvantages of a small number of unique preparations. Three of the preparations to be discussed here, motor nerve terminals, the squid giant synapse, and the posterior pituitary, hold a special place in the study of presynaptic mechanisms. Pioneering studies in all three of these preparations were instrumental in the ascension of the calcium hypothesis for transmitter release. In addition, two other preparations for the investigation of nerve terminal membranes, ciliary ganglion and retinal bipolar neurons, have emerged more recently to provide important additional insights.

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The present discussion is limited to studies in which direct electrical recordings were made from nerve terminals. Because of the difficulty of recording directly, investigators have resorted to various model systems and indirect strategies in the study of presynaptic excitability. Measurements of postsynaptic responses provide an index of presynaptic function which reflects the nature of presynaptic ion channels. By extrapolating from the properties of already characterized ion channels in nerve cell bodies, inferences have been drawn about the properties of ion channels in nerve terminals and how these channels contribute to the regulation of neurosecretion (Edmonds et al., 1990;Finch et al., 1990;Perney et al., 1986; Thompson zt al., 1993;Wheeler et al., 1994). Secretory cells from endocrine tissues, such as the adrenal and pituitary glands, have also received considerable attention in the study of secretion. A number of nerve cell bodies and sensory cells secrete neurotransmitter, and the relation between channels and secretion has been studied extensively in these systems (Haydon, 1988). Membrane and tissue homogenates from nervous systems have been enriched in nerve terminal membrane and examined by avariety of techniques in an effort to understand presynaptic ion channels (Meir et al., 1993; Nachshen and Blaustein, 1982; Umbach et al., 1984). A discussion of work in these systems in addition to the direct recordings from nerve terminals would extend this review beyond a manageable scope. Therefore, no attempt will be made to review the literature on model systems for presynaptic membranes.

II. Motor Nerve Terminals

At neuromuscular junctions on skeletal muscle, electrodes positioned on the nerve terminals can pick up small extracellular voltage signals arising from the presynaptic action potential. At the rat neuromuscular junction, measurements of these signals were used to infer that the nerve terminal membrane can generate action potentials, and that the spread of voltage changes from the axon to the nerve terminal was active rather than passive (Hubbard and Schmidt, 1963). Examination of this issue at the frog neuromuscular junction revealed a number of different extracellular waveforms detected at different positions along the nerve terminal (Katz and Miledi, 1965).A thoughtful examination of these waveforms in terms of appropriate local circuits and cable properties indicated that all of the observed waveforms were consistent with active impulse propagation. The nature of excitability at motor nerve terminals was then neglected for more than a decade following the early work by Hubbard, Katz, and Miledi. But in the 1980s a

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number of investigators returned to this preparation, determined to use extracellular recording techniques to identify ion channels in the presynaptic membrane and to evaluate their distribution within a complex nerve terminal arbor consisting of a terminal heminode, branching axon segments, and boutons.

A. K+ CHANNELS In mouse motor nerve terminals, traditional K+ channel blockers, such as tetraethylammonium (TEA) and Caminopyridine ( M )produced , large changes in the extracellular waveform consistent with what one would expect from a blockade of Ktchannels (Brigant and Mallart, 1982). Further investigation of these signals resolved contributions from two distinct K+ channels, one of which could be classified as a delayed rectifier and the other as a Ca2+-activatedKt channel (Anderson et al., 1988; Mallart, 1985; Penner and Dreyer, 1986).A similar pair of K+ channels were seen in motor nerve terminals of lizard (Lindgren and Moore, 1989) and frog (Hevron et al., 1986). In frog the Ca2+-activatedK+ channel was blocked by charybdotoxin but not by apamin. Since charybdotoxin enhanced stimulus-evoked Ca2+entry and transmitter release, it would appear that nerve terminal Ca2+-activated K+ channels contribute to repolarization of the frog presynaptic action potential (Robitaille and Charlton, 1992). Further tests of a large number of K+ channel blockers in mouse motor nerve terminals led investigators to propose the existence of a third K+ channel that was not Ca2+dependent (Dreyer and Penner, 1987; Tabti et aL, 1989). The two Ca2+-independentK" channels were distinguished functionally on the basis of activation kinetics, with one denoted as fast and the other as slow. Mouse motor nerve terminals also appear to have ATP-sensitive K+ channels, as sulfonylurea reduces part of the fast signal arising from Kf current (Deist et aL, 1992). The ATP sensitivity of this current is supported by the fact that bathing in a glucose-free solution increases the amount of sulfonylurea-sensitivecurrent. In contrast to the appearance of two delayed rectifiers in mouse motor nerve terminals, at lizard neuromuscular junction the motor nerve terminal action potential waveform in TEA and 4AP reflected the influence of two Ca2+-dependentoutward currents (Morita and Barrett, 1990).A rapid component that contributed to action potential repolarization was blocked by apamin. A slower component responsible for an afterhyperpolarization was blocked by charybdotoxin. Thus, the lizard motor nerve terminal contains two distinct Ca2+-activatedK+ channels with different activation kinetics, pharmacological sensitivities, and functional roles.

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In the crayfish walking leg opener muscle, intracellular electrodes can be inserted in the excitor axon within about half a space constant of the nerve terminals (Sivaramakrishnan et aL, 1991a). Current injection into these axons activated outward K+ current. A component of this outward current could be blocked by cadmium, barium, tetraethylammonium, or charybdotoxin, indicating the presence of a Ca2+-activatedK+ conductance. The blockade of the Ca2+-activatedK+ channels was additive with blockade by 4AP or diaminopyridine (DAP); these two aminopyridines were thought to act on a delayed rectifier type of K+ channel. Thus, crayfish nerve terminals may also have this common pair of Kt channels. Blockade of either of these K+ channels broadens the presynaptic action potential and enhances action potential evoked neurotransmitter release, indicating that both of these channels contribute to action potential repolarization Blockade of the Ca2+-activatedK+ channel also simplified the voltage dependence of facilitation of neurotransmitter release (Sivaramakrishnan et al., 1991b) . As the voltage of a conditioning pulse increased, facilitation first went up and then declined. Since the decline at higher conditioning pulse voltage was removed by TEA, it was proposed that this decline was due to Ca2+activated K+ channels.

B. cA2+CHANNELS Blockade of the Kt channels in motor nerve terminals revealed a slow depolarization, which exhibited kinetic behavior and pharmacological sensitivity reminiscent of Ca2+channels studied in nerve cell bodies and muscle (Fig. 1) (Brigant and Mallart, 1982; Gundersen et aL, 1982). Presynaptic Ca2+current increased with increasing CaZt concentration; barium could replace Ca2+ as the charge carrier. The Ca2+ channels of motor nerve terminals can be blocked by varying concentrations of divalent cations such as cadmium, nickel, and cobalt (Fig. 1). Whether the presynaptic Ca2+current is carried by one channel or two has been difficult to ascertain. The Ca2+current in mouse motor nerve terminals exhibited biphasic kinetics (Penner and Dreyer, 1986), with each kinetic component distinguishable on the basis of blocker sensitivity and dependence on extracellular Caz+. However, in rat a monophasic Ca2+ signal was reported (Hamilton and Smith, 1992). In mouse, blockade of Ca2+currents by cadmium revealed two binding sites with differing affinities (Penner and Dreyer, 1986;Wiegand et aL, 1990). Furthermore, the blocking actions of verapamil and u-conotoxin GVIA were additive in a manner suggestive of two different channel types sensitive to one or the other of these blockers (Anderson and Harvey, 1987). However, in rat the depolar-

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01, -5

-4

-3

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log B l o c k w r Concwntratlon (M)

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FIG.1. Presynaptic CaZfcurrent was revealed in rat motor nerve terminals at the extensor digitorum longus muscle following blockade of K+ current by TEA and DAF'. (A) With both TEA and DAP, long lasting Caz' currents could be seen that were blocked by cadmium. (B) When only TEA was added the unblocked K+ current was sufficient to change the voltage and terminate the Ca2' signals after only about 3 ms. These Ca2' spikes were also blocked by cadmium. (C) Nickel was a relatively poor blocker of the motor nerve terminal CaZt channel; 0.1 mM had little effect. (D) Cobalt was also a poor blocker of nerve terminal Ca2+channels. (E)Percentage of inhibition of peak Ca2' current was plotted versus concentration for cadmium, nickel, and cobalt [from Hamilton and Smith (1992) with permission from Elsevier Science].

ization that exhibited a lower Cd2+sensitivity could be blocked by tetrodotoxin, suggesting that it was due to a Na+ channel rather than a Ca2+ channel (Hamilton and Smith, 1992).While there is agreement with regard to the presence of o-conotoxin GVIA-sensitive and presumably N-type Ca2+ channels in rodent motor nerve terminals (Anderson and Harvey, 1987; Hamilton and Smith, 1992), the putative second Ca2+channel cannot be L type as dihydropyridines had no effect (Hamilton and Smith, 1992; Penner and Dreyer, 1986). Mice injected with serum from patients with Lambert-Eaton myasthenic syndrome showed remarkable changes in motor nerve terminal Ca2+current (Smith et d., 1995). The presynaptic Ca2+current was 57% of that in controls. This difference was not likely to be related to changes in the geometry of the neuromuscular junction because the Ca2+current was normalized to the outward capacitative current flowing from the last heminode. In the Lambert-Eaton myasthenic syndrome animals, the Ca2+current was not reduced as the stimulus frequency increased, suggesting that the

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lost Ca2+channel was an inactivating variety. Most surprisingly, Ca2+currents in nerve terminals of mice treated with Lambert-Eaton serum were insensitive to u-conotoxin GVIA and were blocked by the dihydropyridine nifedipine. Thus, the serum from this autoimmune disease appears to target Ntype Ca2+channels in motor nerve terminals. The concomitant emergence of Ltype Ca2+channels may result either from enhanced expression in response to targeted loss of N-type channels or to an unmasking of Ltype channels already present. These results would therefore justify a careful reexamination of control motor nerve terminals with improved blockade of other currents. At lizard motor nerve terminals the dihydropyridine sensitivity of both nerve terminal Ca2+current and transmitter release indicated the presence of a single type of Ca2+channel closely related to the L type (Lindgren and Moore, 1989). Frogs are different from lizards. A strong irreversible blockade of frog motor nerve terminal Ca2+current by wconotoxin GVIA supports a role for N-type Ca2+ channels in synaptic transmission, and dihydropyridine insensitivity argues against a function for Ltype Ca2+channels (Silinskyand Solsona, 1992). Between the uncertainties of extracellular nerve terminal recordings and apparent variations between species, the question of Ca2+channel types in motor nerve terminals remains unresolved. Experiments employing synaptic transmission as the assay for sensitivity to various Ca2+channelblockers also failed to identify the motor nerve terminal Ca2+channels (Augustine et aL, 1987; Olivera et aL, 1994). The motor nerve terminals of chick ciliary neurons have been patch clamped in tissue culture (Brosius et al., 1990). In cocultures with skeletal muscle, nerve terminals as large as 15 pm emanate from chick ciliary neurons and form functional neuromuscularjunctions (Brosius et aL, 1990). Ca2+current recorded from these nerve terminals under voltage clamp was reduced 50% by 4 p M cadmium and blocked by 20 p M cadmium or 5 mM manganese. The presynaptic Cay+current had a relatively weak voltage sensitivity, with an activation range extending from -60 to 0 mV. This broad activation range may in part be due to poor voltage clamp, which was made difficult by z o n a l processes appended to the nerve terminals. The Ca2+current activated in less than 1.4 ms above 0 mV; tail currents deactivated in 1.57 ms. These rapid kinetics are necessary for Ca2+channel gating to follow the rapid voltage changes occurring during the presynaptic action potential. Presynaptic Ca2' current showed no sign of inactivation during pulses lasting 100 ms. Activation of Ca2+current appears to have sigmoidal kinetics, which could be important in rendering secretion sensitive to the duration of the presynaptic action potential (Augustine, 1990). This study in ciliary nerve terminals provided evidence for only a single type of Ca2+channel. The lack of inactivation of this channel is reminiscent

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of the L-type Ca2+channel, but the weak dihydropyridine sensitivity distinguishes the ciliary nerve terminal Ca2+channel from the Ca2+channel of ciliary nerve cell bodies (Stanley and Atrakchi, 1990; Yaw0 and Chuhma, 1993),and indicates that these motor nerve endings contain a different type of Ca2+channel, presumably specialized for secretion (Brosius et al., 1990). Similar studies in Xenopus nerve-muscle cocultures also show promise (Hulsizer et al., 1991;Yazejian et al., 1993). Ca2+currents have high voltage thresholds, but in contrast to the Ca2+currentsin chick motor nerve terminals, some Ca2+current inactivation is evident. The amount of inactivation varied from terminal to terminal. The rapidly inactivating current washed out during whole terminal patch clamp recording. These results together with stronger blockade of the rapidly inactivating component by 0conotoxin GVIA suggested that these motor nerve terminals contain two types of Ca2+channels. These presynaptic Ca2+currents are completely blocked by 400 p M cadmium but not by dihydropyridines. Thus, Xenopus motor nerve terminals contain one Ca2+channel that is very similar to the N type. Another Ca2+channel has properties like those of Ltype Ca2+ channels, with the exception of sensitivity to dihydropyridines. The two patch clamp studies in Xenopus and chick thus provide the most compelling evidence that in different species motor nerve terminals may have either one or two types ofCa2+channel. The extensivevariation in pharmacological sensitivity of extracellularly recorded CaZtsignals suggests that there is no one typical presynaptic Ca2+channel in motor nerve terminals, but rather that the motor nerve terminal is an important site of variation in Ca2+ channel function. Differences in motor nerve terminal Ca2+channels may have important implications for synaptic plasticity. High frequency stimulation reduced the motor nerve terminal Ca2+current in rat neuromuscular junction (Fig. 2) (Hamilton and Smith, 1992). In mouse motor nerve terminals a similar frequency dependent reduction was observed in the slower component of Ca2+current, with maximal effect observed at a frequency of 10 Hz (Penner and Dreyer, 1986).An inactivation of this form could be brought about either by Ca2+accumulation within the nerve terminal o r by the depolarization itself, which could inactivate the presynaptic N-type Ca2+ channels. This activity-induced depression of Ca2+current is very likely to play a role in use-dependent depression of synaptic transmission.

C. PRESYNAF-TIC TRANSMITTERS Activation of purinergic and muscarinic receptors on motor nerve terminals depresses neuromuscular synaptic transmission in many species. In rat the presynaptic Ca2+current was reduced by activation of either purinergic or muscarinic receptors (Hamilton and Smith, 1991, 1992). Effects on

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1Oms

FIG.2. Motor nerve terminal Caz' current such as that shown in Fig. 1 was reduced following high-frequency stimulation. Stimulus frequencies were maintained for 1 min after which the Ca4' current was recorded. Blockade of K' current by TEA alone (A) revealed an effect of frequency that was less dramatic than that observed in the presence of TEA and DAP ( B ) [from Hamilton and Smith (1992) with permission from Elsevier Science].

other presynaptic currents were not seen. Inhibition of presynaptic CaZt current was independent of depression induced by high-frequency stimulation described above, as this form of depression persisted in the presence of purinergic receptor antagonists (Hamilton and Smith, 1992). Both muscarinic and purinergic depression of presynaptic Ca2+current were blocked by pertussis toxin indicating mediation by a G protein. In both rat and frog, use-dependent depression of neuromuscular transmission has been linked to ATP corelease with acetylcholine. Upon release, ATP is broken down to adenosine, which activates purinergic receptors on the nerve terminals (Meriney and Grinnell, 1991; Redman and Silinsky, 1994; Smith and Lu, 1991). In frog the receptor has been identified as A, (Redman and Silinsky, 1993). However, in frog motor nerve terminals activation of these receptors does not modulate Ca2' current or current through other channels (Silinsky and Solsona, 1992). Because purinergic agonists depressed miniature synaptic currents in a reversed Ca2+gradient, it was concluded that this action is independent of ion currents and is thus downstream from the Ca2+entry step of triggered secretion. D. CHANNEL LOCATIONS The motor nerve terminal is geometrically complex. This has raised interesting questions about the propagation of action potentials from the

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myelinated axon into the unmyelinated arbor of branches, varicosities, and boutons. Extracellular depolarization applied to frog motor axons can spread passively to the nerve terminal to induce release (Del Castillo and Katz, 1954; Katz and Miledi, 1977), seemingly obviating the need for excitability in the motor nerve terminal. Yet, extracellular recordings at various sites on the preterminal zone just beyond the final sheath of myelin could all be explained in terms of active propagation of an action potential (Katz and Miledi, 1965). It was concluded that virtually the entire nerve terminal was excitable and capable of conducting action potentials. This conclusion was bolstered by subsequent studies with tetrodotoxin application to various regions. At any location along the nerve terminal, tetrodotoxin application diminished postsynaptic responses arising from more distal but not more proximal regions of the nerve terminal (Katz and Miledi, 1868). A later reexamination of this issue in mouse by Brigant and Mallart (1982) lead to the very different conclusion that the nerve terminal membrane differed substantially from the axonal membrane. In contrast to results from frog, local application of tetrodotoxin failed to affect electrical activity in the terminal. The local circuit used to explain these results included Na+ channels in the preterminal region but not in the terminal arbor. Higher densities of Ca2' and K+ channels in the terminal arbor were proposed to pass current through the distal portions of membrane. Subsequent modeling studies suggested that the large distal K+ currents could mask a small but significant inward Na+ current through more distal nerve terminal membrane (Peres and Andrietti, 1986). Furthermore, a small tetrodotoxin-sensitive inward current was detected in the distal portions of the rodent terminal arbor (Hamilton and Smith, 1992; Konishi, 1985). A careful evaluation of extracellular recordings from lizard with the aid of a computer model also indicated significant densities of Na+ channels throughout the terminal arbor (Lindgren and Moore, 1989). Thus, the pattern of ion channel segregation in motor nerve terminals proposed by Brigant and Mallart (1982) should be viewed as partial rather than absolute. Na+ channels are reduced but not excluded from the nerve terminals of rodent and lizard. The excitability and channel distribution in the nerve terminal is intimately connected with geometry and size. Although depolarizing potentials in axons can penetrate deep into the nerve terminal by purely passive mechanisms (Brigant and Mallart, 1982; Del Castillo and Katz, 1954; Konishi, 1985), the abrupt drop in input impedance at the end of the myelination, and the often long segments and extensive branching that emerge soon thereafter can place a large load on the presynaptic action potential and limit its spread (Revenko et al., 1973). Thus, the distances between nodes of Ranvier become shorter toward the end of a motor axon to boost

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axial currents (Lindgren and Moore, 1989). This effect will manifest to different degrees depending on the geometric complexity of the terminal arbor. Thus, in the longer nerve terminals of amphibians and reptiles a higher density of Na+ channels may be essential for presynaptic action potentials to produce large depolarizations. In the more compact motor nerve terminals of the mouse, an invading action potential can depolarize the nerve terminal with less assistance from excitable membrane within the nerve terminal itself. The problem of channel localization in motor nerve terminals has also been addressed by histochemical techniques, and this approach has met with considerable success. Using fluorescently tagged wconotoxin GVIA, confocal microscopy revealed concentrated ribbons of label at approximately 1 pm intervals coursing perpendicular to the long axis of the terminal (Robitaille et al., 1990). These images showed the Ca2+channels in near perfect register with ribbons of postsynaptic acetylcholine receptor labeled with a-bungarotoxin derivitized with a different fluorophore. Thus, presynaptic Ca2+channels, presumably N type, are located exclusively in the active zones of the synapse. The location of N-type Ca2+ channels delivers Cay+rapidly to the secretion triggering apparatus while limiting Ca2+incursions into other domains within the nerve terminal. Subsequent labeling studies with biotinylated charybdotoxin demonstrated a similar distribution of presynaptic Ca'+-activated K+ channels (Robitaille et al., 1993).Thus, these channels are positioned to respond to Ca2+immediately on entry rather than following diffusion away from the Ca2+channels.This result is consistent with the observation that blockade of the Ca2+-activated K+ channel enhances synaptic transmission (Robitaille and Charlton, 1992; Robitaille et al., 1993).

111. Squid Synapse

The presynaptic terminal of the squid stellate ganglion is probably the largest found in nature (Llinas, 1982). This large size enabled some of the first highquality electrical recordings of presynaptic current. Intracellular recordings following K+ channel blockade in these terminals revealed Ca2+dependent depolarizations, and these signals correlated well with transmitter release (Katz and Miledi, 1967; Kusano et aL, 1967). Voltage clamp of the squid nerve terminal, achieved with a three-microelectrode recording configuration (Llinas et al., 1976), revealed currents carried by Na+, K', and Ca" (Llinas et al., 1981).

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A. CA2' CHANNELS The squid presynaptic Ca2+channel is activated by small depolarizations. Depolarization to only - 55 mV produces discernible Ca2' current (Charlton and Augustine, 1990). Ca2+current peaks at membrane potentials between -10 and 0 mV (Augustine et al., 1985b; Llinas et aL, 1981). Depolarizations to 50 mV extinguished the Ca2+current(Fig. 3). Depolarizations beyond 60 mVfailed to produce outward current through the Ca2+channel, presumably because of the very low intracellular [Ca2'] (Llinas et al., 1981).Tail currents demonstrated that channel activation is half maximal at - 13mV and occurs predominantly within the range from - 20 to 20 mV (Augustine et al., 1985b; Llinas et al., 1981). This is consistent with the bell-shaped plots of current versusvoltage. The voltage threshold for activation of this Ca2' channel is low compared to Ltype and N-type Ca2' channels, as well as most of the other presynaptic Ca2+channels described below. Furthermore, during shortdepolarizations the presynaptic Ca2+channel exhibits little inactivation; pulses on

-30 rnV

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+ 10

1 rns

FIG.3. Presynaptic . . CaZt currents in the squid nerve terminal evoked by pulses from -70 mV to the indicated voltages. Note that the current onset occurs approximately 1 ms after the pulse. Large CaZt tail currents are also evident upon repolarization. Ca2+current was corrected for leak as described by Augustine et uZ. [from Augustine et aZ. (1985b).Reprinted with the permission of The Physiological Society].

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the order of seconds produce significant inactivation to an extent that a p pears to depend on CaZtentry (Augustine et al., 1987).Thus, the squid presynaptic CaZtchannel is capable of triggering sustained secretion in response to small graded depolarizations (Charlton and Atwood, 1977). Double-pulse experiments have been used to estimate an instantaneous current through open channels (Llinas et al., 1981). This instantaneous current was highly nonlinear, suggesting that current through the open presynaptic Ca2+channel deviates from Ohm's law. Increasing extracellular [Ca"] from 3 to 40 mM shifted the voltage of peak Ca'+ current 10 mV positive and increased the magnitude of peak Ca*+currentapproximately fivefold. These properties formed the basis for a rudimentary model for CaZtpermeation that was used in a quantitative analysis of secretion induced by voltage pulses and action potentials (Llinas et al., 1981). Further work on permeation showed that the Cay+channel saturates with increasing extracellular [Ca"] with an apparent dissociation constant of 60 mM (Augustine and Charlton, 1986). Two other divalent cations, Bazt and Sr2+, also permeate the presynaptic Ca2+channel, with Bazf substitution producing larger current than CaZt (Augustine and Eckert, 1984). When special care was taken to permit Ca2+entry only into well-clamped regions of the nerve terminal, optically detected Ca2+signals followed Ca'+ current very closely (Augustine et al., 198513). In common with most other voltage activated channels, this channel exhibited sigmoidal activation kinetics, such that current was not evident until after a lag of 0.5-1 ms following the onset of a depolarizing pulse (Fig. 3); current peaked 13 ms after the start of the pulse (Augustine et al., 1985b; Llinas et aL, 1976, 1981, 1982). Both the latency and the rise time of Ca'+ current decreased with increasing voltage. As a result of this lag in Caet current activation, CaZt entry begins late during the presynaptic action potential and because channel closure takes approximately 0.5 ms, Ca" entry continues for only a short time after the action potential ends (Augustine, 1990; Llinas et al., 1981). The sigmoidal activation kinetics and nearly exponential closure kinetics have been used to formulate a model for the kinetics of the presynaptic Ca2+ channel in which five voltage-sensing subunits interact to gate the channel (Llinas et al., 1981).The sigmoidal activation kinetics of the presynaptic Ca'+ channel plays a key role in rendering synaptic transmission highly sensitive to action potential duration. Small increases in the duration of the presynaptic action potential, brought about by partial blockade of presynaptic KCchannels, produce disproportionally large increases in the amount of Ca'+ entry. A 30% increase in the duration of the presynaptic action potential causes a 190% increase in the amplitude of the postsynaptic current. The linear dependence of postsynaptic current on action potential duration (Fig. 4B) is probably closely related to the linear relationship between postsynaptic

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response and presynaptic Ca2+current (Llinas et al., 1981).These results indicate that the third power dependence of secretion on CaZt concentration (Augustine et al,, 1985a) does not contribute to the enhancement of synaptic transmission during action potential broadening (Augustine, 1990).This linearity can be explained in terms of nonoverlapping domains of high Ca2+ around open Ca2+channels. The presynaptic CaZtchannel of squid can be blocked by the usual inorganic Caz+channel blockers cadmium, cobalt, and manganese (Augustine et al., 1987; Llinas et al., 1981), although 50% block by cadmium required an unusually high concentration of 100pMwhen the concentration of Ca2+was 11 mM (Charlton and Augustine, 1990). The Ltype Ca2' channel antagonist nitrendipine and the N-type Ca2+ channel antagonist uconotoxin GVIA had no effect on squid presynaptic Ca2+currents, suggesting that this channel defies classification as N type or L type. Additional consideration of pharmacological and biophysical properties ruled out the T-type Ca4+channel (Charlton and Augustine, 1990).The presynaptic Ca2+channel has a low threshold like T-type Ca2+channels, but inactivates slowly like Ltype Ca2+channels. A toxin derived from funnel web spider with strong efficacy against Ca2' channels of mammalian purkinje neurons suppressed the squid presynaptic Ca2' current. Sensitivity to this toxin has been proposed as the basis for defining a novel CaZtchannel denoted as P type (Llinas et al., 1989). Since funnel web spider toxin blocks other currents in cortical neurons (Brown et al., 1994) the mammalian Ca2+channelsensitive to the toxinw-Aga IVA may be the closest relative of the squid presynaptic Ca2+channel.

A

B

C -

I

PSC

m

V,W

e

-

- 1 3 0 mV

1 ms

AP duration (%control)

100 110 120 130 AP duration ( % control]

FIG.4. DAP broadens the presynaptic action potential and enhances synaptic transmission at the squid giant synapse. (A) When 25 p M DAP was released from a nearby application pipette, the action potential duration increased as K+ channel blocker concentration grew (Vpr,).The postsynaptic current (PSC) grew in parallel. (B) PSC amplitude increased linearly with the increase in action potential duration (both plotted as percentage of control). For every 1% increase in action potential duration there is a 6.92% increase in PSC amplitude correlation coefficient (r) = 0.99. ( C ) Interval between peak of presynaptic action potential and peak of PSC is described by a linear function with a slope of 0.34% change in interval per 1% change in action potential duration ( r = 0.89) All parameters were normalized by dividing by their mean value measured before DAP treatment [from Augustine (1990). Reprinted with the permission of The PhysiologicalSociety].

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B. K+CHANNELS K+ current in the squid giant synapse nerve terminal exhibits two components, one mediated by a delayed rectifier channel similar to that previously observed in the squid giant axon (Augustine, 1990; Llinas et aL, 1981). The K+ channel other component of K+ current was mediated by a Ca2+-activated (Augustine, 1990; Augustine and Eckert, 1982; Llinas et aL, 1981). The Ca2+-activatedK+ current was suppressed by blockade of Ca2+current with cadmium (Augustine and Eckert, 1982). The Ca2+dependentcomponent activates slowly during depolarizing voltage pulses and can be blocked selectively by extracellular TEA. The delayed rectifier activates rapidly during depolarization, is relatively insensitive to extracellular TEA, and is blocked by injected TEA (Fig. 5 ) . The delayed rectifier is very sensitive to 3,Miaminopyridine (DAP) (Fig. 5) (Augustine, 1990). An apparent dissociation constant for DAP of 7 p M at the presynaptic delayed rectifier channel agrees well with that measured in the squid axon, suggesting that they are the same channel. DAP also blocks the Ca2+-activatedK+ channel in this concentration range. Intracellularly injected TEA blocks both K+ currents. With TEA and DAP, the functions of the two squid presynaptic K+ channels can be tested (Augustine, 1990). The delayed rectifier shows a very steep voltage dependence. The threshold for voltage activation is relatively low; current can be measured with depolarizations to only -60 mV. With sustained depolarization current through the delayed rectifier appeared to decline. However, the decline in current was accompanied by a shift in the reversal potential of tail currents, indicating that K+ current causes accumulation of extracellular K+. Taking the accumulation of K+ into account suggested that the delayed rectifier showed little inactivation during depolarizations lasting on the A

/,re

c

5 --.-

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i.i

,

,

,

70-7 10-6

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10-5

,

10-4

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FIG.5. TEA and DAF’ blockade of K+ current in the squid presynaptic terminal. K+ current was evoked by stepping from -70 to -40 mV. (A) TEA was microinjected (100 pC) into the squid nerve terminal to reduce K+ current. (B) Application of 5 &A4 DAF’ to the bathing solution blocked K+ current almost completely (evoked by stepping from -65 to -30 mV). (C) Dose-inhibition curve representing mean (21SEM) for DAP. Current was measured at the end of 20 ms pulses such as those shown in A and B. The plot was well fitted by a curve representing single-site binding with a KO of 7 pA4 [from Augustine (1990). Reprinted with the permission of The Physiological Society].

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order of seconds. The rapid activation of the delayed rectifier limits the duration of the presynaptic action potential. DAF'and intracellular TEA broaden the presynaptic action potential and enhance synaptic transmission (Augustine, 1990). In contrast, extracellular TEA has no effect on the presynaptic action potential or synaptic transmission, indicating that the Ca2+-activatedK+ channel plays little role in regulating secretion under normal conditions.

C. CHANNEL LOCATIONS An aequorin derivative with low affinity for Ca'+ was used to visualize very high Caz+concentrations within submicron domains thought to surround individual Ca2+channels (Llinas et al., 1992a). Furthermore, the number of these high [Ca2'] domains was similar to the number of active zones of secretion, and their locations were roughly similar (Llinas et al., 1992b). To create the high [Ca2+]thought to be necessary for secretion, these channels would have to be very close to active zones. Imaging of fluorescence with the Ca*+ indicator fura-2 has shown that CaZt entry occurs through presynaptic membrane closely juxtaposed against postsynaptic membrane, a region rich in active zones (Smith et al., 1993). Additional evidence for proximity between Ca2+channels and release sites comes from comparing the effect of injecting slow and fast Ca2+chelators (Adler et al., 1991; Augustine et al., 1991). EGTA binds Ca2+ more slowly than does BAPTA, and injecting EGTA into the presynaptic terminals has no discernible effect on secretion, whereas BAPTA reduces secretion. A comparison of the action of various Ca2+chelators suggested that Caz+channels were very close to the release sites. These results thus suggest that the squid synapse is similar to the neuromuscular junction in that Ca2+channelsare close enough to active zones of neurosecretion to produce the high concentrations needed to activate low-affinity Ca2+ receptors (Robitaille et al., 1990).

D. PRESYNAPTIC TRANSMITTERS Relatively little work has been performed on the actions of neurotransmitters at the squid giant synapse presynaptic terminal. The two neuropeptides FMRFamide and FLRFamide enhance synaptic transmission in the squid without altering the shape of the presynaptic spike (Cottrell et aL, 1992). If this action is presynaptic, then the mechanism probably involves

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changes in the secretion apparatus rather than changes in nerve terminal ion channels. Both of these peptides have been detected in the synapse.

N. Posterior Pituitary The posterior pituitary, or neurohypophysis, consists primarily of endings of axons originating in the hypothalamus. Two nonapeptides, vasopressin and oxytocin, account for most of the secretory output of the posterior pituitary, although other peptides are coreleased and nerve terminals from diverse regions secrete other neurotransmitters. Because of the high density of nerve terminals and the absence of nerve cell bodies, this tissue is well suited for the investigation of neurosecretion. Seminal studies were carried out in the posterior pituitary to establish CaZt entry as the trigger for secretion (Douglas and Poisner, 1964; Nordmann, 1983). The high density of nerve terminals has also been exploited in experiments using optical techniques to record voltage changes. These optical signals arise almost exclusively from nerve terminals and thus provide responses relatively free of contamination from voltage changes in other nonsecreting structures (Salzberg et al., 1983, 1985). Another advantage of the high density of nerve terminals is that dissociation of the posterior pituitary produces a high proportion of secretion-competent synaptosomes (Cazalis et aL, 1987). These synaptosomes, or neurosecretosomes, are suitable for patch clamp recording (Fidler-Lim et aL, 1990; Lemos and Nowycky, 1989). Many of the secretory swellings of the posterior pituitary are as large as 15 pm in diameter (Nordmann, 1977). Thus, it is also possible to make intracellular (Bourque, 1990) and patch clamp recordings (Jackson et aL, 1991; Mason and Dyball, 1986) from intact, undissociated tissue. Because the targets of the secreted neuropeptides are peripheral tissues, the peptidergic nerve terminals of the posterior pituitary have no true postsynaptic cells.

A. CA'+ CHANNELS Optical recordings of presynaptic action potentials in frog posterior pituitary indicated that the nerve terminal membrane contained Ca2+channels that were sensitive to cadmium and nickel (Salzberg et aL, 1983). Patch clamp recordings from neurosecretosomes prepared from the rat posterior pituitary revealed two types of Ca2+channels (Lemos and Nowycky, 1989). One of these channels had a conductance of 25 pS (in 110 mMBa2') and was sensitive to the dihydropyridine agonist BayK. These properties

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prompted a tentative identification of the Ca2+channel as L type. One dihydropyridine antagonist, nicardipine, blocked this channel but another dihydropyridine antagonist, nifedipine, was ineffective (Wang et al., 1993). Since nifedipine blocks Ltype Ca2+channels in most cell bodies, this nerve terminal Ca2+channel may be a unique subtype. The other Ca2+channel of posterior pituitary nerve terminals had a lower conductance of 11 pS (in 110 mM Ba2+)and was dihydropyridine insensitive (Wang et al., 1993). The two Ca2+channels can also be distinguished on the basis of differential sensitivity to oconotoxin GVIA, 10-fold higher concentrations of which were needed to block the L type. This suggests that the low conductance Ca2+channel is N type (Wang et al., 1992b). Aminoglycoside antibiotics at concentrations in the 100 p M range block Ca2+channels resulting in a reduction in optical signals associated with secretion (Parsons et al., 1992). It is not known which Ca2+channel is affected, but based on the evidence discussed below for a greater contribution from N-type Ca2+channels in this system, aminoglycoside antibiotics probably act on this channel. Both the high- and low-conductance nerve terminal Ca" channels had relatively high voltage thresholds for activation close to - 10 mV. The high conductance channel exhibited almost no voltage inactivation, but the low conductance channel (with high wconotoxin GVIA sensitivity) was strongly inactivated with a time constant of 100 ms (Wang et al., 1992b).The inactivation of the N-type Ca2+channel provided a means of separating these two currents. Pulses from holding potentials of -50 mV selectively activated the L type. Ltype and N-type Ca2+channel antagonists have been tested and shown to inhibit depolarization-induced changes in intracellular [Ca2+](Brethes et al., 1987; Stuenkel, 1990), depolarization-induced vasopressin secretion (Dayanithi et al., 1988;von Spreckelsen et aL, 1990), and stimulus-induced changes in optical properties associated with secretion (Obaid et al., 1989). While nicardipine, a dihydropyridine with demonstrated antagonist activity at the nerve terminal Ltype Ca2+channel, blocked rises in intracellular [Ca"] resulting from a slow K+-induced depolarization (Brethes et al., 1987; Stuenkel, 1990), it had little effect on secretion induced by electrical stimulation (van Spreckelsen et al., 1990). Dihydropyridines also failed to alter optically detected secretion (Obaid et al., 1989). Concentrations of wconotoxin GVIA low enough to block the nerve terminal N-type channel and spare the Ltype channel were effective in reducing stimulus-induced secretion (Dayanithi el al., 1988; Obaid et al., 1989; von Spreckelsen et al., 1990). These studies indicated that the N-type Ca2+channel makes a greater contribution in triggering secretion. It is likely that each of the two CaZt channels in the posterior pituitary has some specialized function in triggering secretion responses to different forms of activity, but this speculation awaits definitive experimental testing.

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B. Kt CHANNELS K+ channel blockers broaden the nerve terminal action potential (Gainer et al., 1986; Salzberg and Obaid, 1988) and enhance secretion from the posterior pituitary (Bondy et al., 1987; Bondy and Russell, 1988; Hobbach et al., 1988), indicating the presence of Kt channels sensitive to TEA, 4AP, and dendrotoxin. The posterior pituitary nerve terminal membrane contains three K+ channel types (Bielefeldt et aL, 1992). One of these exhibits rapid activation and inactivation reminiscent of the A current (Bielefeldt et al., 1992; Thorn et al., 1991). This channel has a conductance of 32 pS and is activated in less than 1 ms by strong voltage pulses; during sustained depolarization inactivation proceeds with a time constant of 20 ms (Bielefeldt et al., 1992). A transient outward current with many similar properties has been described in the hypothalamic cell bodies from which these nerve terminals originate (Cobbett et al., 1989). Significant discrepancies exist between properties of this channel reported in pituitary slices and neurosecretosomes. The voltage midpoints for steadystate inactivation were -88 mVin slices and -48 mVin neurosecretosomes. Furthermore, the A current in slices was blocked 90% by 5 mM TEA (Bielefeldt et al., 1992), but 100 mMTEAfailed to reduce the neurosecretosome A current (Thorn et al., 1991). These discrepancies are difficult to reconcile, but the finding that TEA enhances secretion from the posterior pituitary (Bondy et al., 1987; Hobbach et al., 1988) supports the results obtained from slices. The inactivation of the nerve terminal A current has been invoked to explain the enhancement of stimulus-evoked secretion by high frequency stimulation. When action potentials fire in the nerve terminal in rapid succession,greater rises in intracellular Ca2+correlate with an enhancement of secretion (Jackson et al., 1991). Broadening of action potentials also occurred in parallel with the frequency-dependent facilitation of secretion (Fig. 6) (Bourque, 1990; Gainer et al., 1986;Jackson et al., 1991). When the nerve terminal was voltage clamped, spike-likevoltage pulses inactivated the transient component of K+ current with a frequency dependence similar to that of action potential broadening (Fig. 6). Thus, a membrane mechanism for spike broadening similar to that described in molluscan neurons (Aldrich et al., 1979) can account for this important form of plasticity. A high-frequency train of action potentials then brings the membrane to a positive potential often enough to inactivate K+ current, which in turn broadens action potentials. As with the squid presynaptic terminal (Augustine, 1990), broadening action potentials leads to a steep increase in Ca" influx (Jackson et al., 1991),with a final result of enhancement in neuropep tide secretion.

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MEYER B. JACKSON

-2.4

-E .-0

P

-2.0 p'

0

Q:

I

I

I

~

i

1.6

- 0.8 -0.7 -

0.6

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FIG.6. High frequency stimulation facilitates secretion in nerve terminals from the posterior pituitary. (A) Under current clamp, stimulation at various frequencies broadens action potentials. A control action potential (evoked after at least 5 s of inactivity) is shown together with action potentials recorded at the end of a 30 action potential train applied at 1 and 10 Hz. (B)Plot of action potential duration (width at half height) versus frequency of stimulation as in A. (C) Under voltage clamp a depolarizing pulse from -80 to 50 mV elicits K+ current. A control current was recorded after at least 5 s of inactivity. Applying trains of 30 pulses 3 ms in duration and 50 mV in amplitude simulates a train of action potentials. The Kt current recorded after trains of 1 and 10 Hz shows a selective loss of a transient component. (D)The ratio is used as an index of the transient K' current. In control currents the peak is approximately twice as great as the final Kt current, so the ratio is slightly less than 0.5. Prior stimulation with action potential-like pulses, as in C, reduces the ratio of Ifinal/Ipcak in subsequently evoked K' current. The plot of this ratio versus frequency is very similar to the plot of action potential duration versus frequency (B)supporting the hypothesis that KCchannel inactivation underlies the broadening of action potentials by high-frequency stimulation [from Jackson et al. (1991) with permission].

The rat posterior pituitary membrane contains a Ca'+-activated K+ channel (Bielefeldt et aL, 1992; Wang et al., 1992a). The cell bodies of the hypothalamus also have Ca'+-activated K t channels, but not enough detail is available for a useful comparison (Bourque, 1988; Cobbett et aL, 1989). With its 134pS conductance the nerve terminal channel resembles the socalled BK channels found in many other cells. But the posterior pituitary channel differs from nearly all other known BK channels in that it is resistant to charybdotoxin. With the possible exception of tetrandine (Wang and Lemos, 1992), selective blockers of this channel are lacking. In addition to charybdotoxin mentioned above, apamin and dendrotoxin were without

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22 1

effect (Bielefeldt and Jackson, 1993; Bielefeldt et al., 1992; Wang et al., 1992a). The nonselective K' channel blockers TEA and 4AP blocked part of the current through the posterior pituitary BK channel at 0.5 and 2 mM, respectively (Bielefeldt and Jackson, 1993). In neurosecretosomes a similar TEA effect was observed, but 4AP was ineffective at concentrations as high as 7 mM (Wang et al., 1992a). In addition to its striking insensitivity to charybdotoxin, the nerve terminal BK channel differs from the Ca2'-activated Kt channels of most other cells in terms of physiological properties. The channel has a high sensitivity to Ca'' so that even at resting intracellular [Ca"] on the order of 100 nM (Jackson et al., 1991), depolarizing voltage steps induced substantial BK channel activation in the absence of Ca2+entry (Bielefeldt et al., 1992). Furthermore, the effect of intracellular Ca'' developed slowly, so that a component of current dependent on Ca2' entry appeared only after depolarizations lasting 0.5-1 s (Bielefeldt and Jackson, 1993; Bielefeldt et al., 1992). Blockade of Ca2t channels by cadmium had no discernible effect on K+ current during the first 0.5 s. Finally, whole terminal current through the posterior pituitary BK channel inactivated in a manner reminiscent of the nerve terminal A current, but with a slower time constant of approximately 100 ms. Since this channel is activated by voltage with sufficient rapidity to contribute to action potential repolarization, it is likely that the inactivation of this channel makes a contribution to the frequencydependent spike broadening described above (Fig. 6) (Jackson et al., 1991). A study using optical recording methods suggested that the Ca"-activated K' channel of frog posterior pituitary is different in a number of respects (Obaid et al., 1989). Charybdotoxin blocks a strong afterhyperpolarization in frog, indicating that this frog nerve terminal Ca2+-activatedK+ channel differs from the rat channel in drug sensitivity. The afterhyperpolarization was also blocked by o-conotoxin GVIA. The dependence of the afterhyperpolarization on Ca2+entry indicates that this channel is activated much more rapidly by Ca" in frog compared with rat. Similar experiments failed to detect a charybdotoxin-sensitive afterhyperpolarization in mouse posterior pituitary (Obaid et aL, 1989), suggesting that the mouse and rat Ca2'-activated K+ channels are similar with regard to charybdotoxin insensitivity and slow response to Ca". The slow response of the posterior pituitary BK channel to Cazt makes it especially well suited for a role in usedependent depression of secretion (Bielefeldt and Jackson, 1993). Following trains of action potentials lasting more than 5 s, secretion was suppressed (Bicknell et al., 1984). Action potential failure can also be seen following similar trains (Bielefeldt and Jackson, 1993). Block of Ca" entry by cadmium prevents fatigue during

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trains of action potentials, and enhancing CaZt entry with the dihydropyridine agonist BayK causes action potential failure to occur much earlier in a train. Other manipulations of this channel by phosphorylation (discussed below) influence action potential failure similarly (Bielefeldt and Jackson, 1994b). These results thus support the hypothesis that a slowly developing Ca'+ activation of the BK channel contributes to use-dependent depression of secretion, but further comparative studies of the effects of blockers on channels and secretion will be necessary to establish this link more strongly. It should be noted that the delayed activation kinetics of the BK channel by intracellular Cay+plays a key role in allowing a single signal, cellular Ca2+,to trigger both secretion and fatigue in separate windows of time. This dual action thus falls into a common pattern for cellular Ca2+-mediated signaling processes, in which the varied sensitivities of cytoplasmic CaZt receptors engage different responses to different temporal patterns of Ca2+ entry (Kasai, 1993). The posterior pituitary BK channel is subject to a remarkable system of enzymatic controls. The first indication of this appeared in experiments with excised patches which were prepared to study effects of Ca2+at the cytoplasmic membrane face. Within approximately 2 min of patch excision into solutions lacking MgATP, the channel activity of a patch dropped dramatically (Fig. 7). This drop in activity was prevented by excision into a MgATP-containingsolution (Bielefeldt and Jackson, 1994b). MgATP also restored channel activity following rundown. Since nonhydrolyzable ATP analogues and ATP without Mg were ineffective, it was likely that a membrane-bound protein kinase restored the activity of the BK channel. This was confirmed with protein kinase inhibitors. A broad-spectrum protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-3-methylpiperizine(IQMP) , prevented the restoration of channel activity by MgATP, as did a low concentration of staurosporine (Fig. 7). Since staurosporine is more effective in blocking phosphorylation by protein kinase C, it is likely that an enzyme from this family can phosphorylate the channel protein or another closely associated protein to increase the open probability. Inhibitors of protein kinase A and calmodulin-dependent protein kinase had no effect. Furthermore, phosphorylation had no requirement for the cofactor diacylglycerol and could be observed in [Ca2+]as low as 10 nM. The lack of cofactor requirement is consistent with the failure of the diacylglycerol antagonist, sphingosine, to inhibit phosphorylation, indicating that this is a cofactor independent activity of C kinase, as has been described elsewhere (Bazzi and Nelsesteun, 1987). The phosphorylation involved in BK channel activation has the unusual property of responding to membrane potential (Bielefeldt and Jackson, 1994a,b). MgATP is much more effective at restoring channel activity at

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I

2mm

FIG.7. A patch containing several BK channels was excised from a posterior pituitary nerve terminal into a solution containing 1 mM ATP. Patches were held at -40 mV in a solution with computed free [Ca2+]of 250 nM. Channel activity continued after excision as long as ATP was present, but 2 min after initiating perfusion with ATP-free solution, activity was dramatically reduced (wash out). Addition of ATP in the presence of 50 n M staurosporine, a potent C kinase antagonist, produced little change in channel activity. ATP and staurosporine were removed (second wash out trace). Subsequent addition of 1 mM ATP restored channel activity [from Bielefeldt and Jackson (1994b) with permission].

positive membrane potentials. It remains unclear whether the enzyme actually senses voltage changes directly or whether the voltage induces conformational changes in the substrate to accelerate enzymatic catalysis. The voltage dependent phosphorylation provides yet another avenue for usedependent depression of activity, for which the Caz+activation of the BK channel was already implicated (Bielefeldt and Jackson, 1993).The voltage dependent activation of this enzyme provides an additional signal directly linked to electrical activity whereby action potentials can be inhibited. The voltage signal together with the Ca'+ signal thus exert a dual form of regulation over the channel. At the highest potentials experimentally accessible, the rate of phosphorylation, as judged by the time course with which channel activity increases, exceeds the theoretical limit proscribed by diffusion (Bielefeldt and Jackson, 1994a). Although this theoretical estimate has some uncertainty, the comparison does suggest that the protein kinase responsible for restoration of activity is tightly associated with the BK channel. This is supported by other findings, including the observation of successful restoration of channel activity in all but 2 out of 115 excised patches (Bielefeldt and Jackson,

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1994a). Experiments with channels reconstituted into bilayers suggested a similar tight association between protein kinase and channel (Chung et aZ., 1991).Based on a comparison of properties, such as channel conductance, charybdotoxin insensitivity, and protein kinase sensitivity, it is very likely that this reconstituted rat brain BKchannel is the same BKchannel observed in posterior pituitary nerve terminals. Just as restoration of channel activity depends on a protein kinase, rundown of activity depends on a protein phosphatase. Channel rundown was blocked by the protein phosphatase inhibitor, okadaic acid, and based on the concentration dependence of this block, it is likely that rundown is due to a type I protein phosphatase (Bielefeldt and Jackson, 1994b). Rundown was dramatically enhanced by addition of GTPyS to the cytoplasmic face of an excised patch, indicating the presence of a G protein in membrane patches. Since this accelerated rundown was blocked by okadaic acid, and since the inhibition of a kinase could be ruled out with experiments on restoration, it appeared that the protein phosphatase is under direct control of a G protein, as was suggested for a tyrosine protein phosphatase stimulated by hormone in human tumor cells (Pan et al., 1992). The coupling of a G protein to the protein phosphatase makes it very likely that a receptor in the plasma membrane can enhance dephosphorylation and thus reduce channel activity, but these receptors have not as yet been identified. Even when the phosphatase activity was enhanced by GTPyS, the rate of rundown was substantially slower than the theoretical diffusion limit. Furthermore, variation in enzyme number among patches followed the Poisson distribution with no correlation to channel number. These observations provide strong evidence that the protein phosphatase, in contrast to the protein kinase, is separate from the channel and exerts its catalytic action by an intermolecular mechanism (Bielefeldt and Jackson, 1994a). These experiments demonstrated that three distinct proteins play a role in the regulation of the nerve terminal BK channel. The protein kinase, protein phosphatase, and G protein provide mechanisms by which a number of signals can alter the activity of the nerve terminal BK channel. Experiments with action potentials under current clamp tested the role of BK channel modulation during more physiological forms of activity. As mentioned above, pharmacological manipulation of Ca2+entry can alter the time of onset of action potential failure in a train of action potentials (Bielefeldt and Jackson, 1993). Blockade of the enzymes responsible for channel modulation has a similar effect. Inhibition of the protein phosphatase with okadaic acid increases BK channel activity, and this caused action potential failure very early in a burst (Figs. 8C and 8D) (Bielefeldt and Jackson, 1994b). In contrast, blockade of the protein kinase with IQMP

A

6

C

Frc. 8. Action potential failure and inhibition of phosphorylation and dephosphorylation. Currentclamped nerve terminals were stimulated with trains of brief current pulses to evoke action potentials. All recordings shown were made 10 seconds into the stimulus train. (A) Using a patch pipette solution including 500 p M IQMP and 4 mM Mg-ATP, a nerve terminal was stimulatrd at 23 Hz with 0.1 nA, 1 ms pulses. Less than one minute aftrr establishing the whole-terminal recording (upper trace), a significant number of stimuli failed to evoke action potentials 10 s into the train. Five minutes later, after IQMP had time to diffuse into the nerve terminal, identical stimuli produced very few action potential failures 10 s into the train (lower trace). (B) The traction of stimuli that failed to evoke action potentials at 10 s is shown at the beginning of the experiment and 5 min later ( P < 0.05; n = 3 ) . The average stimulus frequency was 24 H L . ( C ) Using a patch pipette filled with 50 iiM okadaic acid and 4 niM Mg-ATP, a nervr terminal was stimulatcd at 14 Hz with 0.1 nA, I ins pulses. Immediately after establishing the recording (upper trace), this lower frequency produced fewer failures 10 s into the train compared with that of A. Five minutes later, after okadaic acid had time to diffuse into the terminal, the samc stimulus protocol elicited many more failures 10 s inro the train (lower trace). ( D ) T h r kaction of stimuli that failed to evoke acdon potentials is shown at the beginning of the experiment and 5 min later ( P < 0.05: n = 5). The average stimulus frequency was 19 11% [from Bielefeldt and Jackson (1994b) with permission].

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reduced activity of the BK channel so that action potentials did not fail during long bursts (Figs. 8A and 8B). The next step will be to test these enzyme inhibitors for effectiveness on stimulus-induced secretion of neuropeptides. Such experiments will more clearly define the physiological relevance of these enzymatic processes. The nerve terminal membrane of the posterior pituitary also contains a delayed rectifier type of Kt channel with a conductance of 27 pS. Because the macroscopic current through this channel was relatively small, it was difficult to detect. However, this channel could be readily distinguished in cell-attached patches on the basis of its slow activation kinetics and failure to inactivate (Bielefeldt et al., 1992). Furthermore, the channel was blocked by 20 nMdendrotoxin in a highly selective manner. Interestingly, only half of the nerve terminals tested showed a dendrotoxin-sensitive component of K' current. It is tempting to suggest that this channel is confined to nerve terminals releasing only one type of neuropeptide, but dendrotoxin has been shown to enhance secretion of both vasopressin and oxytocin (Bondy and Russell, 1988).

C. NA+CHA"ELS The posterior pituitary is sufficiently large that action potentials could not propagate far into the terminal arbor without the aid of Nat channels. Recordings of compound action potentials suggested the presence of Na+ channels within the structure (Dreifuss et aL, 1971; Nordmann and Stuenkel, 1986), and optical recordings showed that the action potential reaches the ends of terminal arbors without attenuation (Salzberg et aL, 1983). The size of action potentials recorded intracellularly also indicated Na+channels within the posterior pituitary nerve terminals (Bourque, 1990;Jackson et aL, 1991). Patch clamp recordings from posterior pituitary slices directly demonstrated Na' channels in membrane excised from the larger swellings (Fig. 9) (Jackson and Zhang, 1995).Comparison of observed and simulated action potentials provided an estimate for Na' conductance density of 39 mS/cm2 (Jackson and Zhang, 1995). Since these swellings are exocytosis competent (Hsu and Jackson, 1995), these results imply little if any channel segregation, such as that suggested in rat motor nerve terminals, where Nat channels were thought to be more concentrated in the preterminal region (Brigant and Mallart, 1982; Konishi, 1985; Peres and Andrietti, 1986). Na+ currents recorded in outside-out patches were well fitted by equations of the same basic form as those used to describe Nat currents in the squid axon (Jackson and Zhang, 1995).A complete parametrization of the

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FIG.9. Voltage-activated currents in outside-out patches from posterior pituitary neme terminals. Voltage pulse sequence is indicated schematically above. (A) Total patch current. (B) Currents recorded from same patch as A following addition of 1 p M tetrodotoxin. ( C ) Nat currents were obtained as a difference between control and tetrodotoxin records. These records were fitted to m3h (Hodgkin and Huxley, 1952) to obtain the quantities plotted . plotted point was an average from three patches in D (mmand h,) and E (7, and T ~ )Each [from Jackson and Zhang (1995) with permission].

posterior pituitary Na+ current along the lines of Hodgkin and Huxley (1952) was obtained from these data for use in a model of action potential propagation. With these parameters, simulated action potentials propagated with high fidelity through virtually all realistic barriers based on morphological studies of the posterior pituitary. This result suggested that, in the absence of inhibition or usedependent depression, the branches and large swellings within the terminal do not prevent action potentials from reaching essentially every secretory element of the structure (Salzberg et al., 1983).At swellings approaching the maximum observed size, action potential propagation does not have a large margin of safety. This makes action potential propagation through large swellings very sensitive to small changes in the conductance of the membrane. The sensitivity to small depolarizations is especially acute because of the unique voltage depen-

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dence of the posterior pituitary Nat channel (Jackson and Zhang, 1995). Because the voltage at which inactivation occurred could be reached with a small depolarization, without pushing the membrane potential to the activation threshold, small depolarizations, such as those produced by GABA (see below), can strongly inhibit secretion by blocking action potential propagation (Fig. 11).

D. NEUROTRANSMITTERS Many neurotransmitters modulate stimulus-induced secretion from the posterior pituitary, including opioid peptides, dopamine, and GABA (Bondy et aL, 1989; Crowley and Armstrong, 1992; Hatton, 1990). For most of these neurotransmitters there has been little investigation of mechanism at the membrane level. However, in the case of GABA, a fairly detailed understanding has been achieved. Activation of GABAA receptors has been shown to inhibit stimulus-induced secretion from isolated posterior pituitary and from neurosecretosomes (Saridaki et aL, 1989). The GABAA recep tors mediating this inhibition have been shown to gate a C1- channel with a conductance of 27 pS (Zhang and Jackson, 1993). The conductance of this channel is essentially identical to that of GABAA receptor-coupled channels in other preparations. This nerve terminal GABAA receptor was blocked by picrotoxin and bicuculline, activated by muscimol, and potentiated by chlordiazepoxide. Thus, by standard pharmacological criteria, the nerve terminal receptor was indistinguishable from GABAA receptors in nerve cell bodies. Based on benzodiazepine sensitivityand zinc insensitivity, it has been suggested that the nerve terminal GABAA receptor contains a y subunit (Zhang and Jackson, 1995b).Although the nerve terminal GABAA receptor has much in common with postsynaptic GABAA receptors, it differs in lacking subconductance states. No evidence for GABAB receptors could be found in posterior pituitary nerve terminals (Saridaki et al., 1989; Zhang and Jackson, 1995b, 1993). The GABABreceptor agonist baclofen failed to change membrane potential or current and failed to modulate voltage-gated Ca" or K+ channels. Thus, it is very unlikely that GABAB receptors play a role in GABA-induced inhibition of secretion from posterior pituitary nerve terminals. Nerve terminal GABA responses were enhanced by physiological concentrations of neuroactive steroids (Zhang and Jackson, 1994). This result was obtained in excised patches as well as whole terminals, indicating a membrane-delimited response and implying an allosteric interaction at the GABAA receptor. This form of modulation was well established for GABAA receptors at cell bodies and dendrites (Paul and Purdy, 1992). However,

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this same action at peptidergic nerve terminals has important implications for system level endocrine function. Modulation of GABAA receptors in the posterior pituitary can explain reciprocal changes in peptide and steroid hormones that are known to occur during a number of reproductive transitions in vertebrates. For example, during the transition from pregnancy to parturition, progesterone falls and oxytocin rises. A reduction in circulating allopregnanolone, a progesterone derivative with strong action at nerve terminal GABAA receptors, would reduce the efficacy of GABAergic inhibition and thus enhance oxytocin secretion. Similar mechanisms could underlie the fluid balance symptoms of premenstrual stress syndrome and variations in sexual interest during cycles. GABA was shown to depolarize the nerve terminal membrane by 15 mV from rest (Zhang and Jackson, 1993). This was demonstrated by using the current through K+ channels in cell-attached patches to report membrane potential (Fig. 10). The reduction of current through single K' channels in cell-attached patches signals a change in membrane potential induced by GABA (Fig. 1OA). Similar results were obtained with muscimol (Fig. 10B) but not with baclofen (Zhang and Jackson, 1995a). These small GABA-induced depolarizations blocked action potentials evoked under current clamp (Fig. 10 D), suggesting that GABA could block action potential propagation. Voltage changes of this magnitude were sufficient to block current induced action potentials (Fig. 10E). A detailed analysis of currents active in this voltage range indicated that the intracellular [Cl-] was 20 mM which gave a Nernst potential for C1- of -48 mV (Zhang and Jackson, 1995a). With a resting potential estimated as -67 mV from the reversal potential of K' channels in cell-attached patches, a 15-mVdepolarization induced by GABA would bring the membrane potential to approximately -52 mV. The membrane potential in the presence of GABA represents a balance between GABA-activated C1- current and the membrane currents that maintain the resting potential. The voltage dependence of the nerve terminal Na' channel described above allows the voltage changes induced by GABA to inactivate without activating, and thus reduce the excitability of the nerve terminal. In order to test the efficacy of small depolarizations in the inhibition of secretion, a model of action potential propagation was developed with all parameters derived from experimental data on the posterior pituitary (Jackson and Zhang, 1995).It has already been noted that this model showed propagation of action potentials with high fidelity under control conditions. With GABAinduced conductance changes added only to the membrane of a swelling, propagation could be blocked by GABA (Figs. 11A and 11B). It should be noted that a higher GABA-induced conductance was required for action

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FIG.10. (A)GABA application to a nerve terminal reduced the current through single Kt channels in cell-attached patches by 1.09 ? 0.26 pA (mean ? SEM; = 7). (B)Muscimol reduced single Kt channel currents by 1.15 ? .06pA ( n = 5).The different sizes shown in A versus B reflect the different K+ channel types in the two experiments selected. The recordings shown in A were from a Ca*+-activatedKt channel [conductance = 134 (Bielefeldt et al., 1992)l;the recordings in B were from a delayed rectifier channel [conductance = 27; (Bielefeldt et al., 1992)l.The dashed lines in A and B indicate the current through the closed channel (a) and through the open channel in the absence (b) and presence (c) of 50 p M GABA or muscimol. ( C ) Single Ke channel amplitudes from A were plotted to give control (D) and GABA (A)current-voltage curves. The shifts in these current-voltage curves were used to estimate mean voltage changes induced by GABA (14.3 mV) and muscimol (13.7 mV). (D)Whole cell current clamp experiments were performed with patch electrodes filled with 25 mM KCl, 105 mM potassium-gluconate, 10 mM sodium gluconate, 10 mM HEPES, 10 mM EGTA, 4 mM Mg-ATP, pH 7.3.With this filling solution and no injected current, GABA induced a depolarization of 15.9 ? 1.3 mV (mean ? SEM; n = 5).Injecting a 250 PA current pulse for 2 ms generated an action potential before GABA application, but not while GABA was present. After GABA removal, the membrane potential recovered, and current injections once again generated action potentials. E Depolarization alone prevented action potential generation. The membrane was depolarized under current clamp by injecting steady positive current. Action potentials were generated with current pulses as in D. A depolarization of 7 mV did not block action potentials, but a depolarization of 16 mV did. Similar results were obtained in five terminals for the experiments of both D and E [Reprinted with permission from Zhang and Jackson (1993).GABA activated chloride channels in secretory nerve endings. Science 259, 531-534. Copyright 1993 American Association for the Advancement of Science].

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FIG.11. Voltage versus time is shown for simulations of action potential propagation down the axon and through a spherical swelling. Action potentials were initiated with 0.3 n A of current in the left most compartment for 0.2 ms. Because depolarizing Ga altered the membrane potential from rest, the simulations were run for 25 ms prior to action potential initiation to allow the membrane to stabilize. Numbers above traces indicate output from the locations in the diagram above. Gu was added to the swelling membrane only. The swelling diameter was 10 p m for all simulations of this figure. (A) With a depolarizing G,, reversing at -48 mV, 1.58 mS/cm2 reduced the action potential amplitude in the swelling, but did not block propagation. (B).Increasing C,, to 1.6 mS/cm2 blocked propagation. (C)With a shunting G,, reversing at the resting potential, 2.35 mS/cm2 reduced the action potential amplitude in the swelling, but did not block propagation. (D) Increasing G,I to 2.37 mS/ cm2 blocked propagation [from Jackson and Zhang (1995) with permission].

potential block when only shunting of the membrane was allowed and there was no alteration of the membrane potential (Figs. 11C and 11D). Thus, the maintenance of a C1- Nernst potential positive to rest improves the inhibitory efficacy of GABA. To block simulated action potentials (Fig. 11), the GABA-induced conductance had to exceed GABA responses observed experimentally. However, propagation block could be achieved with this model under two

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conditions, which may be relevant to different physiologicalmodes of inhibition (Jackson and Zhang, 1995). Neuroactive steroid-potentiated GABA responses were large enough to block propagation at large swellings. Thus, when focal GABA release at a single large swelling is coincident with high circulating neurosteroid, a strong inhibition of secretion should result. Without neurosteroid GABA could still inhibit secretion, but not if release was highly localized. Release of GABA within a large region could block propagation by activating receptor along a 0.5-mm long segment of axon. These two conditions for propagation block probably represent very different physiological modes of presynaptic inhibition. Under conditions in which circulating neuroactive steroid concentrations are high, such as pregnancy and the luteal phase of the menstrual cycle, the effectivenessof focal GABA in the inhibition of neurosecretion could be very important in providing a stringent control of neuropeptide secretion. At the other extreme, in the absence of neuroactive steroid, intense activity of GABAergic fibers would be necessary to allow GABA to spill out of synaptic regions and produce a cloud of GABA over a large volume. In addition to GABA, opioid peptides and dopamine also modulate secretion from the posterior pituitary (Bondy et aL, 1989; Crowley and Armstrong, 1992; Hatton, 1990). The actions of opioid peptides are mediated by a K receptor and the actions of dopamine are mediated by both D, and D2 receptors. V. Ciliary Ganglion

The ciliary ganglion of the chick receives cholinergic inputs from the ipsilateral accessory oculomotor nucleus. During synaptogenesis the oculomotor axons innervating ciliary neurons form large calices that partially envelop the postsynaptic cell (Landmesser and Pilar, 1972). As the synapse matures the calix breaks up into a cluster of small boutons, but the large size of the intermediate calix can be exploited to record ionic currents through the presynaptic membrane. Direct recording from these presynap tic calyciform nerve terminals with simultaneous recording from the postsynaptic ciliary cell revealed both electrical and chemical synaptic transmission (Martin and Pilar, 1963). A. CA2+ CHANNELS Whole-terminal patch clamp recording has revealed Ca2+current in ciliary ganglion nerve terminals. The presynaptic Ca2+current was blocked

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by cadmium and reduced by monotoxin GVIA (Stanley and Atrakchi, 1990; Stanley and Goping, 1991; Yaw0 and Momiyama, 1993). In contrast, dihydropyridines had no effect; nifedipine failed to reduce the presynaptic CaZt current and BayK produced no enhancement. A small component of w-conotoxin GVIA-resistant Ca2+current was proposed to represent a second presynaptic CaZt channel (Yawo and Momiyama, 1993). The wconotoxin GVIA-sensitive Ca2+channel makes the predominant contribution to synaptic transmission in the ciliary ganglion (Stanley and Atrakchi, 1990), but when synaptic transmission was enhanced by elevated extracellular [Ca2+]and Kt channel blockade, the w-conotoxin GVIA-resistant Ca2+ channel could also support detectable synaptic transmission (Yawo and Chuhma, 1994). Biophysical characterization of presynaptic Ca2+current in this preparation has been difficult because of poor space clamp, variable health of cells, and difficulty in distinguishing between presynaptic terminals, postsynaptic cell bodies, and Schwann cells. Yaw0 and Momiyama (1993) identified cells with a gap junction-impermeable fluorescent dye, ion currents, and simultaneous intracellular recording of postsynaptic response. These recordings showed large-amplitude Ca2+currents indicative of good health. Presynaptic Ca2+current peaked at 0 mV and declined sharply with increased depolarization. The current had a relatively high threshold of -30 mV and activated in a narrow voltage range. Replacement of CaZt by Ba2+reduced current through CaZtchannels. Furthermore, a weak inactivation of Caz+ current was reduced by Ba2+replacement, suggesting that inactivation was related to Ca2+entry. Ca2+current seen in the presence of oconotoxin GVIA showed stronger inactivation during depolarizing pulses as well as steady-state inactivation when holding potential was varied between -120 and -80 mV. Inactivating CaZfcurrent was also recorded in lizard ciliary ganglion nerve terminals (Martin et aL, 1989).The inactivation described by Yaw0 and Momiyama (1993) was not observed by Stanley and co-workers (Stanley and Atrakchi, 1990; Stanley and Goping, 1991). Although the experimental details of cell identification and health could explain these discrepancies, it is also possible that other as yet unidentified experimental conditions also make a difference. Single Ca2+channel currents have been recorded in ciliary ganglion nerve terminals in cell-attached patches (Stanley, 1991). CaZt channels could be detected only in the transmitter-release face of the terminal adjacent to the postsynaptic cell. Ca2+channels were absent from the external face. Furthermore, patches were found to contain clusters of Ca2' channels, and it is tempting to speculate that these clusters are situated near the active zones of secretion, as has been described above for motor nerve terminals and squid. In simultaneous measurements of singlechannel activ-

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ity and acetylcholine release, a correlation was noted between the acetylcholine signal and single-channel events. Based on this correlation, it was suggested that the opening of a single presynaptic Ca2' channel can evoke release of a single acetylcholine-filledvesicle (Stanley, 1993).A close association of Ca2' channels and the secretion apparatus would be essential for this to take place. The channel conductance of 11-14 pS in 110 mM barium indicates a similarity with N-type Ca" channels. This fits with the wconotoxin GVIA sensitivity of the presynaptic Ca2+channel (Stanley, 1991). However, Ntype Ca2' channels in other systems show more inactivation compared to the ciliary ganglion presynaptic Ca2' channel. Thus, this Ca2+channel has properties that set it apart from the Ca2' channels characterized in the cell bodies of other neurons. The Ca2' channels in the oculomotor nucleus neurons from which the ciliary calices originate have yet to be characterized.

B. OTHERCHANNELS There has been relatively little work on other aspects of the excitability of the calyciform nerve terminals of ciliary ganglion. In a voltage clamp study of K' current, partial block was obtained with TEA and with cobalt (Bennett and Ho, 1992). It was proposed that the TEA blocked current through a delayed rectifier type K+ channel. Cobalt reduced current through a Ca2'-activated K+ channel indirectly by preventing Ca2+entry through Ca2+channels. A Ca2'-activated K+ channel was also invoked to explain small hyperpolarizing voltage fluctuations (Fletcher and Chiappinelli, 1992b). Based on observations that these spontaneous events could be blocked by TEA and increased in frequency with caffeine, it was suggested that a transient increase in intracellular Ca2' gated a small number of K' channels to hyperpolarize the membrane. An inwardly rectifying cation channel has been inferred from voltage responses to hyperpolarizing current pulses (Fletcher and Chiappinelli, 1992a). This current was blocked by Cs+ and has many properties in common with the H current described in nerve cell bodies. The channel is activated by hyperpolarization to produce an excitatory current carried by both K' and Na'.

C. NEUROTRANSMITTERS Recordings from the calyciform nerve terminals of ciliary ganglion have revealed direct actions of adenosine, substance P, and opioid peptides.

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Adenosine was shown to inhibit synaptic transmission by reducing secretion in ciliary ganglion (Bennett and Ho, 1991). Voltage clamp studies in the nerve terminal showed that adenosine reduced Kf current. Since the adenosine response resembled the block of K+ current by cobalt, it was pointed out that this effect could be the result of adenosine modulation of Ca2+ current (Bennett and Ho, 1992).Adenosine also blocked stimulus-induced intracellular [Ca2'] changes measured with fura-2 (Yaw0 and Chuhma, 1993), and this effect was prevented by an A, receptor antagonist. A weak reduction of Ca2' current was also observed and it is unclear how such a small effect on current could produce such a large effect on intracellular [Ca2+].Because no effect of adenosine on intracellular [Ca2'] was observed following treatment with wconotoxin GVIA, it was proposed that adenosine selectively reduces the o-conotoxin GVIA sensitive Ca2+channel. Opioid peptides activate receptors on both the nerve terminals and cell bodies of oculomotor neurons (Chiappinelli et al., 1993).A K opioid receptor agonist was found to hyperpolarize the nerve terminal membrane (Fletcher and Chiappinelli, 1993). Based on the voltage dependence and Cst sensitivity of this effect, it was concluded that K opioid receptor activation caused inhibition of the inwardly rectifying cation channel described previously (Fletcher and Chiappinelli, 1992a). In the cell bodies from which the ciliary nerve terminals originate the opioid peptide responses were quite different, indicating a segregation of function between cell bodies and nerve terminals (Chiappinelli et al., 1993). In contrast, cell bodies and nerve terminals responded similarly to substance P. VI. Goldfish Retina Bipolar Cells

Bipolar (oncenter) neurons in the retina of goldfish receive rod and cone photoreceptor input. These signals are transmitted electrotonically along 25-35 pm lengths of axon to nerve terminals 8-12 pm in diameter (Ishida et al., 1980).In response to light, oncenter bipolar neurons undergo a graded depolarization of approximately 10-20 mV, leading to enhanced release of excitatory amino acid transmitter (Tachibana and Okada, 1991). Enzymatic dissociation of goldfish retina yields bipolar neurons with intact morphology so that patch electrode recording from the terminal bulb is possible (Tachibana and Kaneko, 1987). A. CA~'CHANNELS Depolarizing pulses applied either to the cell body or the nerve terminal of bipolar neurons activate Ca2+current that can be blocked by cadmium

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(Heidelberger and Matthews, 1992) as well as low concentrations of nickel and cobalt (Tachibana et al., 1993). The current depends on extracellular Ca2+(Heidelberger and Matthews, 1992) and is increased by replacement of Ca2+by strontium or barium (Tachibana et al., 1993). This Ca2+channel is sensitive to dihydropyridines but is completely insensitive to wconotoxin GVIA (Heidelberger and Matthews, 1992; Tachibana et al., 1993). BayK enhances the bipolar nerve terminal CaZtcurrent, nitrendipine reduces it, and nifedipine and nicardipine block it completely. The pharmacological properties described above support classification of the bipolar neuron Ca2+channel as L type. These studies together with the biophysical properties indicate that this channel is the only type of Ca2+channel found in bipolar neurons (Heidelberger and Matthews, 1992; Tachibana et al., 1993).Virtually no Ca2+channel inactivation was observed in the bipolar neurons, even when the current was carried by Ca2+ions (Fig. 12A). The bipolar neuron Ca2+channel is activated at voltages 1020 mV more negative than is typical for the L-type Ca2+channels of cell bodies (Heidelberger and Matthews, 1992) (Fig. 12B).The lack of inactivation results in virtually identical current-voltage plots when currents o b tained with voltage steps and voltage ramps were compared (Fig. 12C). A slightly higher threshold reported by Tachibana et al. (1993) compared with that reported by Heidelberger and Matthews (1992) probably reflects a voltage difference between the cell bodies from which the recordings of the former study were made and the nerve terminal membrane through which the current flowed. The threshold for Ca2+current activation is just above the resting potential of the bipolar neuron, so that the 10- to 20-mV depolarization caused by illumination elicits substantial Ca2' entry. The lack of inactivation of the Ca2+current means that secretion will continue for as long as the depolarization initiated by photoreceptor activation is sustained. Using catfish horizontal cells to detect transmitter release (excitatory amino acid), an excellent correspondence was observed between Ca2+ current and secretion (Tachibana et aL, 1993). Using capacitance to follow secretion as change in area of the nerve terminal membrane, it was shown that CaZccurrent can produce a very brief transient of high Ca2+concentration necessary for triggering exocytosis (von Gersdorffand Matthews, 1994). The low affinity of the CaZt trigger mechanism and the observation of secretion in the presence of slow Ca2+chelators once again imply close proximity between Ca'+ channels and active zones. Measurements of cytoplasmic Ca2+concentration with fura-2 showed that Ca2+changes are faster and larger in the nerve terminal compared to those in the cell body. Local application of Ca2+and CaZt channel antagonists provided additional support for a higher density of Ca2' channels in the nerve terminal membrane (Heidelberger and Matthews, 1992).Imaging

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FIG.12. Calcium current in nerve terminals of goldfish retina bipolar neurons was recorded under voltage clamp. (A) Pulses from -60 to -10 mV activated inward Ca2’ current which showed essentially no inactivation during the 150 ms pulse. (B) Peak current from pulses such as those shown in A plotted versus voltage (no leak subtraction). (C)Voltage was ramped at a rate of 100 mV/s to give the smooth CaZt current--voltage plot shown. Current evoked by voltage pulses in the same cell is shown with triangles [from Heidelberger and Matthews (1992). Reprinted with the permission of The Physiological Society].

of fura-2 fluorescence showed that depolarizing pulses produce much larger Ca2+changes in the nerve terminal. When the axon was severed, Ca2+ current virtually disappeared in recordings from the cell body (Tachibana et al., 1993). These experiments suggest that Ca*+ channels are almost exclusively localized to the nerve terminal and absent from the cell body.

B. NEUROTRANSMITTERS Amacrine cells release GABA onto the nerve terminals of bipolar neurons. GABA activates a C1- channel in bipolar neurons, and the strongest

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responses were observed when GABA was applied to the nerve terminal. This observation together with the recording of GABA responses in membrane patches excised from the nerve terminal membrane indicate a nerve terminal location for the GABA receptors (Tachibana and Kaneko, 1987). These studies showed that the nerve terminal GABA receptor could be desensitized by sustained application of saturating concentrations of GABA. Within approximately 1 s, the response decayed to 20-30% of peak. Activation of the receptor is cooperative, with a Hill coefficient of 2.6 and apparent affinity of 2.6 p M (Ayoub and Matthews, 1991). Although the biophysical nature of this response resembles responses mediated by GABAA receptors, the pharmacological signature differs significantly. One standard GABAA receptor antagonist, picrotoxin, blocked C1- current elicited by GABA, but another standard GABAA receptor antagonist, bicuculline, had no effect (Matthews et al., 1994). Related pharmacological anomalies have been noted for GABA-activated C1- channels in bipolar neurons from retina of other species (Feigenspan el al., 1993; Qian and Dowling, 1993). The pharmacological profile of this receptor can be traced to the p subunit, which exhibits retina-specific expression (Cutting et al., 1991; Shimada et al., 1992). The unique properties of this nerve terminal GABA receptor contrast strikingly with those of the rat posterior pituitary, where the GABAA receptor is very similar to receptors in nerve cell bodies (Zhang and Jackson, 1993, 1995b). However, the properties that made the GABA receptor in bipolar nerve terminals unique were also observed in GABA receptors in retina neuron cell bodies (Feigenspan et aL, 1993; Qian and Dowling, 1993), indicating these to be retina-specific rather than nerve terminal-specific characteristics. The unique pharmacology of the retina GABA receptor prompted a proposal of classification as GAB& rather than GABAA receptors. However, because of the similar functional properties, and the sequence homology of the p subunit with other subunits of the GABAAreceptor gene family, the classification of the retina GABA-gated C1- channel as a subtype of the GABAAreceptor is preferable (Matthews et al., 1994). In an entirely different action at the nerve terminals of bipolar neurons, GABA reduced voltage-activated Ca2+current and blocked depolarizationinduced increases in intracellular [Ca*+] (Heidelberger and Matthews, 1991).A similar response had been described in nerve terminals of amphibian bipolar neurons (Maguire et al., 1989). This response to GABA could only be seen when GTP was included in the intracellular solution, indicating that coupling between receptor and channel depended on a G protein (Heidelberger and Matthews, 1991). These mechanistic features show a strong resemblance to responses of nerve cell bodies to baclofen (Dolphin and Scott, 1987). But in bipolar neurons the nerve terminal GABA response

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was not mimicked by the GABAB receptor agonist baclofen, nor was it blocked by the GABAB receptor antagonist 2-hydroxysaclofen (Heidelberger and Matthews, 1991).Thus, this receptor appears to be a novel retinal variety of GABAB receptors. It is interesting that both of the presynaptic GABA receptors operate by classical ionic mechanisms but exhibit novel pharmacological sensitivities. Activation of the nerve terminal GABAB receptor reduces voltage-activated Ca2+current, but this inhibition can be overcome by larger depolarizations (Heidelberger and Matthews, 1991; Matthews et al., 1994). Thus, bipolar neurons could continue to relay visual responses to retinal ganglion cells if the stimuli became stronger. For this reason it is likely that the GABAB receptors of bipolar nerve terminals play a role in visual adaptation (Matthews el al., 1994). Substance P, metenkephalin, and somatostatin are also released by amacrine cells, and these substances reduce Ca2+entry into bipolar nerve terminals in much the same way as GABA, with a positive shift in the current voltage curve (Ayoub and Matthews, 1992). As was found with the GABAinduced reduction in Ca2' current, the effect of substance P was dependent on GTP and could be blocked by GDPPS. Dopamine exerts the opposite effect on nerve terminal Ca2+current and enhances depolarization-induced increases in cytoplasmic Ca2+ (Heidelberger and Matthews, 1994). This action was mimicked by forskolin and a membrane-permeable CAMPanalog, suggesting that dopamine exerts its action by activating adenylate cyclase.

VII. Honorable Mention

The literature reviewed in this chapter illustrates the remarkable impact certain preparations with unique technical advantages have had in advancing our understanding of presynaptic excitability. Work in these preparations is likely to continue to provide valuable insights, but each has significant limitations, and new systems for electrical recording from nerve terminals could easily accelerate progress. It is thus well worth evaluating studies that describe new preparations for presynaptic electrophysiology. Multinucleate fused PC12 cells produce very large growth cones following treatment with nerve growth factor. Microelectrode recordings from these growth cones revealed TEA-sensitive K+ current and cadmiumsensitive Ca2+current (O'Lague et al., 1985). Single channel recordings from patches excised from these growth cones revealed Na' channels, Ca2'activated K+ channels, and delayed rectifier K+ channels.

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In gerbel cochleus, efferent nerve terminals on hair cells have been patch clamped. These recordings revealed large conductance Ca*+-activated K' channels that could be blocked by TEA, charybdotoxin, and aminoglycoside antibiotics (Takeuchi and Wangemann, 1993;Wangemann and Takeuchi, 1993). There has been a preliminary presentation of patch clamp recordings from mossy fiber terminals in the hippocampus (Markram et aL, 1992). While this preparation could be of immense value in characterization of channels, dissociation separates the presynaptic and postsynaptic cells so that evaluation of the roles of the channels in secretion cannot be as easily carried out. In slices of the hippocampus, many investigators have succeeded in visualizing the mossy fiber terminals, but none have been able to patch clamp them consistently. Perhaps investigators should pursue the development of extracellular recording techniques of presynaptic electrical signals in mammalian brain slices. TEA has been shown to broaden the presynaptic spike volley in the CA1 field of the hippocampus. These studies also showed no change in presynaptic spikes in association with long-term potentiation (Laerum and Storm, 1994). Auditory brain stem contains large nerve terminals, and investigators have recently obtained promising results in both rat and chick. In rat brain stem slices containing the superior olivary complex, large nerve terminals called calices of Held have been patch clamped. These recordings revealed action potentials and large, brief afterhyperpolarizations (Forsythe, 1994). Voltage clamp recordings revealed a tetrodotoxin-sensitive Na' current and a 4AP-sensitive K+ current. In chick brain stem slices VIIIth nerve afferents on nucleus magnocellularis neurons contain large nerve terminals that have been patch clamped (Sivaramakrishnan and Laurent, 1994). Ca2' currents in these nerve terminals appeared to flow through two distinct channels that could be blocked selectively by w-conotoxin GVIA and wagatoxin IVA. Since each of these blockers reduced excitatory postsynaptic current, both Ca" channels appear to contribute to synaptic transmission.

VIII. Summary and Conclusions

Based on functional characterizations with electrophysiological techniques, the channels in nerve terminals appear to be as diverse as channels in nerve cell bodies (Table I ) . While most presynaptic Ca2+channels superficially resemble either N-type or Ltype channels, variations in detail have necessitated the use of subscripts and other notations to indicate a nerve terminal-specific subtype (e.g., Wang et d., 1993).Variations such as these

TABLE I IONCHANNELS IN NERVE TERMINAL MEMBRANES Rodent motor newe

Lizard motor nerve

Squid stellate

Rat posterior pituitary

Chick ciliary ganglion

Ca2+channels

One or two L like and/or N like

L type

p type

Both L like and N like

N like but noninactivating

Ca2'-activated K+ channel

Charybdotoxin sensitive

Two types: charybdotoxin sensitive and apamin sensitive

Present

Charybdotoxin insensitive

Present

Delayed rectifier K' channel

Fast and slow

Fast and slow

Present

Dendrotoxin sensitive

A-current K+ channel Acetylcholine (muscarinic)

Goldfish retina bipolar cells L like

Present

1Ic.

GABA

GABA,

T~CI

GABA, J I c a

Purine Other

FMWamide

Opioid Dopamine

A, Substance P Opioid

Substance P Opioid Dopamine Somatostatin

Note. This table summarizes many of the results reviewed in this chapter, indicating which channel types have been identified in which preparation. Presynaptic receptors are also indicated.

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MEYER B. JACKSON

pose a serious obstacle to the identification of presynaptic channels based solely on the effects of channel blockers on synaptic transmission. Pharmacological sensitivity alone is not likely to help in determining functional properties. Crucial details, such as voltage sensitivity and inactivation, require direct examination. It goes without saying that every nerve terminal membrane contains Ca2+channels as an entry pathway so that Ca2' can trigger secretion. However, there appears to be no general specification of channel type, other than the exclusion of T-type Ca2+channels. T-type Ca" channels are defined functionally by strong inactivation and low threshold. Some presynaptic Ca2+channels inactivate (posterior pituitary and Xenopus nerve terminals), and others have a somewhat reduced voltage threshold (retinal bipolar neurons and squid giant synapse). Perhaps it is just a matter of time before a nerve terminal Ca2' channel is found with both of these properties. The high threshold and strong inactivation of T-type CaZtchannels are thought to be adaptations for oscillations and the regulation of bursting activity in nerve cell bodies. The nerve terminals thus far examined have no endogenous electrical activity, but rather are driven by the cell body. On functional grounds, it is then reasonable to anticipate finding T-type Ca2+channels in nerve terminals that can generate electrical activity on their own. The rarity of such behavior in nerve terminals may be associated with the rarity of presynaptic T-type Ca2+channels. In four of the five preparations reviewed in this chapter-motor nerve, squid giant synapse, ciliary ganglion, and retina bipolar neurons- evidence was presented that supports a location for Ca2+channels that is very close to active zones of secretion. All of these synapses secrete from clear vesicles, and the speed and specificity of transduction provided by proximity may be a common feature of these rapid synapses. In contrast, the posterior pituitary secretion apparatus may be triggered by higher-affinity Cast receptors and lower concentrations of Ca2+ (Lindau et al., 1992). This would correspond with the slower performance of peptidergic secretion, but because of the large stimuli needed to evoke release from neurosecretosomes, the possibility remains that the threshold for secretion is higher than that reported. While the role of Ca2+as a trigger of secretion dictates a requirement for voltage-activated Cast channels as universal components of the presynaptic membrane, the presence of other channels is more difficult to predict. Depolarizations caused by voltage-activated Na' channels activate the presynaptic Ca2+channels, but whether this depolarization requires Na+ channels in the presynaptic membrane itself may depend on the electrotonic length of the nerve terminal. Variations in density between motor nerve terminals may reflect species differences in geometry. The high Na' chan-

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nel density in the posterior pituitary reflects the great electrotonic length of this terminal arbor. Whether Na+ channels are abundant or not in a presynaptic membrane, K+ channels provide the most robust mechanism for limiting depolarization-induced Ca2+entry. K+ channel blockers enhance transmission at most synapses. In general, K+ channels are abundant in nerve terminals, although their apparent lower priority compared to Ca2' channels in the eyes of many investigators leaves us with fewer detailed investigations in some preparations. Most nerve terminals have more than one K+ channel type, with different activation kinetics and pharmacological sensitivities. One of the presynaptic K' channels is often a classical delayed rectifier which presumably functions in action potential repolarization. Every nerve terminal examined thus far contains some variety of Ca2+activated K+ channel. Since the activation of a K+ channel will reduce excitation, this may reflect a widespread mechanism of limiting neurosecretion. The presence of Ca'+-activated K+ channels ensures a close link between the triggering of secretion and the limiting of secretion. The presence of other types of K+ channels with delayed rectifier or A-current characteristics appears to vary widely among nerve terminals and probably reflects other forms of usedependent modulation.

Acknowledgments

I thank Drs. Dean Smith, George Augustine, and Gary Matthews for many helpful discussions and comments.

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Smith, D. O., and Lu, Z. (1991).Adenosine derived from hydrolysis of presynaptically released ATP inhibits neuromuscular transmission in the rat. Neurosci. Lett. 122, 171-173. Smith, S. J., Buchanan, J., Osses, L. R., Charlton, M. P., and Augustine, G. J. (1993). T h e spatial distribution of calcium signals in squid presynaptic terminals. J. Physiol. (London) 472, 573-593. Smith, D. O., Conklin, M. W., Jensen, P. J., and Atchison, W. D. (1995). Decreased calcium currents in motor nerve terminals of mice injected with serum from patients with LambertEaton myasthenic syndrome. J. Physiol. (London) in press. Stanley, E. F. (1991). Single calcium channels on a cholinergic presynaptic nerve terminal. Neuron 7,585-591. Stanley, E. F. (1993). Single calcium channels and acetylcholine release at a presynaptic nerve terminal. Neuron 11, 1007-1111. Stanley, E. F., and Atrakchi, A. H. (1990). Calcium currents recorded from a vertebrate presynaptic nerve terminal are resistant to the dihydropyridine nifedipine. Aoc. Null. Acad. Sci. U.S.A. 87, 9683-9687. Stanley, E. F., and Coping, G. (1991). Characterization of a calcium channel in a vertebrate cholinergic presynaptic nerve terminal. J. Neurosci. 11, 985-993. Stuenkel, E. L. (1990). Effects of membrane depolarization on intracellular calcium in single nerve terminals. Brain Res. 529, 96-101. Tabti, N., Bourret, C., and Mallart, A. (1989). Three potassium currents in mouse motor nerve terminals. pfuegers Arch. 413, 395-400. Tachibana, M., and Kaneko, A. (1987). y-aminobutyric acid exerts a local inhibitory action on the axon terminal of bipolar cells: Evidence for negative feedback from amicrine cells. Pmc. Natl. Acad. Sci. U.S.A. 84, 3501-3505. Tachibana, M., and Okada, T. (1991). Release of endogenous excitatory amino acids from ON-type bipolar cells isolated from the goldfish retina. J. Neurosci. 11, 2199-2208. Tachibana, M., Okada, T., Arimura, T., Kobayashi, K, and Piccolino, M. (1993). Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina. J. Neurosci. 13, 2898-2909. Takeuchi, S., and Wangemann, P. (1993).Aminoglycoside antibiotics inhibit maxi-Kf channel in single isolated cochlear efferent nerve terminals. Hear. Res. 67, 13-19. Thompson, S. M., Capogna, M., and Scanziani, M. (1993). Presynaptic inhibition in the hippocampus. Trends Neurosci. 16, 222-226. Thorn, P. J., Wang, X.M., and Lemos, J. R. (1991). A fast, transient Kt current in neurohypophysial nerve terminals of the rat. J. Physiol. (London) 432, 313-326. Umbach,J. A., Gundersen, C. B., and Baker, P. F. (1984). Giant synaptosomes. Nature (London) 311, 474-477. von Gersdorff, H., and Matthews, G. (1994).Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature (London) 367, 735-739. von Spreckelsen, S., Lollike, K., and Treiman, M. (1990). Ca" and vasopressin release in isolated rat neurohypophysis: differential effects of four classes of Ca'+ channel ligands. Bruin Res. 514, 68-76. Wang, G., and Lemos, J. R. (1992). Tetrandrine blocks a slow, large-conductance, Ca'+activated potassium channel besides inhibiting a non-inactivating Ca2+current in isolated nerve terminals of the rat neurohypophysis. P'uegers Arch. 421, 558-565. Wang, G., Thorn, P., and Lemos, J. R. (1992a). A novel large-conductance Ca"-activated potassium channel and current in nerve terminals of the rat neurohypophysis. J. Physiol. (London) 457, 47-74. Wang, X., Treistman, S. N., and Lemos, J. R. (1992b). Two types of high-threshold calcium currents inhibited by omega-conotoxin in nerve terminals of rat neurohypophysis. J. Physiol. (London) 445, 181-199.

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Wang, X., Treistman, S. N., and Lemos, J. R. (1993). Single channel recordings of N-type and L-type Ca2+currents in rat neurohypophysial termina1s.J Neurophysiol. 70, 1617-1628. Wangemann, P., and Takeuchi, S. (1993). Maxi-K+channel in single isolated cochlear efferent nerve terminals. Hear. Res. 66, 123-129. Wheeler, D. B., Randall, A., and Tsien, R. W. (1994).Roles of N-type and Qtype Ca2+channels in supporting hippocampal synaptic transmission. Science 264, 107-1 11. Wiegand, H., Uhlig, S., Gotzsch, U., and Lohmann, H. (1990).The action of cobalt, cadmium and thallium on presynaptic currents in mouse motor nerve endings. Neurotoxicol. Teratol. 12, 313-318. Yawo, H., and Chuhma, N. (1993).Preferential inhibition of omega-conotoxin-sensitivepresynaptic Ca2' channels by adenosine autoreceptors. Nature (London) 365, 256-258. Yawo, H., and Chuhma, N. (1994). monotoxin-sensitive and -resistant transmitter release from the chick ciliary presynaptic terminal. J PhysioZ. (London) 477, 437-448. Yawo, H., and Momiyama, A. (1993).Reevaluation of calcium currents in pre- and postsynaptic neurones of the chick ciliary ganglion. J. Physiol. (London) 460, 153-172. Yazejian, B., Meriney, S. D., and Grinnell, A. D. (1993). Simultaneous patch clamp recordings of presynaptic calcium currents and postsynaptic responses at a vertebrate synapse. SOC. Neurosci. Abst. 19, 255.4. Zhang, S. J., and Jackson, M. J. (1993). GABA activated chloride channels in secretory nerve endings. Science 259, 531-534. Zhang, S. J., and Jackson, M. B. (1994). Neuroactive steroids modulate GABA, receptors in peptidergic nerve terminals. J. Neuroendocrinol. 6, 533-538. Zhang, S. J., and Jackson, M. B. (1995a). GABAAreceptor activation and the excitability of nerve terminals in the rat posterior pituitary. J. Physiol. (London) 483, 583-595. Zhang, S. J., and Jackson, M. B. (1995b). Propertes of the GABA, receptor of rat posterior pituitary nerve terminals. J. Neurophysiol. 73, 1135-1144.

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MONOAMINE NEUROTRANSMlllERS IN INVERTEBRATES AND VERTEBRATES: AN EXAMINATION OF THE DIVERSE ENZYMATIC PATHWAYS UTILIZED TO SYNTHESIZE AND INACTIVATE BlOGENIC AM1NES

B. D. Sloley and A. V. Juorio' Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 and 'Neuropsychiatric Research Unit, Department of Psychiatry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 413

I. Introduction 11. A Critical Review of Methods Used for the Analysis of Catecholamines, Indoleamines, Their Metabolites, and Related Enzymes in Biological Tissues 111. Distribution of Monoamines in Invertebrates and Vertebrates A. Acoelomates B. Pseudocoelomates C. Protostomes D. Deuterostomes N. Synthesis of Monoamines in Invertebrates and Vertebrates A. Tyrosine Hydroxylase B. Tryptophan Hydroxylase C. Aromatic L-Amino Acid Decarboxylase D. Dopamine-P-hydroxylae V. Storage, Release, and Reuptake of Monoamines VI. Catabolism of Monoamine Neurotransmitters in Invertebrates and Vertebrates A. Invertebrates B. Vertebrates VII. Conclusions References

I. introduction

Monoamines, also referred to as biogenic amines or simply amines, occur in the central and peripheral nervous systems of both invertebrates (Florey, 1967; Cottrell and Laverack, 1968; Welsh, 1972; Robertson and Juorio, 1976;Axelrod and Saaverdra, 1977;Evans, 1980;Brown and Nestler, 1985;Anctil, 1989;van Marle, 1989) and vertebrates (reviewedby Holzbauer and Sharman, 1972; Weiner and Molinoff, 1989; Green, 1989). These comINTERNATIONAL REVIEW OF NEUROBIOLOCY, VOL. 38

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Copyright 6 1995 by Academic Press, Inc. All rights of reproduction in any form resewed.

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pounds, which include the catecholamines dopamine (DA), noradrenaline (norepinephrine, NA) , and adrenaline (epinephrine, EPI), the indolealkylamine 5-hydroxytryptamine (serotonin, 5-HT) and the phenolamine p octopamine (OA) (see Fig. 1for structures), often act as true neurotransmitters and are associated with the regulation of specific hormonal (Smelik et al., 1983; Orchard, 1985),immunological (Brinton, 1987), and behavioral processes (Meyerson et al., 1979). In the central nervous system, these amines are found within the cell bodies, axons, and terminals of specific neurons (Dahlstrdm and Fuxe, 1964, 1965; Dymond and Evans, 1979; Bishop and O’Shea, 1982; Beltz and Kravitz, 1983). Within the brain they are released on stimulation to act at postsynaptic and/or presynaptic receptors and are often removed from the synaptic cleft by reuptake into the presynaptic terminal. In addition to their well-described neurotransmitter functions, monoamines also function as neurohormones and true hormones. The mammalian hypophyseal-portal transport of DA to the pituitary is now well established (Fuxe and Hdkfelt, 1969; Gudelsky and Porter, 1979) and potential OH

OH

HCa2NH2

I

OH

WPAMINE

NORADRENAUNE

6 mw m2a2w2

Ea2NH2

H

I

OH

p GTOPAMINE

5-HYDROXWRYPTAMINE

FIG 1. Structure of some important monoamine neurotransmitters.

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release sites for 5-HT into insect hemolymph (Davis, 1985; Sloley et al., 1986; Lange et al., 1988) have now been demonstrated. Vertebrate hormonal systems influenced by monoamines include secretion of gonadotropins (Peter et al., 1986),growth hormone (Chang et al., 1990) and prolactin (Cowen et al., 1990; Frawley and Neill, 1983). Monoamines are versatile as they can have either stimulatory or inhibitory influences on select endocrine systems. For example, DA can stimulate growth hormone release from goldfish pituitary (Chang et aL, 1990), whereas gonadotropin secretion is inhibited by DA (Peter et aL, 1992). The release of amines from nerve terminals elicits responses at target membranes by means of multiple forms of specific receptors linked to various second-messenger systems resulting in biochemical modification of the target tissues (reviewed by Nishizuka, 1986; Agranoff, 1989; Northup, 1989). In accordance with their important and intimate role in the regulation of neuroendocrine systems, monoamines have long maintained a central position in theories concerning the etiology of human psychiatric and neurodegenerative disorders including schizophrenia, depression, and Parkinson’s disease. With relevance to behavioral disorders, it has been demonstrated that a multitude of behavioral paradigms can be influenced by amines, including feeding and satiety (Curzon, 1990), aggression (Insel et al., 1990), and nociception (Richardson, 1990). In addition, chemicals which cause the selective degeneration of dopaminergic, noradrenergic, and serotoninergic neurons in mammals are well documented and are used as models for the study of human neurodegenerative diseases (Jenner et al., 1986; Singer et al., 1987). Although extensive studies of vertebrate models have provided considerable insight into how monoamines regulate neuroendocrine and behavioral systems, the sheer complexity of interactions between one monoamine and other monoamines, peptides, amino acids, steroids, and additional neuroactive compounds make interpretations of such studies difficult. As an alternative, invertebrate systems present the researcher with the advantage of studying large, single identified neurons, and they have been extensively used as models for the study of aminergic neurotransmission. However, the interpretation of studies which compare invertebrate and vertebrate tissues must take into account the fact that the biochemistry and pharmacology of invertebrate animals is very different from vertebrates. Of particular importance is the fact that although the mechanisms involved in the synthesis, storage, and release of amines appears to be quite similar throughout the vertebrate and invertebrate phyla, controversy exists concerning the catabolism of amines in a number of animals (Sloley and Downer, 1990; Sloley et aL, 1990, 1992). Furthermore, neurotoxic

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substances, including t%hydroxydopamine, l-methyl-4phenyl-l,2,3,6tetrahydropyridine (MPTP), and 5,7-dihydroxytryptamine, which are effective in vertebrates, have actions that are different and limited in many invertebrate systems (Lange et aZ., 1988; Sloley and McKenna, 1993). The importance of monoamines, both as factors involved in human nervous disorders and as compounds fundamental to the regulation of growth and reproduction in agriculturally important species, has aroused an increased desire for a precise understanding of the roles of these neuroactive substances in many diverse animals. The aim of this chapter is to provide a critical assessment of methods used to measure amines and their metabolites in biological tissues and a comparative examination of amine synthesis, storage, and degradation in invertebrates and vertebrates. Due to the extensive differences in monamine catabolism in the different phyla emphasis is on the different metabolic routes used by a number oforganisms to inactivate aminergic neurotransmitters. We believe that these differences in catabolic routes between invertebrates and vertebrates complicate attempts to pharmacologically manipulate monoamine systems in invertebrate animals with drugs developed for mammalian models and contribute to the many varied responses described. Hopefully, this chapter will provide the reader with an appreciation of how diverse monoamine metabolism is in the various metazoan phyla.

11. A Critical Review of Mehods Used for h e Analysis of Catecholamines, Indoleamines, Their Metabolites, and Related Enzymes in Biological Tissues

Prior to examining the literature regarding the presence or absence of amines and their associated metabolic pathways in various animals one must appreciate the difficulties involved in unequivocally establishing the identities of these particular compounds in biological tissues. A large variety of analytical procedures have been developed and utilized over the years to determine the presence of amines and their metabolic pathways in animals. To a great extent it is this diveristy of methods that has contributed to the many conflicting and contradictory results which exist in the literature. The ability to critically evaluate the methods used to identify amines and their metabolites is essential to providing accurate findings. Initial work, although crude by today's standards, involved the best procedures available at the time. No doubt today's methods will appear crude in the very near future. It is generally accepted, however, that those procedures which did not involve some form of solvent partition (solvent extractions or chromatographic separations) of amines and metabolites

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were prone to problems with misidentification. Even when highperformance liquid chromatographic separation is included in the procedure the misidentification of compounds is still a major problem. The identification of the presence of aminergic neurotransmitters and their metabolites has involved fluorimetric estimation of amines in tissue homogenates, radioenzymatic labeling of amines which are then separated and quantified, liquid chromatographic separation of amines followed by electrochemical or fluorimetric quantification, or gas chromatographic separation in concert with various forms of mass spectrometry. Gas chromatography in conjunction with mass spectrometry (GC/MS) is the most likely technique to provide unequivocal identification of these compounds. Due to the specificty of the GC/MS technique the use of this procedure usually gives estimates of concentrations of these substances which are lower than those of any of the other methods (Durden and Phillips, 1980;Juorio and Durden, 1984). The disadvantage of the GC/MS technique is that it is time consuming and very expensive. Alternatively, because of selectivity, low cost, and versatility, the use of high-performance liquid chromatography with electrochemical detection (HPLC/ED) has become the method of choice when estimating and identifylng biogenic amines and their metabolites. Unfortunately, HPLC/ED is not without disadvantages. Because of previously published, less stringent interpretations of complex HPLC/ED chromatograms from novel animals, it is now more difficult to exclude the presence of a substance from among many unknown peaks than it is to confirm its presence. The misidentification of compounds resulting from less than critical interpretations of chromatograms is a recurring problem. For example, by altering separation and detector conditions we recently demonstrated that the measurement of large amounts of NA sometimes reported in gastropod tissues may be artifactual (Sloley et al., 1990). In addition, reports of NA in the vertebrate retina which were determined by means of radioenzymatic or HPLC methods have now been disputed through use of gas chromatographic-negative ion chemical ionization mass spectrometry (Macfarlane et aL, 1989). Identification of compounds by means of HPLC requires careful evaluation of the presumed substance and authentic standards under a number of separation conditions. In addition, an examination of the way the compound behaves under varied detector conditions also aids in confirming the identification of biological substances. It must be realized, however, that it is not always possible to establish compound identification by altering detector characteristics alone. For example, when using electrochemical detectors, the acidic, y-glutamyl and N-acetylated metabolites of DA and 5-HT have almost identical electrochemical characteristics in some systems (Sloley et aL, 1990). Studies in which unusual concentrations of

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amines or their metabolites are reported and HPLC separation and detection conditions are not manipulated should be evaluated with caution. When less selective systems, such as fluorimetric or radioenzymatic methods, are used without the selective separation of products, specificity and misidentification problems are dramatically increased (Williams et al., 1987). Histofluorescence technique has been used to demonstrate the presence of catecholamines and indolamines in nervous tissue of vertebrates and invertebrates for some time (Dahl et al., 1962; Falck and Owman, 1965; Frontali and Norberg, 1966; Matus, 1973; Tansey, 1980). While it is an excellent method for establishing the neuroanatomy of these systems, the technique is limited and often cannot distinguish between different catecholamines or indolamines and lacks fine resolution. Recently, immunohistochemistry has provided an excellent method for the determination of the neuroanatomy of aminergic systems in vertebrates and invertebrates. Interestingly, this technique indicates that the immunoreactive amines are not only found at the terminals of neurons but are also found in the axons and cell bodies, indicating that amine synthesis occurs throughout the cell. When used in conjunction with other analytical techniques, such as HPLC/ ED, agreement between immunohistochemical and quantitative analyses can often be seen (Sloley et aL, 1986). Immunohistochemical techniques do have their limitations. For example, although specific antibodies for tryptamine which have relatively little cross-reactivity with 5-HT exist, the shear preponderance of 5-HT in most nervous tissues makes it difficult to resolve a tryptamine-specific signal (Dabadie and Geffard, 1993). This difficultywith cross-reactivityapplies to both immunohistochemical localization and radioimmunoassays. Studies investigating the activity of enzymes capable of synthesizing or catabolizing amines also encounter problems with the specific identification of metabolic products, especially when the separation and identification of labeled or tagged products is not carefully performed. Despite almost two decades of report to the contrary, the assumption that monoamine oxidase (MAO) is responsible for the catabolism of monoamines in the nervous tissue of all animal phyla pervades the literature (see Ui-Tei et al., 1994; Hetherington et al., 1994). Almost all early work regarding the measurement of MA0 activity involved a one-step extraction which separated the labeled acidic metabolites resulting from MA0 activity from their parent amines. The possibility that other catabolic routes of amines might also produce relatively acidic or hydrophyllic and extractable metabolites was often dismissed. Other techniques used for evaluating MA0 activity, including measurement of enzymatic products such as H 2 0 2and aldehydes or oxygen consumption, have now been demonstrated to be relatively

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nonspecific. The result is that “MAO” activity has been demonstrated in a number of tissues in which it has been later found to be either absent or, at most, a minor catabolic route. The advent of HPLC/ED has permitted the simultaneous analysis of a number of products of amine catabolism including sulfates, N-acetylated amines, and y-glutamyl and sugar conjugates in biological tissues (Battelle et ul., 1988; Kennedy, 1978; Sloley et uL, 1990; Sloley and Goldberg, 1991; Sloley, 1994). Since the substrates for these reactions can be specificallylabeled and the resulting labeled products resolved by HPLC, very selective studies of amine catabolism can now be performed. With the implementation of such techniques a number of previously ignored metabolic pathways have gained acceptance as the major catabolic routes for amines in a number of animals. Once the presence of a monoamine neurotransmitter has been documented in one animal species it is usually demonstrated to be present in other closely related species. The relative concentrations of these compounds, however, are often reported to be quite different. When attempting to explain such differences in the relative concentrations of monamines between similar animals investigators often invoke the term “species differences” to account for the discrepancies between their subjects and those of other researchers. While one may be tempted to discount this reasoning as a convenient way of dismissing technical difficulties, striking differences in the concentrations of monoamines do exist in the tissues of relatively closely related animals. For example, monoamine concentrations in the brain and pituitary of goldfish ( Curussius uurutus) and garfish (Lepisosteus osseus) collected and measured at the same time and with the same equip ment are substantially different (Table I). The same holds true when comparing monoamine concentrations in the nervous tissues of certain invertebrates. For example, Hymenopteran insects, such as bees and wasps, can have very different monoamine profiles in their nervous tissues (Table 11).

111. Distribution of Monoaminas in Invertebrates and Vertebrates

A. ACOELOMATES

Aminergic neurotransmitters have been reported to be present in a number of primitive metazoans including the tissues of various coelenterates (Venturini et ul., 1984; reviewed by Martin and Spencer, 1983; Anctil, 1989;Carlberg and Anctil1993). DA has been identified in the hydromedusan Cnidarian Polyorchis penicillutus (Chung et al., 1989) where it appears to act as an inhibitory neuromodulator (Chung and Spencer 1991). The

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TABLE I CONGENTRATIONS OF NORADRENALINE, DOPAMINE, AND ~-HYDROXYTRWTAMINE I N THE BRAIN AND PITUITARY OF THE GOLDFISH ( CARAssius Auurus) AND THE GAR(LEPISOSTEUS OSEUS) DETERMINED AT THE SAME TIMEON THE SAMEEQUIPMENT Tissue

Species

Noradrenaline

n

Dopamine

SHydroxytryptamine

ng/g Hypothalamus

6

1127 5 69

494 C 21

528 t 16

6

795 t 66

2052 C 199

2322 C 182

Carassius auratus

6

1471 t 144

154 t 12

240 t 5

Lepisosteus

6

359 2 23

703 t 155

585 ? 90

Carmsius auratus @isosteus osseus

Telencephalon

0sseu.s

pg/cLg protein ~~

Telencephalon

Carassius auratus Lepisosteus

~

~

14

ND

5

ND

0.1

0.4 t 0.1

41.6 t 5.4

3.8 C 0.6

2.4

?

osseus

Note. Values are the means 2 the standard errors based on n determinations. ND, Not detected. Procedures are identical to those described in Sloley et al. (1992).

concentrations of DA determined in nerve-rich tissue are quite low (0.1-0.2 femtomol/mg wet wt) suggesting low concentrations within the cells or a diffuse distribution of the DA-containing cells (Chung and Spencer, 1991). TABLE I1 CONCENTRATIONSOF DOPAMINE AND 5-HYDROXYTRWTAMINEIN THE CEREBRAL GANGLIA OF THREEHYMENOPTERAN INSECTS DETERMINED AT THE SAME TIMEON THE SAMEEQUIPMENT (%/Cerebral ganglion) Species

n

~~

Dopamine

5-Hydroxytryptamine

~

Apis mellfera

7

9.8 5 0.7

Bombus sp.

5

28.5 t 6.5

4.4 t 0.4

Vespula maculfrons

5

7.0 -C 1.5

40.3 t 6.1

2.7 t 0.6

Note. Values are the means t the standard errors based o n n determinations. Procedures are identical to those described in Sloley and Owen (1982).

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261

Using HPLC/ED Pani and Anctil(l994) reported that several body parts of the anthozoan Renilla koellikeri contain large amounts of NA, DA, 5-HT, EPI, 3,4-dihydroxyphenylalanine(DOPA), 3,4dihydroxyphenylaceticacid (DOPAC),5-hydroxyindoleacetic acid (5-HIAA),and other related metabolites. Thus, it seems that anthozoans may differ considerably in their amine complement from hydrozoans. However, exact identification of some of these substances is questionable as a number of these compounds (NA, EPI, DOPA) elute close to or within the solvent front and have retention times that can vary up to 10%. In addition, a number of unknown peaks are also observed within the reported chromatograms. Personal experience involving HPLC/ED studies of the hydromedusan Polyorchis indicate the presence of a number of unknown substances in these animals which can confound the precise identification of amines and related metabolites. A more rigid and exacting procedure is required to establish the identification of amines, their metabolites, and the unknown materials present in these animals. Using GC/MS the presence of a number of catechol and indole metabolites including 5-hydroxy-DOPA and cysteinylDOPA has been reported in sea anemones (Carlberg et aL, 1982; reviewed by Carlberg and Anctil, 1993). This discovery suggests that further amine-related neurotransmitter candidates exist in lower invertebrates. In platyhelminthes the presence of 5-HT (Chou et aL, 1972; Ribeiro and Webb, 1984; Gustafsson et al., 1985) and catecholamines (Bennet and Bueding, 1971;Bennet and Gianutsos, 1977;Gustafsson and Eriksson, 1991) has been established. In the parasitic digeneans Schistosoma mansoni and S. japonicum, 5-HT appears to be the dominant biogenic amine, whereas in Fasciola hepatica catecholamines are dominant (Bennet and Bueding, 1971; Bennet and Gianutsos, 1977; Gianutsos and Bennet, 1977). These compounds are known to affect motility, carbohydrate metabolism, and adenylate cyclase and protein kinase activities in these animals (Mansour, 1979; Tomosky et al., 1974).

B. PSEUDOCOELOMATES In more advanced pseudocoelomates, studies have indicated that amines are often concentrated in the nervous tissues. The presence of catecholamines in nematodes has been described in several species (Wright and Awan, 1978) and the presence of DA, 5-HT, and OA has been determined in the nematode Cuenmhabditis ekgans (Horvitz et al., 1982). Studies of the nematode Phocanema decipiens (Goh and Davey, 1984) indicate the presence of NA in the cephalic papillary ganglia, nerve ring, and associated nerves of this animal. Additional neuroanatomical evidence indicates that NA is

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B. D. SLOLEY AND A. V. JUORIO

a neurohormone and it is believed that this compound is involved with the ecdysial process in these nematodes (Goh and Davey, 1984).

C. PROTOSTOMES With the branching of the major phylogenetic lines into the protostomes and deuterostomes a generalized divergence in the complement of amines in the nervous tissue is observed. The aminergic neurotransmitters usually associated with protostome invertebrates are DA, 5-HT, and OA. Relatively low levels of NA are found in most of these animals (Kerkut, 1973;Robertson and Juorio, 1976; Walker and Kerkut, 1978). This observation holds true for annelid, insect, crustacean, and some mollusc central and perpheral nervous tissues. It is suggested that OA rather than NA is the aminergic neurotransmitter involved in the peripheral neurotransmission of most protostomes (Evans, 1980). High concentrations of 5-HT in the nervous tissue are known in a number of annelids including polychaetes Uost et al., 1981; Anctil et aZ., 1990),oligochaetes (Sloley, 1994),and Hirudinae (Lent, 1984). In addition, the nervous system of the earthworm Lumbncus terrestris is known to contain high concentrations of OA (Robertson, 1975; Tanaka and Webb, 1983) and DA, whereas NA concentrations are relatively low (Sloley, 1994). In the peripheral tissues of the earthworm 5-HT levels are relatively low and NA and DA are essentially absent (Sloley, 1994). High concentrations of amines have been described in the nervous and peripheral tissues of all molluscs so far examined. NA, DA, OA, and 5-HT have been found in the nervous tissue of cephalopods (Florey and Florey, 1954; Blashko and Hope, 1957; Bertaccini, 1961; Cottrell, 1967;Juorio and Killick, 1973;Juorio and Barlow, 1976; Tansey, 1980; Kime and Messenger, 1990) and the presence of DA and 5-HT has been determined in bivalves (Dahl et al., 1962; Sweeny, 1963; Satchel1 and Twarog, 1978) (Table V). With respect to gastropods, NA, DA, OA, and 5-HT have been reported in both nervous and peripheral tissues (Dahl et al., 1962;Osborne and Cottrell, 1970;Walker 1984;Juorio and Killick, 1972a;Weinreich et aZ., 1973; Straub and Kuhlmann, 1984; Franchini et aZ., 1985; Ottaviani et aZ., 1988; Sloley et al., 1990). While it is generally accepted that high concentrations of DA and 5-HT occur in gastropod tissues, controversy exists over the presence of NA (Hetherington et aL, 1994). It is our contention that the concentration of NA in gastropod nervous tissues is low (Sloley et al., 1990). Studies of arthropods which indicate the presence of DA, 5-HT, and OA in the nervous tissue of chelicerates (Batelle et al., 1988;Wong and Kaufman,

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1981; Sparks et al., 1994; Kaufman and Sloley, submitted for publication), crustaceans (Laxmyr, 1984;Juorio and Sloley, 1988;Cournil et aL,1994),and insects (Evans, 1980;Sloley and Owen, 1982) are well documented. Furthermore, it is now believed that 5-HT can act as a neurohormone in arthropods as the presence of superficial varicosities containing 5-HT has been demonstrated on some nerves of insects (Davis, 1985;Sloley et al., 1986;Lange et al., 1988) and the lobster (Beltz and Kravitz, 1983). In chelicerates, however, 5HT has rarely been found in any quantity (Sparks et al., 1994, Kaufman and Sloley, submitted for publication). Interestingly, it has been demonstrated that 5-HT is used as a neurotransmitter or neurohormone which regulates aspects of movement and posture in crustacea (Kravitz et al., 1980; Livingstone et al., 1980). One possible reason for the low levels of 5-HT determined in chelicerates may be that these animals lack a muscular extensor system for their limbs and rely instead on a hydraulic system. As a result chelicerates may have lost the requirement for 5-HT in movement and thus express 5-HT at lower levels in their nervous tissue. NA concentrations in arthropods are generally low or found in association with tissues other than the nervous system including venom glands and venoms (Owen and Bridges, 1982) and sclerotized cuticle (Czapla et al., 1988). For example, a number of Hymenopteran insects contain high concentrations of NA in their venoms (Owen and Bridges, 1982; Owen and Sloley, 1988). This observation indicates that although the nervous tissues of such insects do not appear to contain much, if any, NA they still possess the capacity to synthesize this compound in peripheral locations. Insects also incorporate enormous amounts of DA into the cuticle during sclerotization (Wright, 1987) and high levels of DA metabolites are found in insect tissues prior to ecdysis (Bodnaryk and Brunet, 1974;Hopkins et aL, 1982; Sloley and Downer, 1987). Furthermore, the ability of insect neuronal cell lines to produce L-DOPA but not DA (Ui-Tei et aL, 1994) suggests that L-DOPA is either a potential neurotransmitter in insects or that many insect cells retain the capacity to produce catechols ultimately destined for sclerotization. Recently, 5-hydroxy-DAand 6-hydroxy-DAhave been determined in the heads of Drosophila (Watson et al., 1993). Whether these compounds act as invertebrate neurotransmitters or compounds involved with cuticle sclerotization remains to be determined. Certainly the presence of a compound such as 6-hydroxy-DA in insect tissues which is known to be a substance toxic to mammalian dopaminergic neurons warrants further study. This is especially relevant to studies of neurodegenerative disorders such as Parkinsonism which has an etiology which suggests that exposure to or ingestion of environmental factors at an early age might be involved in the onset of this disease.

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B. D. SLOLEY AND A. V. JUORlO

D. DEUTEROSTOMES In contrast to protostomes, deuterostome nervous tissues are usually considered to include NA, DA, and 5-HT as their major aminergic components. Interestingly, although high concentrations of NA and DA have been found in all deuterostome nervous tissues examined so far, 5-HT appears to be absent from some echinoderms. For example, high concentrations of the catecholamines NA and DA have been determined in the nervous and gastric tissues of starfish and sea urchins (Cottrell and Pentreath, 1970; reviewed by Pentreath and Cobb, 1972;Walker, 1984). However, in contrast to vertebrates, starfish (Asteroideu) have very low or undetectable concentrations of 5-HT, whereas tryptamine levels are relatively high (Juorio and Robertson, 1977; Khotimchenko and Deridovich, 1988; Sloley and Juorio, 1990). This is thought provoking because 5-HT is considered important in regulating the development of some echinoderm embryos. In mammalian brain NA, DA, and 5-HT are the predominant amines. Tyramine, 2-phenylethylamine, EPI, OA, and tryptamine, while potentially important, are considered trace amines in mammalian nervous tissues (Boulton and Juorio, 1982). In contrast, the brains of other vertebrates, including those of amphibians, reptiles, and birds, have been reported to contain high concentrations of EPI (Anton, and Sayre, 1964; Bogdanki et al., 1963; Juorio and Vogt, 1967;Juorio, 1973, 1992). This suggests that EPI has a relatively important function in the regulation of processes important to these animals. Suprisingly, high concentrations of EPI are not found in the brains of teleost fish (Sloley et ul., 1992). As previously mentioned, biogenic amines are not relegated to an entirely central action in vertebrates. These compounds are often found in high concentrations in peripheral tissues and are associated with a number of peripheral functions in both vertebrates and invertebrates. In mammals hypothalamic DA is released into the hypophyseal-portal system and acts as a neurohormone to regulate pituitary function (Gudelsky and Porter, 1979). NA, which is a major neurotransmitter associated with the vertebrate sympathetic nervous system and is released as a neurohormone from the adrenal medulla, is also present in high concentrations in chromaMin tissues (v. Euler and Fange, 1961; Nilsson, 1990), vas deferens (Sjbstrand, 1965; Celuch and Sloley, 1988), and other peripheral tissues (Holzbauer and Sharman, 1972). Similarly, 5-HT is found in high concentrations in mast cells (Benditt and Wong, 1957; Erspamer, 1966; Celuch and Sloley, 1989).

N. Synthesis of Monoamines in Inverbbmtes and Vetiebrotes The synthesis of catecholamines, phenolamines (Fig. 2), and indolealkylamines (Fig. 3) in invertebrates and vertebrates appears to involve similar

NEUROTRANSMIITERS IN INVERTEBRATES AND VERTEBRATES

p -TYRAMINE

265

p -0CTOPAMINE

NH2

DIHYDROXYPHENYLAUNINE

1

@- @ AADC

DBH

a2a2-2

7 a 2 w 2

OH

DOPAMINE

NORADRENALINE

FIG.2. Schematic diagram of the pathway for catecholamine and phenolamine production from tyrosine. The reactions are catalyzed by the following enzymes: TH, tyrosine hydroxylase; AADC, aromatic amino acid decarboxylase; DBH, dopamine-B-hydroxylase.

mechanisms (Sekeris and Karlson, 1966; Evans, 1980). The production of both catecholamines and indoleamines involves the hydroxylation of aromatic amino acids by hydroxylases specific to either tyrosine or tryptophan followed by a relatively nonspecific decarboxylation reaction. Further modifications include additional hydroxylation, Nacetylation, or methylation steps. With regards to the biosynthesis of aminergic neurotransmitters in nervous tissue, the enzymes tyrosine hydroxylase, tryptophan hydroxy

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B. D. SLOLEY AND A. V. JUOFUO

TRYPTOPHAN

SHYDROXYTRYPTOPHAN

I

AADC

M YDROXYTRYPTAMINE FIG.3. Schematic diagram for the production of 5-hydroxytryptamine form tryptophan. The reactions are catalyzed by the following enzymes: TPH, tryptophan hydroxylase; AADC, aromatic amino acid decarboxylase.

lase, aromatic amino acid decarboxylase, and dopamine-P-hydroxylase are the most important.

A. TYROSINE HYDROXVLASE In the case of DA, tyrosine is converted to L-DOPAby means of tyrosine hydroxylase and the resulting L-DOPAis then converted to DA by means of aromatic L-amino acid decarboxylase (AADC) (Blaschko, 1973). In nervous tissue tyrosine hydroxylase is believed to the rate-limiting step in this pathway (Levitt et al., 1965) and there is little accumulation of DOPA as AADC

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rapidly converts the newly synthesized DOPA to DA. The activity of tyrosine hydroxylase is pterin dependent and appears to be closely regulated by means of phosphorylation-dephosphorylation reactions (Kaufman and Nelson, 1988). The phosphorylation reactions are dependent on calciumand cyclic AMPdependent protein kinases (El Mestikawy et al., 1988). Tyrosine hydroxylase activity can also be regulated by cyclic GMP (Roskoski, 1988). Due to the instability of the phosphorylated tyrosine hydroxylase in crude tissue extracts and the ease with whkh the natural pterin cofactor is oxidized, tyrosine hydroxylase is often difficult to measure accurately. Tyrosine hydroxylase appears to be present in all tissues which synthesize DA and the structure of this enzyme may be well conserved between distant phyla as antibodies to mammalian tyrosine hydroxylase appear to react specifically with insect (Orchard, 1990) and gastropod (Goldberg, 1995) tyrosine hydroxylase, In all immunohistochemical studies the immunoreactive tyrosine hydroxylase is found throughout the cytoplasm of immunoreactive cells. The conversion of tyrosine into DOPA has been demonstrated in the Octopus ganglia, supporting the presence of tyrosine hydroxylase in this species (Juorio and Barlow, 1973). Furthermore, the tyrosine hydroxylase inhibitor, a-methyl-ptyrosine, is effective in depleting DA in vertebrates (Spector et al., 1965), insects (Sloley and Orikasa, 1988), and molluscs (Sloley et al., 1990), which also suggests a common pathway.

B. TRWTOPHAN HWIROXYLASE The synthesis of 5-HT also appears to be very similar in vertebrates and invertebrates with tryptophan being converted to 5-hydroxytryptophan by tryptophan hydroxylase and the 5-hydroxytryptophan subsequently converted to 5-HT by AADC. Again, the hydroxlation step is the ratelimiting step in the synthesis of 5-HT (Freedman et d., 1972) and little 5hydroxytryptophan accumulates in the nervous tissue. Inhibition of tryptophan hydroxylase by 0-methyltryptophan decreases 5-HT concentrations in both vertebrates (Sourkes et al.,1970) and insects (Sloley and Orikasa, 1988). However, in contrast, pchlorophenylalanine which inhibits mammalian tryptophan hydroxylase and substantially reduces 5-HT concentrations in mammalian nervous tissue, appears to be ineffective in reducing 5-HT concentrations in insects in uivo (Sloley and Downer, 1990) although it inhibits insect tryptophan hydroxylase activity in uitro (Sloley and Yu, 1987). In addition, although pchlorophenylalanine slows the accumulation of 5HT in embryos of the gastropod Helisoma triuolvis it does not reduce the concentration of this amine (Diefenbach et al., 1995). Unlike tyrosine

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B. D. SLOLEY AND A. V. JUORIO

hydroxylase, no selective antibody to mammalian tryptophan hydroxylase has been tried on invertebrate systems and immunological comparisons of vertebrate and invertebrate tryptophan hydroxylase do not exist. Studies using in vitro techniques have been used to compare tryptophan hydroxylase activity from insect and rat nervous tissues (Sloley and Yu, 1987). This work indicates that insect tryptophan hydroxylase, like rat tryptophan hydroxylase, is pterin dependent, extremely liable, and has a molecular weight of about 54,000-60,000. Unlike rat tryptophan hydroxylase, the insect tryptophan hydroxylase has a lower pH optimum.

C. AROMATICLAMINOACIDDECARBOXYLASE: The presence of AADC activity appears to be fairly ubiquitous in the animal kingdom although properties of the enzyme may'differ in some phyla (Bowsher and Henry, 1986). The prevailing view, so far, has been that AADC is not the rate-limiting enzyme for the synthesis of DA or 5-HT (Brodie et al., 1962; Dairman et al., 1971; Bowsher and Henry, 1986). Some recent investigations, however, have suggested that AADC is a regulated enzyme and that, in consequence, its activity depends on the homeostasis of the system considered. In the rat retina, AADC activity is increased in reponse to light (Hadjiconstantinou et al., 1988) and this change is mediated by DA D, and a2receptors (Rossetti et al., 1989, 1990). In addition, changes in the activity of AADC have been observed in the rat and mouse striatum following blockade or activation of DA receptors (Hadjiconstantinou et aL, 1993; Zhu et at., 1992, 1993, 1994). These changes in AADC activity appear to be the consequence of two different mechanisms. A rapid onset mechanism involving second-messenger systems and independent of protein synthesis (Zhu et al., 1992) and a second mechanism that occurs after 2 h or at later times and is associated with de novo protein synthesis (Hadjiconstantinou et al., 1993). For a complete discussion see the review by Zhu and Juorio (1995). Insects appear to use two forms of AADC to synthesize amines (Yu and Sloley, 1987). One form appears to have a role in the formation of neurotransmitter DA and its activity can be induced to aid in providing the peripheral DA ultimately destined for cuticle sclerotization. The other form of AADC which has a substrate preference for ptyrosine may be involved in the synthesis of neurotransmitter OA. In mammals, solvents such as benzene can substantially stimulate AADC activity toward L-DOPA and this may contribute to the effects observed with solvent abusers (Juorio and Yu, 1985). In contrast, insect AADC activities are inhibited by benzene (Yu and Sloley, 1987).

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269

Among the molluscs the presence of AADC was observed throughout the central nervous system of Aplysia (Weinreich et al., 1972). The observation that starfish are able to produce NA, DA, and tryptamine, but not 5-HT Uuorio and Robertson, 1977; Khotimchenko and Deridovich, 1988; Sloley and Juorio, 1990), suggests that although these animals are able to decarboxylate amines they may no longer possess an active tryptophan hydroxylase.

D. DOPAMINE-P-HYDROXXASE The conversion of DA to NA in mammals is catalyzed by the enzyme dopamine-P-hydroxylase (Kaufman and Friedman, 1965). Insects and gastropods contain little, if any, NA in their nervous systems (Dahl et al., 1962, Sloley and Owen, 1982; Sloley et aL, 1990). In contrast, the ganglia of cephalopods contain relatively large concentrations of NA (Cottrell, 1967; Juorio, 1971;Juorio and Killick, 1972a;Juorio and Philips, 1976). Since the formation of OA, which is found in all of these animals, also requires a P-hydroxylase and as a number of insects produce NA in their venoms (Owen and Bridges, 1982), it appears that P-hydroxylases are possessed by most invertebrates. The expression of NA rather than OA would then be expected to depend on the presence of tyrosine hydroxylase in conjunction with a P-hydroxylase in particular neurones (Robertson and Juorio, 1976). The possibility also exists that neurones which possess both an active P-hydroxylase and a selective DA uptake system could also express NA.

V. Storage, Release, and Reuptake of Monoarnines

Storage and release of monoamine neurotransmitters appear to be very similar in vertebrates and invertebrates. The nervous and neuroendocrine tissues of vertebrates (Carlsson et al., 1963; Cook et al., 1991), insects (Orchard, 1983;Meola, 1984),ascidians (Nilsson et al., 1988),and other invertebrates possess electrondense vessicles at synapses and other release sites which contain and store amines and other neurotransmitters. Immunohistochemical studies indicate that amines are synthesized throughout the neuron and are present in the cell body, axon, and dendrites (Beltz and Kravitz, 1983; Lange et al., 1988). Release of amines appears to be from localized sites, such as synapses and varicosities, suggesting that only a relatively small portion of the amine contained within the nervous tissue is secreted at any one time. Release of amines in vertebrates and invertebrates

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B. D. SLOLEY AND A. V. JUORIO

appears to be elicited by similar mechanisms and reserpine can interefere with storage of amines in mammals (Shore et aL, 1955;Holzbauer and Vogt, 1956;Bertler, 1961), molluscs (Juorio, 1971;Juorio and Killick, 1972b),and insects (Sloley and Owen, 1982). It is generally accepted that a considerable portion of the released amine is inactivated by removal from the synapse by reuptake at the presynaptic membrane. Reuptake is considered to be a significant amine inactivation process in both vertebrates and invertebrates and a number of studies in molluscs suggest that reuptake is more important than enzymatic catabolism (Myers and Sweeny, 1973; Osborne et aL, 1975). In mammals, amines that are catabolized to acids are rapidly removed from the tissue by a transport system which can be inhibited by probenecid (Sharman, 1967; Elchisak et al., 1977). As a result, concentrations of the endogenous neurotransmitter amine usually greatly exceed those of the metabolites. Reports in which nervous tissue concentrations of the neurotransmitter metabolite exceed those of the endogenous neurotransmitter should be evaluated with caution. Exceptions to this situation do exist. These include determinations of monoamine concentrations in nervous tissue which has been subjected to considerable postmortem handling without freezing, tissue which has been thawed, and tissues, such as the anterior pituitary of mammals, which are not innervated by aminergic systems but receive amines from distant sources. In addition, samples which result from tissue dialysis often have concentrations of amine metabolites which exceed those of the parent amines (Harvey et al., 1993) as the dialysates must pass through glial cells, which have grown to surround the site of implantation, prior to entering the dialysis tubing.

VI. Catabolism of Monoamine Neurotmnsmitterr in Invertebrates and Veriebrates

The catabolism of aminergic neurotransmitters in vertebrates has been thoroughly investigated. As a result of the comprehensive studies in mammals little emphasis has been placed on the catabolism of amines in invertebrates or lower vertebrates. The lack of extensive invertebrate studies has lead many researchers to make the assumption that amine catabolism in invertebrates is similar to that of vertebrates. Nothing could be further from the truth. Unfortunately, those few studies which have been conducted concerning amine catabolism in invertebrates provide a plethora of contradictory evidence. This chapter attempts to consolidate the evidence concerning monoamine catabolism in central and peripheral tissues of invertebrates and vertebrates. However, the reader must appreciate that few of

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271

the cited studies, especially those on lower invertebrates, have ever been repeated and confirmed. We suggest that any researcher attempting to examine amine catabolism in novel animals or tissues perform their own initial studies using labeled substrates and extensive separation systems instead of assuming that the literature concerning their model is correct. As extensive, careful, and reproducable research has thoroughly examined amine catabolism in mammalian models, a short review of this process should provide a basis for comparison with the alternate routes to be described later. In mammalian nervous tissue it is generally accepted that DA, NA, and 5-HT are predominantly deaminated by monoamine oxidase (MAO) which can occur in at least two forms (MAOAand MAO,). M A 0 catalyzes the conversion of amines to aldehydes which are then rapidly metabolized to the acid by an ubiquitous aldehyde dehydrogenase. DA is converted to DOPAC, NA to 3,4-dihydroxyphenylethyleneglycol, and 5-HT to 5-HIAA. For a recent review of MA0 activity see Berry et al. (1994). In the central nervous system of mammals both DA and DOPAC can also be metabolized by catechol-Omethyltransferase (COMT) respectively producing 3methoxytyramine (3-MT) and homovanillic acid (HVA) (Sharman, 1973; Guldberg and Marsden, 1975). One of the greatest problems associated with comparative studies of amine metabolism is the presence of a large number of reports which use relatively nonspecific enzymes assays to examine MA0 activity. In many cases the identification of M A 0 activity is based entirely on the ability of a tissue to produce metabolites which are isolated by crude extractions. Thus, any reaction which alters an amine from a relatively basic to a relatively acidic compound could be identified as M A 0 activity. In vertebrates this nonspecificity is academic as almost all amine metabolites result from the activity of MAO. However, recent work which involves chromatographic separation and resolves the MAOderived metabolities of amines from other related metabolites has demonstrated the presence of a number of heretofore unknown catabolic routes for amines in invertebrates. A summary of the catabolic routes for DA and 5-HT is provided in Figs. 4 and 5, respectively.

A. INVERTEBRATES 1. Some Initial Comments Concerning Monoamine Catabolism in Invertebrates The presence of a number of different enzymatic routes for monoamine metabolism which do not rely on M A 0 or COMT activity has now been established in several groups of invertebrates. Nacetylation in insects and,

D H Y D R O X Y P H E MXI ~

HOMOVANILLK:ACID

FIG.4. Schematic diagram for the catabolism of dopamine in some animals. The reactions are catalyzed by the following enzymes: MAO, monoamine oxidase; C O W , catechol-Omethyltransferase;NAT, Nacetyltransferase; PST, phenolsulfotransferae; y-GT, y-glutamyltransferase.

NEUROTRANSMITTERS IN INVERTEBRATES AND VERTEBRATES

w I

I

H

273

0(2*2-3

H

N-ACETYL 5HYDAOXURYPTAMtNE WYDROXYINDOLEACETK ACID

\ 01Z01ZmCOCH3

'"'"

I

ti MELATONIN

FIG.5. Schematic diagram for the catabolism of 5-hydroxytryptamine in some animals. The reactions are catalyzed by the following enzymes: MAO, monoamine oxidase; NAT, N acetyltransferase; yGT, y-glutamyltransferase; HIOMT, hydroxyindole-Omethyltransferase.

possibly some crustacea and y-glutamyl conjugation in gastropods, oligochaetes, and Linzulus are important examples. The reasons for the existence of these alternate pathways remain obscure. It has been suggested that since many invertebrates live in anoxic conditions and have relatively poor oxygen transport systems they have developed routes of inactivation for monoamines which avoid the high energy and oxygen requirements necessary for M A 0 activity. Alternatively, it may be that these metabolic pathways are simply more suited to the properties of the particular organs of excretion possessed by each group of animals. Unfortunately, the limited and

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B. D. SLOLEY AND A. V. JUORIO

contradictory evidence concerning amine catabolism in invertebrates that has been published does not address the excretion of amine metabolites. This makes it difficult to assess the relative importance of physiological and anatomical differences in this area. The presence of an Nacetyltransferase responsible for monoamine metabolism in insects was demonstrated over 20 years ago (Dewhurst et aZ., 1972; Nishimura et al., 1975). Recent work regarding amines and their metabolites in insects which involved measurements performed using highperformance liquid chromatography with electrochemical detection has confirmed this observation. However, as the major catabolites of DA [DOPAC, Nacetyldopamine (NADA),and y-glutamyl-DA] have similar oxidation and retention characteristics in these systems (Sloley el d.,1990; Sloley and Goldberg, 1991), the positive identification of these compounds requires effort. Figure 6, which examines DA metabolism in a tick, illustrates just how difficult it is to completely separate DOPAC, NADA, y-glutamylDA, and DA from each other and from other interfering compounds. To add to the difficulties, DOPAC, NADA, y-glutamyl-DA, and DA cannot be easily distinguished from each other on the basis of oxidation characteristics. Similar problems are encountered when examining the metabolites of 5-HT. Again, it must be emphasized that misidentification of compounds is a frequent and recurring problem when numerous standards and separation conditions are not employed. When examining tissues from a previously uncharacterized source, it is imperative that studies using labeled substrates in conjunction with a number of purified and identified standards and separation systems capable of resolving these various metabolites are used until the model system is well understood.

2 . Coelenterates Although 5-HT, DA, and NA have been reported to occur in tissues of various coelenterates (Venturini et al., 1984; reviewed by Martin and Spencer, 1983; Anctil, 1989; Pani and Anctil, 1994), there is little material concerning the route for inactivation of aminergic neurotransmitter candidates in these animals. Recently, the presence of acidic metabolites derived from biogenic amines has been reported in the sea pansy (R. koellikm’) (Pani and Anctil, 1994) suggesting that MA0 may be the major enzymatic pathway for the inactivation of amines in coelenterates. We are unaware of any studies specifically designed to measure the production of amine metabolites from either endogenous or exogenous substrates in these animals. A summary of amine catabolism in Cnidarians and other lower invertebrates is provided in Table 111.

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B

FIG.6. Chromatographic demonstration of DOPAC production in the tick Amblyomma hebraeurn. (A) Chromatograph of a standard mixture of 5 ng each of dopamine (DA), Nacetyldopamine (NADA), dihydroxyphenylacetic acid (DOPAC), y-glutamyldopamine (y-GLUT-DA),and 2.5 ng isoproterenol (ISP) as internal standard. (B) Chromatogram derived from heomlymph of saline-injected tick. (C) Chromatogram derived from hemolymph of tick 5 min after injection of DA showing a large quantity of DA and relatively little DOPAC. (D) Chromatogram derived from hemolymph of tick 30 min after injection of DA showing the almost complete removal of DA and the production of a large quantity of DOPAC. Note how closely NADA, DOPAC, and y-GLUT-DA chromotograph in this system which is optimized for the separation of these compounds (From Kaufman and Sloley, submitted for publication).

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3. Platyhelminthes Monoamine oxidase activity and its involvement in the catabolism of 5-HT in the cestode Hymenolepis diminuta has been reported (Moreno and Barrett, 1979; Ribeiro and Webb, 1984). The formation of 5-HIAA from 5-HT and no N-acetyltransferase (NAT) activity toward 5-HT has been reported in H. diminuta (Ribeiro and Webb, 1984). The possible presence of other catabolic pathways, such as sugar, amino acid, sulfate, and other conjugation processes, has not been investigated in these animals.

4. Pseudocoelomates M A 0 activity has been reported in the nematode Ascaridia galli using a relatively nonspecific assay (Mishra et al., 1983). However, more recent work has suggested that Trichostrongylus colubnfmis and Brugia pahangi metabolize monoamines by means of Nacetylation with no MA0 activity being present (Frandsen and Bone, 1987; Isaac et al., 1990) and it was postulated that some of the N-acetylated products might be incorporated into the cuticle as part of the sclerotization process (Frandsen and Bone, 1987).Thus, controversy concerning amine catabolism in nematodes exists and further work must be conducted to clarify the situation. A summary of amine catabolism in Pseudocoelomates and other lower invertebrates is provided in Table 111.

5 . Annelids a. Polychaetes. Although the presence of 5-HT has been established in the nervous tissue of Nods virens (Jost et al., 1981) and NA and DA have been reported in the brain of Ophryotrochapuerilis (Pfannenstiel et al,, 1990), the route of catabolism of these amines in polychaetes has not been investigated. The monoamine oxidase inhibitor iproniazid has been reported to increase intracellular amines in polychaetes incubated for up to 21 days in the presence of the drug (Pfannenstiel et al., 1990). However, as the increase in monoamines was measured by the rather insensitive in situ glyoxylic acid fluorescence technique and the animals did not exhibit physiological responses, such as sex changes or shedding of oocytes characteristic of elevated amine levels, the effectiveness of this treatment remains questionable. b. Oligochaetes. Little work has been performed concerning amine catabolism in oligocheates. Recently, an examination of 5-HT catabolites produced by earthworm nervous tissue was performed (Sloley, 1994). This work demonstrates the production of y-glutamyl conjugates of endogenously produced 5-HT from earthworm nervous tissue in culture and from exogenous 5-HT in vivo. Radioactive 5-HT (Fig. 7) or glutamic acid were both demonstrated to be incorporated into the putative y-glutamyl conju-

TABLE 111 ROUTESOF AMINECATABOLISM DETERMINED IN CENTRAL NERVOUS SY~TEM AND PERIPHERAL TISSUES OF SOME LOWER INVERTEBRATES Animal Cnidaria Renilla koellikeri Platyhelminthes Hymenolepis

Schistocerca

Tissue

Substrates

Products

Enzyme

Reference

various

DA, 5 H T

DOPAC, 5-HIAA, NA5HT

MAO"

Pani and Anctil (1994)

Whole body Whole body

5HT Tyramine, NA, DA, but not 5HT Tryptamine, 5 H T

5-HIAA Oxygen utilization

MA0 MA0

Ribeiro and Webb (1994) Moreno and Barrett (1979)

Radioactive metabolites in toluene

MA0

Nimmo-Smith and Raison (1968)

MA0 NAT NAT

Mishra et al. ( I 983) Frandsen and Bone (1987) Isaac et al. (1990)

YGT

Sloley (1994)

Whole body

Pseudocoelomates Ascaridia Trichastrongylus Brugia

Whole body Whole body Whole body

Tyramine, NA, DA, 5-HT DA, OA, 5 H T

Aldehydes NADA present N-acetylated mines

Annelida Earthworm

CNS and PT

DA, 5 H T

y-glutamyl DA, y-glutamyl 5 H T

Note. CNS, central nervous system; PT, peripheral tissue, DA, dopamine; 5 H T , 5hydroxytryptamine; OA, octopamine; DOPAC, dihydroxyphenylacetic acid; SMT, Smethoxytyramine; HVA, homovanillic acid; SHIAA, 5hydroxyindoleacetic acid NADA, Nacetyldopamine; NASHT, Nacetyl-5hydroxytryptamine; yGDA, yglutamyldopamine; yG5HT. yglutamyl-5hydroxytryptamine;MAO, monoamine oxidase; COMT, catechol-Omethytransferase;NAT, Nacetyltransferase; PST, phenolsulfotransferase; yGT, yglutamyltransferase. a The presence of endogenous metabolites determined. Actual enzyme assay not performed.

EL

0

[14’C (CPM)

n “41C (CPM)

I

0 u

W

I

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279

gate further supporting this observation. Little or no 5-HIAA or Nacetyl5-hydroxytryptamine (NA5-HT) was produced. This suggests that y-glutamyltransferase is the major enzyme used to catabolize 5-HT in oligochaetes (summarized in Table 111). c. Himdinea. The presence of amines in the nervous system of the medicinal leech is well established (McCaman et al., 1973). In the leech, 5-HT is known to integrate feeding behavior (Lent and Dickinson, 1984) and the concentrations of this compound are modified by a selective neurotoxin (Lent, 1984). We are unaware of any studies concerning the catabolism of amines in these animals. 6. Molluscs a. Cephalopods. The presence of DOPAC and 5-HIAA has been demonstrated directly in a number of cephalopod ganglia (Juorio and Killick, 1972b;Juorio and Philips 1976; Kito-Yamashita et al., 1990). Inhibition of MA0 produced a substantial reduction in their levels (Juorio and Killick, 1972b; Juorio and Barlow, 1973; Juorio and Philips, 1976). In contrast, neither the presence of HVA in Octopus cerebral ganglia nor its in uitro formation following incubation with labeled L-DOPAwere observed (Juorio and Killick, 1972a;Juorio and Barlow, 1973). The presence of a pargylinesensitive M A 0 activity, which could not be differentiated into either MA0 types A or B, was observed in squid ganglia (Youdim et aL, 1986). This evidence suggests that MA0 is the major catabolic enzyme involved in the inactivation of amines in cephalopods. A summary of amine catabolism in cephalopods and other molluscs is provided in Table IV. b. Bivalves. Very little work concerning amine catabolism in bivalves has been performed. The presence of M A 0 activity has been described in the nervous system and tissues of the freshwater bivalve Anodonta q p e a (Hiripi and Salanki, 1971). In addition, low M A 0 activity and the absence of NAT activity has been reported in the retractor muscle of Mytilus edulis (Hayashi et al., 1977). The freshwater clam Anodonta grandis produces 5HIAA from 5-HT injected into the foot muscle (Table V) . However, analysis

FIG.7. Chromatographic demonstration of radioactive y-glutamyl-5-hydroxytryptaminederived from radioactive 5-hydroxytryptamine injected into the earthworm Lumbricus terrstris. (A) Chromatographic traces of 10 ng each o f dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), yglutamyl-5-hydroxytryptamine(y-GLU-5-HT),and 5-hydroxytryptamine (5-HT) produced by high-performance liquid chromatography (HPLC) with electrochemical detection. (B) HPLC separation of radioactive [ 2-"C] 5-hydroxytryptamine used for injection into earthworms. (C) HPLC separation of radioactive compounds obtained from an extract of an earthworm which had been injected with radioactive [2-"C]5-hydroxytryptamine (1.5 pCi/g) 1 h prior to dissection (reprinted from Sloley, 1994, with permission).

TABLE IV RVUTESOF AMINECATABOLISM DETERMINED IN CENTRAL NERVOUS SBTEMAND PERIPHERAL TISSUES OF SVME Mouuscs AND ECHINODERMS Animal Mollusca Gastropods

Tissue

Substrates

Products

Enzyme

CNS, PT

DA, 5-HT

yGDA, yG5-HT

CNS CNS

5HT DA, 5 H T

Conjugates Absence of or low M A 0 metabolites

CNS

DA, 5 H T

DOPAC, 5-HIAA

Bivalves

Muscle

OA

Foot muscle CNS

5-HT

Cephalopods

CNS

Tryptamine, 5-HT Radioactive metabolites

Echinodermata Starfish

CNS, PT

YGT

MA0 Some MAO, no NAT

5-HIAA DOPAC, 5HIAA

DA, 5-HT

~~~~~

MA0

Nicotra (1982), Nicotm et al. (1986)

MA0 ~

~

McCaman ef al. (1985), Sloley et al. (1990). Sloley and Goldberg (1991) Goldman and Schwartz 1977) Juorio and Killick (1972a), Hayashi et al. (1977) Osborne and Cottrell (1970). Guthrie et al. (1975) Hayashi d al. (1977) B. D. Sloley (unpublished results) Juorio and Killick (1972a), Kite Yamashita et al. (1990) Youdim d al. (1986).

MA0

Radioactive metabolites

5HT, PEA ~~

Reference

~~

Note. CNS, central nervous system; PT, peripheral tissues, DA, dopamine; 5HT, Shydroxyuyptamine; OA, octopamine; DOPAC, dihydroxyphenylacetic acid; SMT, 3-methoxytyramine; HVA, homovanillic acid SHIAA, 5hydroxyindoleacetic acid; NAJM, Nacetyl dopamine; NASHT, Nacetyl5hydroxytryptamine; yGDA, y-glutamyldopamine; yG5-HT, yglutamyl-5hydroxyuyptamine; PEA, phenylethylamine; MAO, monoamine oxidase, COMT, catechol-Omethytransferase; NAT, Nacetyltransferase; PST, phenolsulfotransferase; yGT, y-glutamyltransferase.

28 1

NEUROTRANSMITTERS IN INVERTEBRATES AND VERTEBRATES

of incubations of the visceral ganglion of A. grundis with 5-HT by means of HPLC/ED demonstrates the production of only a small amount of 5HZAA. The visceral ganglion of A. grandis also produced two other unknown electrochemically active metabolites from 5-HT which are not y-glutamyl5-HT or NA5-HT (B. D. Sloley, unpublished results). This work suggest that although M A 0 activity may be involved in amine catabolism in bivalve nervous tissue, the situation is more complex than previously believed and further work may produce some interesting results. It is interesting to note that it has been postulated that routes of monoamine catabolism, which require less oxygen and energy than M A 0 may have been developed by animals which inhabit anoxic environments. The reduction in oxygen demand provided by such routes might be advantageous to animals who are oxygen limited. However, as bivalves, which often inhabit the most anoxic of environments, appear to posses M A 0 activity toward 5-HT, it is difficult to reconcile this argument. Alternatively, it could be that bivalves are an exception. Although many bivalves do inhabit anoxic regions, their welldeveloped gills may partially offset the requirement for systems which require less free oxygen. c. Gastropods. Controversy exists regarding the catabolism of DA and 5-HT in gastropods. Some work (Cardot, 1966;Osborne and Cottrell, 1970; McCaman and Dewhurst, 1971; Marsden, 1973; Osborne, 1976) has suggested that M A 0 or COMT activity or the metabolites of monoamines formed by the activity of these enzymes could be found in extracts of gastropod brain, heart, and other tissues. However, other work has indicated a general lack of acidic metabolites and low MA0 activity in gastropods (Kerkut et al., 1966;Juorio and Killick, 1972a; Guthrie et al., 1975; Hayashi et aL, 1977). Later work suggested that in Aplysia 5-HT was catabolized by

TABLE V EFFECT OF INJECTION OF 5-Hkl)ROXYTRWTAMINE(5-HT) (5 p G / G , 1 H ) INTO THE FOOTMUSCLE OF THE FRESHWATER BWALUE ANODONTA GRANDIS ON CONCENTRATIONS OF 5-HMIROXWRWTAMINEA N D SOME RELATED METABOLITES IN THAT TISSUE Treatment

n

5-HT (pg/g)

5Hydroxyindoleacetic acid

Nacetyl5-HT

y-Glutamyl5-HT

Saline 5-hydroxytryptamine (5 CLg/g, 1 h)

6 6

2.8 t 0.4 95.6 f 14.7

0.09 L 0.02 1.6 t 0.3

ND ND

ND ND

Note. Values are the means ? the standard errors based on n determinations. Procedures are identical to those described in Sloley and Coldberg (1991). ND, Not detected, detection limits are less than 50 ng/g. Other, as yet unidentified, metabolites resulting from 5-HT injection were observed in the chromatographic traces.

282

B. D. SLOLEY AND A. V. JUORIO

a conjugation reaction (Goldman and Schwatrz, 1977). Recently, it has been determined that y-glutamyl conjugation is the major catabolic route of amines in tissues from a number of gastropod species (McCaman et al., 1985; Sloley et al., 1990; Sloley and Goldberg, 1991). 7. Arthropods a. Chelicerates i. Merostomata. The catabolism of OA and tyramine in the visual system of Limulus has been carefully examined (Battelle et al., 1988). This work suggests that the major catabolic route for amines in this tissue is yglutamyl conjugation. As recent studies of oligochaete annelids and of gastropods also indicate that y-glutamyl conjugation is the major catabolic route for amines in these animals, there appears to be no reason to dispute the determination of this metabolic route in merostomes. A summary of amine catabolism in Limulus and other arthropods is provided in Table VI. ii. Arachnids. MA0 activity has been measured in the ticks BooPhilus microplus (Holden and Hadfield, 1975) and Amblyomma hehaeum (Wong and Kaufman, 1981). Recent work has demonstrated that injection of DA into adult A . hebraeum results in a rapid conversion of DA to DOPAC (Fig. 6). There is no production of NADA and little if any y-glutamyl-DA is present (Kaufman and Sloley, submitted for publication). The conversion of DA to DOPAC in the tick can be inhibited by the MAOB inhibitor deprenyl (100% inhibition at doses of 5-10 pg/g) much more effectively than the MAOAinhibitor clorgyline (100% inhibition at doses exceeding 300 pg/g) suggesting that tick MA0 may have some properties of the Btype enzyme. In addition, when 5-HT is injected into the tick it is almost completely converted into 5-HIAA. Again this process is inhibited by deprenyl indicating that this animal uses a form of M A 0 similar to MAOBto inactivate both DA and 5-HT. Oddly enough, studies on mites suggest that OA may be N-acetylated (Sparks et al., 1994). It would be interesting to know if these animals catabolize DA and 5-HT by one enzymatic route and OA by another. 6. Crustaceans. It has been known for some time that amine catabolism in crustaceans does not involve M A 0 (Hayashi et al., 1977). One investigation suggests that inactivation of 5-HT in lobsters is by means of sulfate conjugation (Kennedy, 1978) but other work indicates that crustaceans Nacetylate amines (Hayashi et al., 1977; Dubbles and Elofsson, 1989). Other investigators have developed procedures for measuring 5-HT and 5-HIAA in lobster hemolymph (Fadool et al., 1988) but the chromatographic separation and identification of 5-HIAA (5-HIAAis in the solvent front) is tenuous. We suggest that the evidence strongly indicates that oxidative deamination is not the route of amine inactivation in crustacea. Recent work with the

TABLE VI ROUTES OF AMINECATABOLISM DETERMINED IN CENTRAL NERVOUSSEXEMAND PERIPHERAL TISSUE OF %ME ARTHROPODS Animal

Tissue

Substrates

Products

Enzyme

Reference

Merostomata Limulus

CNS

OA

y-Glutamyl OA

Y-GT

Batelle et al. (1988)

Arachnida Ticks

PT

Tryptamine, DA, 5HT

Indoleacetic acid, DOPAC, 5HIAA

MA0

Wong and Kaufman (1981); K a h a n and Sloley, submitted for publication

Crustacea Lobster Crayfish

CNS CNS

DA, 5 H T 5HT

Sulfates of DA, 5-HT, OA NA5-HT

PST, M A 0 or COMT NAT, no M A 0

Kennedy (1978) Dubbels and Elofsson (1989)

Insecta Cockroach

CNS, PT

DA, 5HT, OA

NADA, NASHT, Nacetyl OA

NAT

PT

DA

DA-3Osulfate

PST

CNS PT PT CNS CNS CNS

5HT 5HT Benzylamine DA, 5 H T 5HT DA, 5 H T

5HIAA NA5-HT, sugar conjugation Benzoic acid NADA NA5-HT NASHT HVA, 5HIAA

MA0 NAT MA0 NAT NAT MA0,COMT

Sloley and Downer (1984, 1990). Downer and Martin (1987), Murdock and Omar (1981), Hayashi et al. (1977) Bodnaryk and Brunet (1974), Sloley and Downer (1987) Pandey and Habibulla (1980) Trimmer (1985) El-Sebae et al. (1979) Dewhurst et al. (1972) Evans and Fox (1975a,b) Nagao and Tanimura (1988)

Flies Drosophila Honey bee Cricket

Note. CNS, central nervous system; PT, peripheral tissue, DA, dopamine; 5HT, Bhydroxytryptamine;OA, octopamine; DOPAC, dihydroxyphenylacetic acid; 3MT, 3methoxytyramine; HVA, homovanillic acid; SHIAA, 5hydroxyindoleacetic acid NADA, Nacetyl dopamine; NASHT, Nacetyl-5hydroxytryptamine; y-GDA, y-glutamyldopamine; yCSHT, y-glutamyl-5hydroxytryptamine;MAO, monoamine oxidase; COMT, catechol-Omethytransferase;NAT, Nacetyltransferase; PST, phenolsulfotransferae; y-GT, yglutamyltransferase.

284

B. D. SLOLEY AND A. V. JUORIO

Pacific shore crab Hemigrapsus nudus indicates that this animal does not produce 5-HIAA, y-glutamyl-5-HT, or NM-HT from exogenously injected 5-HT, although it is capable of catabolizing this compound (B. D. Sloley, unpublished data). c. Insects. Insects provide a special case when examining amine catabolism. Insects use catecholamines, phenohmines, and indoleamines as neurotransmitters, components of venoms, and as tanning agents in cuticle and egg case sclerotization. Thus, the demand for amines and their metabolites varies between tissues and between developmental stages. Amine catabolism in insect nervous tissue and adult peripheral tissues has occasionaly been attributed to M A 0 (El Sebae et al., 1979; Pandey and Habibulla, 1980; Flanagan, 1984; Nagao and Tanimura, 1988). Careful evaluation of these papers suggests, however, that assays for M A 0 or the presence of 5-HIAA were not specific enough to resolve acidic metabolites from N-acetylated compounds. In fact, none of these authors reported attempting to determine whether N-acetylated compounds could interfere with their assays. Other much more extensive work demonstrates that Nacetylation is the major route of amine catabolism in the nervous tissue of a number of insects (Dewhurst et al., 1972; Evans and Fox, 1975a,b; Hayashi et al., 1977; Evans et al., 1980;Mir and Vaughan, 1981;Murdock and Omar, 1981; Sloley and Downer, 1984,1990; Downer and Martin, 1987;Trimmer, 1985;Martin and Downer, 1989; Linn et al., 1994).The presence of N-acetylated metabolites of 5-HT in insect tissues is transient and once N-acetylated 5-HT appears to be sugar conjugated (Trimmer, 1985; Sloley and Downer, 1990). Although large amounts of NA, DA, and 5-HT are present in hymenopteran venom (Owen and Bridges, 1982; Owen and Sloley, 1988), an examination of the possible catabolism of these compounds by the venom gland and associated tissues has not been attempted. Involvement of DA and its related metabolites, NADA, hrp-alanyl-DA, and DA-3-Osulfate, in cuticular tanning has been thoroughly investigated. Prior to ecdysis DA is synthesized and stored as DA-30sulfate and other conjugates which do not appear to interact with DA receptors and are highly hydrophyllic (Bodnyark and Brunet 1974;Sloley and Downer, 1987). At ecdysis the DA-3-Osulfate is converted to other DA metabolites and, with other DAderived compounds, is incorporated into the cuticle. Some of these DA metabolites include DOPAC (Barrett, 1990), NADA quinone (Sugumaran et al., 1988), UP-alanyldopamine (Hopkins et al., 1982), and N-P-alanylnorepinephrine(Czapla et al., 1988). In some insects sugar conjugates of amines, benzoic acid, and diphenols are involved in the production of egg cases by the colleterial gland (Takahashi, 1971; Lake et al., 1975; Kawasaki and Yago, 1983). The complex genetics of amine metabolism in sclerotization and melanization of Drosophila have been reviewed (Wright,

NEUROTRANSMITTERS IN INVERTEBRATES AND VERTEBRATES

285

1987) and this work indicates that at eclosion, amine metabolism can involve the use of amine oxidation, Nacetylation, /3-alanyl conjugation, sugar conjugation, and/or a number of other processes. Selective incorporation of these tanning elements results in the particular melanization patterns characteristic of different insect species. 8. Echinoderms All work concerning amine catabolism in echinoderms suggests that monoamine oxidase is the principal enzyme involved (Nicotra, 1982; Nicotra et al., 1986; summarized in Table IV). 9. Chmdata Several studies have indicated the presence of 5-HT (Welsh and Loveland, 1968; Sakharov and Salimova, 1982; Nilsson et al., 1988) in ascidians but we are unaware of any work concerning the catabolism of this amine in these animals. The catecholamines NA and DA, but not the indoleamine 5-HT, have also been reported in the cerebral ganglion of Ciona intestinalis (Osborne et al., 1979), but again there are no catabolic studies.

B. VERTEBRATES As previously mentioned, in contrast to invertebrates the catabolism of monoamines in mammalian vertebrates has been well studied. The general concensus is that oxidative deamination by MA0 is the predominant route for monoamine inactivation in all vertebrate nervous tissue. Lesser pathways resulting in Omethylation and/or sulfation of dopamine are also present in vertebrates and the synthesis of melatonin from 5-HT requires Nacetylation. No vertebrate nervous tissue has been shown to use y-glutamyl conjugation, sulfation, N-acetylation, or sugar conjugation as the primary route of amine inactivation. Although M A 0 is used be all vertebrates, there are subtle and interesting variations in monoamine catabolism between lower and higher vertebrates.

1. Lower Vertebrates In teleost fish the catabolism of DA is the subject of some controversy (Winberg and Nilsson, 1993). The majority of evidence suggests that a single form of MA0 with properties similar to MAOAis responsible for the catabolism of DA (Figueroa et al., 1981; Edwards et al., 1986). The metabolites produced by teleost nervous tissue or pituitary in vivo and in vitro are DOPAC and to a much lesser extent 3-MT and HVA (Saligaut et al., 1992; Dulka et al., 1992;Sloley et al., 1992).A number of researchers have claimed

286

B. D. SLOLEY AND A. V. JUOFUO

that COMT rather than MA0 is the major route of DA inactivation in the nervous tissue and pituitary of certain teleosts (Saligaut et al., 1990; Nilsson, 1989). Unfortunately, these claims are difficult to substantiate as later work performed by the same authors contradicts this proposal. For example, Saligaut et al. (1990) reported concentrations of 3-MT in trout nervous tissue which approach those of DA. In contrast, they later report that 3MT concentrations are undetectable and that the only measurable DA metabolite is DOPAC (Saligaut et aL, 1992). To add to the confusion, work by Nilsson (1989) claimed that HVA is the major DA metabolite found in carp brain with levels (75-125 ng/g) often approaching or exceeding those of DA. In contrast, the same author (Nilsson, 1990) reported brain HVA concentrations of 10-20 ng/g in the brain of the same species of fish. In this work these HVA concentrations are only 10% those of DA. These studies suggest that effects such as those described in mammals where postmortem COMT activity results in elevated 3-MT and HVA concentrations (Westerink and Spaan, 1982) must be carefully considered. The continued postmortem activity of COMT may be especially relevant when working with poikilotherms. Studies suggest that the two M A 0 isoenzymes (MAO, and MAO,) exist in terrestrial tetrapods but only one form (MAO, like) can be detected in teleosts and aquatic amphibia (Figueroa et aL, 1981; Hall and Uruena, 1983; Edwards et aL, 1986). In a number of tissues of the premetamorphic bullfrog MAO, activity predominates. During metamorphosis, however, there is an increase in MA0 activity due, predominantly, to an appearance of MAO, activity (Hall and Uruena, 1983). Overall the evidence indicates that DA and 5-HT catabolism in lower vertebrates is similar to that of mammals except that teleosts and aquatic amphibia do not express much MAO, activity. However, since MPTP, a dopaminergic neurotoxin which appears to require MAOBactivity to be active (Lewin, 1984), is effective in fish (Poli et al., 1990; Sloley and McKenna, 1993) it may be that teleost M A 0 possesses properties of both MAOAand MAOBas suggested by Yoshino et al. (1984). The catabolism of amines in lower vertebrates is reviewed in Table VII.

2. Birds and Mammals It has been demonstrated that vertebrates use a number of enzymatic systems to inactivate aminergic neurotransmitters (Sharman, 1973; Brown et al., 1984). A summary of the catabolic routes used for monoamine inactivation in vertebrate tissues is provided in Table VII. As previously indicated, mammalian nervous tissues catabolize DA, NE, and 5-HT predominantly by oxidative deamination using MA0 (EC 1.4 :3.4, amine :oxygen oxidoreductase; deaminating, flavin containing) (Rivett et al., 1982; Yu, 1986)

TABLE VII ROUTESOF AMINECATABOLISM DETERMINED IN CENTRAL NERVOUSS'ISTEM AND PERIPHERAL TISSUE OF SOME VERTEBRATES Animal

Tissue

Substrates

Products

Enzyme

Mammals

CNS, PT CNS CNS, PT CNS

DA, 5 H T DA DA 5-HT

DOPAC, 5-HIAA 3MT, HVA DA-.%sulfate NA5-HT

MA0 COMT PST NAT

Reference Yu (1986) Rivett et al. (1982), Westerink and Spaan (1982) Yu et al. (1985) Brown et al. (1984)

Birds

CNS

5HT

5-HJAA

MA0

Hall et al. (1984)

Amphibia

CNS, PT

Phenylethylamine 5-HT

Phenylacetic acid 5HIAA

MA0

Nicotra and Senatori (1984), Senatori and Nicotra (1985)

Fishes

CNS, PT CNS, FT ' CNS Pituitaxy

DA, SHT, DA, Kynuramine DA

DOPAC, 5-HIAA .%MT,HVA 4Hydroxyquinolene DOPAC, .%MT,HVA

MA0 COMT MA0 MAO, COMT

Sloley et al. (1992), Hall aria Uruena (1983) Nilsson (1989, 1990), Saligaut el al. (1989, 1992) Figueroa et al. (1981), Edwards et al. (1986) Sloley el al. (1992). Dulka el al. (1992)

Note. CNS, central newous system; PT, peripheral tissues, DA, dopamine; 5HT, 5Hydroxytryptamine; DOPAC, dihydroxyphenylaceticacid SMT, 3methoxytyramine; HVA, homovanillic acid; SHIAA, 5hydroxyindoleacetic acid; NA5-HT, Nacetyl-5hydroxyuyptamine;MAO, monoamine oxidase; COMT, catechol-Omethyltransferase;NAT, Nacetyltransferase; PST, phenolsulfotransfere.

288

B. D. SLOLEY AND A. V. JUORIO

to produce aldehydes. The two forms of M A 0 (MAO, and M O B ) are differentiated by their substrate preferences and sensitivities toward specific M A 0 inhibitors. MAOApreferentially oxidizes 5-HT and is very sensitive to the MA0 inhibitor clorgyline, whereas MAO, preferentially oxidizes 2-phenylethylamine and is sensitive to the M A 0 inhibitor deprenyl. The conversion of DA to DOPAC can be catalyzed by either MAOAor MAOB. The aldehydes produced by MA0 activity are rapidly metabolized to the acid by an ubiquitous aldehyde dehydrogenase. In the central nervous system of mammals both DA and DOPAC can also be metabolized by COMT respectively producing 3-MT and HVA (Sharman, 1973, Guldberg and Marsden 1975). The action of COMT can produce postmortem elevations of 3-methoxytyramine concentrations in rat (Westerink and Spaan, 1982). Phenolsulfotransferase (Yu et al., 1985) is another enzyme which may have a minor role in catabolizing DA in the mammalian central nervous system. There are two major routes for 5-HT metabolism in mammalian brain, pineal gland and retina (Brown et al., 1984). Catabolism of 5-HT by MAO, is generally regarded as the predominant pathway for inactivation of 5-HT in the central nervous system (Sloley et al., 1993),whereas Rr acetylation is considered to be the rate-limiting step in the production of melatonin from 5-HT in the pineal gland (Klein and Weller, 1970) and retina (Pang et al., 1977). In addition, 5-methoxytryptamine and 5-hydroxytryptophol are other relatively minor 5-HT metabolites reported to be produced in mammalian nervous tissues. The peripheral catabolism of amines in mammals also involves extensive utilization of M A 0 but a number of additional conjugation reactions with other compounds, including sugars, have been reported (Bartlet and Gilbert, 1971). Little work has been conducted concerning amine catabolism in birds. The concensus reached, based on reviews of the literature, is that, like mammals, birds possess two forms of MA0 and, based on selective inhibition of activity, these forms correspond to MAO, and MAO, (Hall et al., 1982, 1984; Hall and Uruena, 1983). This is supported by the demonstration of DOPAC, HVA, and 5-HIAA in areas of the bird brain (Juorio and Vogt, 1967; Ahtee et al., 1970).

VII. Conclusions

It is now well established that monoaniines have diverse and important roles as both stimulators and inhibitors of cellular mechanisms. They can act as neurotransmitters, neuromodulators, neurohormones, and true hor-

NEUROTRANSMITTERS IN INVERTEBRATES AND VERTEBRATES

289

mones. This diversity of function suggests that monoamines have had a long time, evolutionarily speaking, to become involved in a large number of varied responsibilities. Indeed, in a phylogenetic sense, monoaminergic systems appear to be very old with evidence for their use now being established in nervous systems as primitive as those found in the Cnidarians. All metazoan nervous systems so far examined have a complement of biogenic amines which is distributed in particular cells characteristic of neurons. This complement of amines, however, is not consistent over the many phyla. Among the protostomes, DA, 5-HT, and OA appear to be the most prevalent amines with respect to concentrations within the nervous tissues. Evidence indicates that NA can also be synthesized by some of these animals although it is rarely found in high concentrations in the nervous tissues of most. In the chelicerates studied so far 5-HT concentrations also appear to be very low. In contrast, deuterostomes possess high concentrations of NA, DA, and 5-HT in their nervous tissues, the only known exception being starfish, whose nervous tissue appears to have relatively little, if any, 5-HT. As the adaptive loss of expression of certain amines does occur, it would be interesting to determine whether the genetic information required for the production of certain amines, such as NA in insect, crustacean, or gastropod nervous tissue and 5-HT in tick and starfish nervous tissue, is expressed at much reduced levels or exists at all. If a particular amine is to be expressed in a particular neuron the conditions involving the synthesis, storage, and release of monoamines appear to follow similar patterns among all metazoans. Slight differences are observed with respect to synthetic enzyme optima and storage and release parameters but these differences are consistent with the varied homeostatic conditions found within animals adapted to environments which contain different salt, pH, and temperature regimes. Once a monoamine is released as a chemical messenger all metazoans appear to use similar mechanisms to perceive and transduce the signal into a physiological response. Certainly, the ability of amines to affect common second-messenger systems, such as adenylate cyclase and inositol phosphate, appears to be well conserved. As expected, some specialization in the recognition of particular monoamines has occured and these receptors are now able to differentiate between stucturally similar neurotransmitters. For example, arthropod OA receptors appear to differ from mammalian NA receptors although they do have many common agonists and antagonists and probably have common phylogenetic ancestries. Such adaptations are not unexpected as they could provide optimal and selective message recognition and would be expected to occur over extended divergencies from a common ancestral stock.

290

B. D. SLOLEY AND A. V. JUORIO

By far the greatest differences between the monoamine systems determined in the various phyla are found among the enzymatic routes used for the inactivation of amines. MAO, which is the prevalent route for amine catabolism among the deuterostomes, is absent or at most a minor route in most, but not all, protostomes. Strong evidence exists for a general absence of M A 0 in annelid, gastropod, insect, and crustacean tissues. Among the protostomes only ticks and cephalopods provide strong evidence for the presence of MA0 activity and in each of these cases it is either MAOBor M A 0 with properties of both the A and B type enzyme which is present. Evidence exists that annelids, gastropods, and merostomes use yglutamyl conjugation to metabolize and inactivate their monoamines. Insects use Nacetylation and sugar conjugation for this purpose but those that use DA for sclerotization purposes also use sulfation and other conjugation reactions for storage of DA prior to incorporation into the cuticle following ecdysis. The crustaceans studied so far do not use MA0 to inactivate amines and although it has been suggested that they sulfate or N-acetylate their amines, further work must be done to resolve those contentions. The presence of diverse inactivation mechanisms for amines in invertebrate animals used as models for studies of monoamine neuronal and neuroendocrine systems has profound implications. One can no longer assume (indeed, never should have assumed) that M A 0 inhibitors, selective neurotoxins, or other pharmacological agents will work in the same manner in these animals as they do in mammals. Careful pharmacological evaluations of these invertebrate models must be conducted prior to attributing modifications of physiology or behavior to any specific aspect involving the selective manipulation of any monoaminergic system.

Acknowledgments

The authors thank The Alberta Heritage Foundation for Medical Research and Saskatchewan Health for financial support during the preparation of the manuscript.The comments and support of Dr. R. E. Peter, Dean of Science, University of Alberta are gratefully acknowledged.

References

Agranoff, B. W. (1989).Phosphoinositides. In "Basic Neurochemistry" (G. Seigel, B. Agranoff, R. W. Albers, and P. Molinoff, eds.), 4th Ed., pp. 333-347. Raven New York.

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NEUROTRANSMlllE R SYSTEMS IN SCHIZOPHRENIA

Gavin P. Reynolds Deportment of Biomedical Science, The University of Sheffield, Sheffield, S 1 0 2TN, United Kingdom

Introduction Etiology: Genetic and Environmental Neuropathology Neurochemistry of Schizophrenia A. Dopamine Systems B. Nondopamine Neurotransmitter Systems V. Dopamine A. Presynaptic Dopamine Function B. D2 Receptors C. DI Receptors D. Dopamine Dsand D4 Receptors VI. Acetylcholine MI. 5-Hydroxytryptamine A. Presynaptic 5Hydroxytryptamine Function B. Postsynaptic 5-HT2 Receptors C. Other 5 H T Receptors VIII. Noradrenergic Systems IX. GABA X. Neuropeptide Systems A. Neurotensin €3. Cholecystokinin (CCK) C. Somatostatin D. Opioid Peptides XI. Glutamate Systems A. Presynaptic Glutamatergic Markers €3. Glutamate Receptors C. IT Receptors XII. Conclusions A. A Neurochemical Pathology of Schizophrenia B. Prospects for Drug Treatment References I. 11. 111. N.

1. Introduction

For many years, the idea that schizophrenia might be understood in terms of neurochemical dysfunction has done much to stimulate research INTERNATIONAL REMEW OF NEUROBIOLOGY, VOL. 38

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into the disease. Surprisingly, early suggestions that aberrant neurochemistry may underlie schizophrenia emerged from Freud and Jung, although the effect of the development of their psychoanalytical teachings in the middle decades of this century inhibited biological approaches to the understanding of psychiatric disease. Nevertheless, the concurrent advances in chemical analytical methods and in our understanding of neurotransmitter biochemistry and pharmacology, along with the introduction of the phenothiazine antipsychotic drugs in the 1950s, provided substantial opportunity for proposing and testing neurochemical hypotheses of psychiatric disorders. Precedents were established by the application of biochemical techniques in defining several inborn errors of metabolism and in understanding the neurochemical basis of Parkinson’s disease. It was the observation that dopamine was severely depleted in the striatum of Parkinsonian patients (Ehringer and Hornykiewicz, 1960) that led directly to the initial trial of the highly successful L-3,lMihydroxyphenylalanine (L-DOPA)therapy. Certainly the question of the biological basis of schizophrenia is an important one. The disease is one of great social and economic impact. It has a lifetime incidence of a little less than 1 % and, following its appearance in late adolescence or early adulthood, a chronic course of drug treatment, often with hospitalization, is the fate of most sufferers. Key symptoms include hallucinations (particularly auditory hallucinations), delusions, and abnormal experiences, such as the feeling that control of one’s thoughts has been lost, perhaps to some outside agency. The patient loses empathy with others, becoming withdrawn and demonstrating inappropriate or blunted moods. These symptoms may vary greatly; in fact, no single core symptom is seen in all patients. The variability in symptom profile has led to the classification of subtypes of the disease, with implications in terms of differences in etiology or pathology. Thus, Crow (1980) defined two syndromes on the basis of the relative proportions of positive or negative symptoms. Patients having primarily positive symptoms (delusions, hallucinations, incongruous affect) are described as type I, while those with negative symptoms (withdrawal, loss of drive, flattened affect) have the type I1 syndrome. It is the type I syndrome that best responds to classical antipsychotic drugs, which are far less effective at ameliorating negative symptoms. Conversely, the syndrome of negative symptoms has occasionally (if inconsistently) been associated with identifiable abnormalities of the brain. There is still some dispute as to whether these subtypes relate to distinct disease processes or whether, at the other extreme, they are different expressions of a single disorder. This does not imply that the categories are entirely distinct; patients may show overlap between the two types and may change symptoms, and hence, syndrome, during the course of the disease. Subsequently, systematic study has defined three discrete syndromes, reality dis-

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tortion, psychomotor poverty, and disorganization, and neuroanatomical correlates for these schizophrenic syndromes have been identified by imaging studies (Liddle, 1987).

II. Etiology: Generic and Environmental

The symptoms seen in schizophrenia can occur in various other diseases of the brain, including epilepsy and Huntington’s disease, as well as in some metabolic disorders. Along with the wide variation of the symptoms shown by schizophrenic patients, this might suggest schizophrenia to be the product of several disease processes. Certainly, many etiological factors, both genetic and environmental, have been proposed. That the disease is partially genetically transmitted is demonstrated by a concordance of up to 50% between monozygotic twins. Environmental factors may determine whether or not the disease appears in individuals with a genetic predisposition to schizophrenia, although an alternative explanation is that either environmental or simple genetic etiologies are separately responsible in different cases. Using families in which a clear hereditary transmission is present, searches have been undertaken for genes of major effect. These have usually been initiated following single observations of families in which chromosomal abnormalities have been associated with syndromes that included schizophrenic symptoms.A report apparently linking schizophrenia with markers on chromosome 5 in seven Icelandic and British families was not subsequently reproduced in a range of other European and American pedigrees, and the original study is now considered to be a false positive result (reviewed by Owen and Mullan, 1990). Linkage to chromosome 11 has proven more positive but can still only account for a small proportion of hereditary disease. Association with genes on the pseudoautosomal part of the sex chromosomes has also been postulated, again with some supportive evidence, although in a review of genetic studies Kendler and Diehl (1993) concluded that no positive linkage of any genetic locus to schizophrenia has emerged. The dopamine D2 receptor has attracted some interest as a candidate gene, particularly since it is coded for on chromosome 11, and some recent results are mentioned under Section V.B. However, investigations of such candidate genes as possible sites of genetic abnormality in schizophrenia have generally been developed from naive hypotheses that have usually yielded negative or inconsistent data in simpler biochemical studies. This reflects the lack of evidence for a consistent, and primary, neurochemical abnormality in the disease.

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Genetic studies in schizophrenia are complicated by the difficulties of a circumscribed diagnosis; many pedigrees in which schizophrenia is prominent also contain individuals with affective disorder and other psychiatric diagnoses. Results are often sensitive to whether or not such cases are included as “schizophrenic,” although the concept of psychosis as a continuum, in which manic-depressive and schizoaffectivediseases are not genetically differentiated from schizophrenia, has proven useful in some analyses. Of the possible nongenetic causes, several pre- or perinatal events are postulated to be responsible for the development of the disease. One such correlate is the occurrence of obsteric complications, although whether this represents etiological factors or whether it reflects other developmental problems has attracted some debate (Waddington, 1993). Another proposed environmental cause is viral infection of the mother and/or fetus; it seems likely that this can contribute to a proportion of schizophrenic cases (e.g., O’Callaghan et aL, 1991). The interest in such early events as predisposing factors to the development of schizophrenia has been stimulated by the substantial accumulation of evidence for subtle morphological abnormalities of the brain in schizophrenic patients. These abnormalities appear to be established before the onset of the symptoms of the disease and provide evidence for a neurodevelopmental pathogenesis. But it is important to note that a neurodevelopmental problem predisposing to the later onset of schizophrenia need not exclude a genetic etiology of the disease.

111. Neuropathology

Over the past 10 years the application of quantitative histopathological techniques to autopsy tissue and the development of methods for brain imaging in wiuo have established evidence for the presence of neuronal deficits in schizophrenia. Indicators of such deficits have been identified primarily in the frontal cortex and the temporal lobe, regions of the brain concerned with attention, mood, social interaction, and various other complex behavioral functions that may be disturbed in schizophrenia. These abnormalities have been reviewed elsewhere (Roberts, 1991; Waddington, 1993) and include ventricular dilatation, particularly of the temporal horn of the lateral ventricles, reflecting diminished volumes of the limbic system structures in the medial temporal lobe: the amygdala, hippocampus, and parahippocampal gyrus of the entorhinal cortex. Macroscopic indicators of structural deficits in these regions are reflected by microscopic changes

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in neuronal numbers and distribution. One such finding is the abnormal clustering of cells in lower layers of the entorhinal cortex; these p r e a cells appear to have failed to migrate to the outer laminae of this medial cortical region Uakob and Beckmann, 1986), a phenomenon which has been interpreted as reflecting arrested neural development. The absence of gliosis in regions of the brain where neuronal deficits occur has also been interpreted as an indicator that the neuropathology is of developmental origin (Roberts and Bruton, 1990); neuronal damage is almost invariably associated with glial cell proliferation. Similarly, the indicators of functional deficits in the frontal cortex (hypofrontality) that have emerged from imaging of blood flow and energy metabolism in schizophrenic patients (Weinberger, 1987) appear to have neuropathological correlates in the deficits of certain types of neurons in frontal regions (e.g., Benes el al., 1991). Several neuropathological studies have indicated an asymmetry of the abnormalities in schizophrenia in which the left hemisphere is more severely affected; for example, the reported increase in lateral ventricular volume is most apparent in the left temporal horn (Crow et aL, 1989). Although many pathological and neurochemical studies of the brain have not been undertaken bilaterally, those that have do provide some evidence for the original postulate that schizophrenia is a disorder of the left temporal lobe (Flor-Henry, 1969). Only recently have neurochemical approaches been employed to provide a better understanding of these neuronal abnormalities; past neurochemical hypotheses of schizophrenia were developed primarily from pharmacological observations and cannot easily relate to neuronal pathology. While it is through their actions on neurotransmitter systems that the antipsychotic drugs bring about their effects, it is only by understanding the relationship between neuronal abnormalities and neurotransmitter systems that we shall understand how these drugs bring about a reversal of (some of) the effects of the morphological abnormalities in schizophrenia.

IV. Nwrochemistry of Schizophrenia

The history of neurochemical research in schizophrenia has reflected, with a predictable regularity, advances in neurochemistry and pharmacology that have contributed to our understanding of human brain function. Thus, the identification of the relationship between hallucinogenic drugs and monoamine neurotransmitters and the discovery of the endogenous opiates and of the neuropeptides have all given rise to new theories of

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a neurochemical etiology of schizophrenia-theories often founded on insecure premises and leading to much effort in the pursuit of artifactual results. Nevertheless, identiijrlng a neurochemical dysfunction in schizophrenia has been a major target of biological psychiatry. Apart from some hypotheses involving endogenous or exogenous neurotoxins in the brain, and occasional indications of amino acid, lipid, or other metabolic abnormalities, neurochemical research in schizophrenia has concentrated on dysfunction of neurotransmitter systems, which will be the subject of this chapter.

A. DOPAMINE SYSTEMS Dopamine has occupied a central and primary role in neurochemical and neuropharmacological research in schizophrenia. It was a combination of clinical observation and neuropharmacological studies that provided the evidence on which the dopamine hypothesis of schizophrenia was established. This hypothesis, which remains the basis both for the understanding of antipsychotic drug action and for the development of new antipsychotics, originally developed from the observations that high doses or chronic administration of amphetamine to humans can induce a psychosis indistinguishable from acute paranoid schizophrenia (Randrup and Munkvad, 1965).Amphetamine acts by increasing dopamine release; other dopamine agonists may also have similar psychotogenic effects. Further support was provided by the finding that antipsychotic drugs block dopamine receptors (Carlsson and Lindqvist, 1963) and, although other neurotransmitter receptors may also be affected by these drugs, it is their antagonist action at the D2 subtype of dopamine receptors that correlates particularly well with their clinical efficacy (Seeman et al., 1976).

B. NONDOPAMINE NEUROTRANSMITTER SY~TEMS Although the dopamine hypothesis remain central to much research into psychopharmacological mechanisms, it is becoming apparent that a primary role for dopamine in the etiology of schizophrenia may be absent. Nevertheless, neurochemical research in this area is as active as ever, stimulated by several useful hypotheses. One major source for these neurochemical theories is the recent rapid advance (discussed previously) in identifying neuropathological abnormalities in the disease; understanding and defining the neurotransmitters directly or indirectly affected by these neuronal deficits is thus a major goal of schizophrenia research.

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A second stimulus to neurochemical research in schizophrenia is that of antipsychotic pharmacology. Although established in the dopamine hypothesis, drug development for schizophrenia is now addressing actions at a range of other neurotransmitter systems (Reynolds and Czudek, 1995). As mentioned previously, D2 receptor antagonists are effective as antipsychotics, but their blockade of striatal dopaminergic function leads to unwanted motor side effects. These include parkinsonism, an acute effect that responds to lowering the antipsychotic dose or to inclusion of a muscarinic acetylcholine antagonist in the drug regimen, and tardive dyskinesia which develops chronically, affecting about 30% of patients (particularly the elderly) and for which the pathogenesis is unknown. Thus, the search for new treatments is partly driven by the need for drugs which do not cause such movement disorders. There are two further targets for antipsychotic drug development: to provide relief for those patients who do not respond adequately to treatment with classical antipsychotics and to diminish the negative symptoms that respond less well to such treatment. Several pharmacological approaches are employed in the pursuit of these targets. Greater specificity in antidopamine drugs for the limbic and cortical systems, rather than the nigrostriatal pathway, should minimize extrapyramidal symptoms, as should drugs which also bind at receptor sites that act counter to the effects of striatal dopaminergic receptors. Alternatively, the new identification of nondopaminergic receptor sites that mediate an antipsychotic action may both avoid the problem of these side effects and provide a mechanism for greater antipsychotic efficacy. Such receptors have yet to be identified. Nevertheless, there is an anomalous drug that provides a surfeit of pharmacological clues for such research. This is clozapine, which was withdrawn shortly after its introduction as an antipsychotic in the 1970s following several fatalities due to drug-induced agranulocytosis. Nevertheless, it had become apparent that clozapine offered a unique antipsychotic action, with an action on negative symptoms and what is effectively an absence of extrapyramidal side effects, most notably a lack of tardive dyskinesia following long-term treatment (reviewed by Fitton and Heel, 1990).Perhaps the most important aspect of clozapine's action (and, at present, its major clinical indication) is its efficacy in schizophrenic patients who do not respond to treatment with classical antipsychotics; approximately 50% of such patients demonstrate an improvement after 6 months of treatment with clozapine. A growing interest in its clinical potential has culminated in the recent reintroduction of the drug in several countries. The pharmacological profile of clozapine contrasts strongly with those of selective dopamine D2antagonists; its affinities for a adrenoceptors, histamine, muscarinic, and several subtypes of 5-hydroxytryptamine (5-HT) receptors all exceed that for dopamine sites. This does not exclude its

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antipsychotic effect from involving an antagonist action at dopamine receptors, although PET studies show that in tli710 it occupies approximately 50% of D2 sites which is somewhat lower than the 70-80% occupancy exhibited by other antipsychotic drugs (Farde et al., 1992). Clozapine’s pharmacology indicates several neurotransmitter systems through which it may induce its unique effects, although it should be emphasized that any involvement these systems may have in the mechanism of clozapine’s efficacy remains hypothetical. These transmitter systems can be divided into two separate groups: those that interact with dopamine systems and thereby modulate the effects of dopaminergic blockade, and those that are presumed to be independent of dopamine in relation to their role in clozapine’s action. While the various neurotransmitters implicated in this way need not necessarily have any primary involvement in schizophrenia, they have at the very least provided some direction and stimulus to neurochemical research into the disease.

V. Dopamine

A. PRESYNAPTIC DOPAMINE FUNCTION The many biochemical studies of the past 20-30 years have provided no conclusive evidence to support a general overactivity of dopamine neurons in schizophrenia. The major dopamine metabolite, homovanillic acid (HVA), has been determined in both plasma and CSF, and such measurements have been considered to reflect, at least in part, dopamine function. There is evidence from animal experiments that CSF concentrations of HVA reflect cortical dopamine metabolism (Elsworth et al., 1987), while HVA in plasma has been found to correlate with its striatal concentrations (Bacopoulos et al., 1979a). Generally, CSF HVA concentrations have been found to be diminished in schizophrenics, particularly in those patients with cerebral atrophy (Van Kammen et al., 1983; Doran et al., 1987). Although this finding has been interpreted as indicative of a dopaminergic hypometabolism occurring in patients with more negative symptoms, it is conceivable that the finding is artifactual in the sense that it may relate to the increased volume of CSF in such patients (discussed in Reynolds, 1989). Certainly, postmortem studies provide no support for this hypothesis since they show HVA to be elevated in the cortex in schizophrenics, a finding which reflects the increase in dopamine turnover in this region following chronic treatment with antipsychotic drugs (Bacopoulos et al., 1979b).

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Although the measurement of plasma HVA has not contributed much to our understanding of the neurochemistry of schizophrenia, it has provided interesting results of relevance to mechanisms of antipsychotic drug action. Treatment with antipsychotic drugs has substantial effects on plasma HVA which appear to reflect changes in brain dopaminergic activity. Following an initial increase after drug administration, in the longer term a decrease in plasma HVA concentrations can be seen which has been found to correlate with an improvement in clinical symptoms following treatment with antipsychotics (Picker et al., 1984). Since plasma HVA derives in part from subcortical brain dopamine, it is conceivable that this change reflects the development of tolerance to dopaminergic blockade in striatal and limbic brain regions which, in turn, may indicate one possible mechanism of antipsychotic effect. Are there abnormalities in subcortical dopamine systems in schizophrenia? Neurochemical studies of the brain postmortem have attempted a more direct answer to this question. Inevitably such studies have concentrated on the regions of the striatum and accumbens, in which dopamine innervation is greatest. However, few consistent changes in any presynaptic marker of dopamine systems have been identified. There have been occasional reports of increased dopamine in the caudate and accumbens nuclei in schizophrenia (e.g., Mackay et al., 1982) although this has not been observed by other groups (Reynolds, 1987). Other markers for dopaminergic terminals have not shown consistent changes in these regions; while increased activity in schizophrenia of synaptosomal dopamine uptake in caudate and accumbens nuclei has been reported (Haberland and Hetey, 1987), direct measurement of dopamine uptake sites by radioligand binding to striatal tissue was unable to identify any abnormality (Czudek and Reynolds, 1990). The regions of the striatum are primarily concerned with motor function which is not considered to be affected to any great extent in schizophrenia (Fig. 1). More strongly implicated are the mesocortical and mesolimbic dopamine pathways, innervating regions of the brain that are thought to be involved in the symptoms and neuropathology of the disease. As levels of dopamine are much lower in these regions, they have received substantially less attention, although the neuropathological findings that have emerged in recent years have served to demonstrate the potential value of neurochemical studies in medial temporal and cortical regions. Of the structures in the medial temporal lobe it is the amygdala that receives the greatest dopaminergic innervation. An elevation in dopamine concentrations in the amygdala is found in schizophrenia (Reynolds, 1983), and this effect is also observed for HVA but not for a range of other neurotransmitter markers (Reynolds, 1987). This increase is significantly greater in the left hemisphere, although other regions do not show any asymmetry in dopa-

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parahippocampal gyrus

FIG 1. Dopaminergic projections in the human brain and their relationship with sites implicated in the neuropathology and pathophysiology of schizophrenia. (Left) sagittal view of dopamine projections from substantia nigra and ventral tegmental area to ( 1 ) striatum, (2) nucleus accumbens, (3) amygdala, and (4) c o tex. ~ (Right) coronal sections illustrating striatal and medial temporal regions; dotted lines o n saggittal view indicate where these sections are taken. [Reproduced from G. P. Reynolds (1992) TrendsPhannacol. Sn. 13,116-121, with permission from Elsevier Trends Journals.]

minergic markers. It seems likely that this change may also be secondary to neuronal deficits, bearing in mind the neuropathological evidence for greater left hemispheric abnormalities mentioned previously. One result in support of this view is discussed under Section IX.

Interest in the dopamine hypothesis received a substantial stimulus in the late 1970s when it was discovered that there was an increase in the numbers of D2 receptors in brain tissue taken from schizophrenic patients at autopsy (Owen et aL, 1978). Some years of dispute over the origin of this increase in receptors followed; was it related to the disease process or was it an effect of prior drug treatment? Drugs blocking D2receptors induce, after some weeks of administration, an upregulation of D2 receptor density (Clow el aL, 1980), and it was argued that the antipsychotic treatment inevitably received by most schizophrenic patients was responsible for the increase in brain D2 receptors (Reynolds et aL., 1981; Mackay et aL., 1982). The introduction of positron emission tomography (PET) imaging studies

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had been expected to provide a consistent answer to this problem by permitting assessment of radioligand binding to dopamine receptors in untreated living patients. The first major PET study of neuroleptic-naive patients reported an increase in ["C]methylspiperone binding to D, recep tors (Wong et al., 1986); this finding was, however, not confirmed by other groups sometimes using other ligands (e.g., Farde et al., 1987).The hypothesis that the difference between these results relates to different receptor specificities of the radioligands used is discussed under Section V,D; however, many now accept that the increase in D2 receptors is primarily a response to drug treatment and probably does not occur in young, untreated schizophrenic patients. A further observation added to the interest generated by the study of D2 receptors in schizophrenia: striatal samples from about 70 different schizophrenic subjects demonstrated a bimodal distribution for D2 receptors (Seeman et al., 1984); one group had near normal levels and the other elevated receptor density. This result is open to several possible interpretations relating to the presence of drug-induced parkinsonism or dyskinesia or to subtypes of the disease process. However, in the light of the indications that increased D4receptors relate to prior drug treatment, it seems possible that only a subgroup of patients is responding to antipsychotic drugs by upregulation of D, receptors. Modern molecular biological techniques have demonstrated subtypes of the D2 receptor based on peptide length. D2 is found in a long (DZA) and a short (D2B) isoform, and a recent report found these twosforms demonstrated a difference in their affinity for antipsychotic drugs including clozapine (Malmberg et al., 1993). It is notable that the D2B isoform, with a higher affinity for clozapine, is expressed in regions associated with a somewhat lower innervation by dopaminergic neurons. Whether this might impart some regional pharmacological selectivity has yet to be seen; however, it is notable in the light of the recent observation that clozapine has a higher affinity at D2-like receptors in the human frontal cortex in comparison to its action at striatal D2 sites (Mason and Reynolds, 1994). While these findings are of potential value in understanding mechanisms of action, there is no evidence that a substantial abnormality in the distribution of the two receptor subtypes occurs in schizophrenia. Nevertheless, molecular biological studies into the D2 receptor have culminated in some interesting findings. As mentioned previously, the receptor is coded for on chromosome 11, which has been implicated in genetic aspects of schizophrenia. Some initial studies of the D2 receptor gene indicated no abnormalities associated with the disease (e.g., Sarkar et aL, 1991), nor was the D2 region associated with the chromosome 11 linkage (Su et al., 1993). However, a search for changes in the protein structure of the D2 receptor in a Japanese population revealed a point

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mutation with a higher frequency in schizophrenics, particularly those with a family history, than in a control population (allele frequency 13.5 and 1.8%,respectively) (Arinami et al., 1994). This variant, which was not found in several studies in Western populations, is on an intracellular loop of the receptor, which may have functional consequences by affecting G protein interactions. Others have suggested abnormalities of dopamine receptor G protein interactions in schizophrenia. Seeman et al. (1989) reported a reduction in the interactions between D2and D1receptors in the brain in schizophrenia (and in Huntington’s disease), interactions thought to be mediated by G proteins. The possible influence of antipsychotic drug treatment on these results cannot be totally excluded, nor is the anatomical (co)localization of the D1 and D2 receptors fully resolved (see, e.g., Surmeier et al., 1993). However, the result is interesting in the light of a report (Okada et at., 1990) of diminished G proteins in the striatum in schizophrenia.

C. D1RECEPTORS The D1 subgroup of dopamine receptors is not thought to directly mediate the antipsychotic effects of drugs used to treat schizophrenia, although several of these drugs, including clozapine, do have D1antagonist properties in addition to their Dz action. Clozapine is the antipsychotic that demonstrates the greatest relative D1 site occupancy, as shown by in vivo PET studies of a radiolabeled D1 antagonist in human brain (Farde et al., 1992);clozapine occupies 36-5276 of DI sites. Flupenthixol may displace similar proportions of ligand from these sites, although this drug is associated with substantially higher D2receptor occupancy (Farde et aL, 1992). There is no evidence that D1blockade is an important feature of clozapine’s action, although it may enhance the effects of D2 antagonism to achieve an antipsychotic response below the threshold for extrapyramidal effects. The interaction between D1 and D2 receptors is a complex one; in some animal behaviors DI facilitates Dz-mediated neurotransmission, in others the receptors work in opposition. Nevertheless, the role of DI sites in mediating dopaminergic function has indicated their potential involvement in the dopamine hypothesis. However, post-mortem studies of the receptor have not provided consistent support for any dysfunction of DI in schizophrenia. A report of diminished striatal D, receptor density in the disease (Hess et al., 1987) has not been confirmed in other investigations (Pimoule et al., 1985; Czudek and Reynolds, 1988). An earlier study indicated a functional abnormality of the DI receptor. Using agents that act directly on the second-messenger system of the D1 receptor, Memo et al., (1983)

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observed an increase in brain tissue adenylate cyclase stimulation in schizophrenia, an effect they interpreted as a facilitated coupling between the D1 receptor and the cyclase. How this might relate to the abnormal DI-D2 interaction observed by Seeman et al. (1989) is unclear but intriguing.

D. DOPAMINE D3AND D4RECEPTORS In 1990, a new dopamine receptor, termed D3,was discovered by cloning following the identification of its mRNA (Sokoloff et aL, 1990). In comparison to D2,D3generally has a higher affinity for dopamine and other agonists but a slightly lower affinity for most antagonists. Unlike the D2 receptor, found primarily in the striatal regions of the brain, the D3 receptor is expressed mainly outside the striatum, notably the mesolimbic terminal regions of the nucleus accumbens and olfactory tubercle. Since the pharmacology of D3resembles that of D2fairly strongly, at least in terms of antipsychotic drug affinity, it has been suggested that this D3 receptor may be more important in mediating antipsychotic drug action, while blockade of the D2receptor is primarily responsible for extrapyramidal symptoms. The presence of D3receptor mRNA in the substantia nigra suggests that D3may also have some autoreceptor function (Sokoloff et aL, 1990). Study of the D3 receptor is hampered by the presumably very low amounts of the protein in brain tissue; however, determination of its mRNA has yielded some interesting results supporting its proposed role in antipsychotic mechanisms. As would be expected, mRNA for D2is increased following classical antipsychotic drug administration. This is also true to a greater extent for D3 although, unlike D2, D3 mRNA is also increased following high doses of sulpiride and clozapine (Buckland et al., 1992, 1993). How closely these findings are paralleled by changes in receptor density has yet to be determined. Seeman and Schaus (1991) labeled dopamine receptors in the striatum with t3H]quinpirole, an agonist at D2and D3receptors, and observed an apparent D3component. A very recent autoradiographic study of D3receptor density in schizophrenia identified an increase, greater than that for D2,in regions of the pallidum, accumbens, and caudate, although the influence of prior drug treatment could not be excluded (Gurevich et aL, 1994). Genes for two further dopamine receptors have now been identified. D4 is another DTlike receptor, while D5 is more closely related to D,. The D4 receptor resembles the D2 and D3 proteins with approximately 40% homology and is expressed in regions that include the amygdala and frontal cortex (Van To1 et aL, 1991), parts of the brain implicated in the functional pathology of schizophrenia (see above). Furthermore, its potential impor-

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tance in antipsychotic action is particularly indicated by the fact that clozapine is reported to have a 15-fold greater affinity for D4 than for the D2 site, while other antipsychotic drugs generally have a preference for D2. Seeman (1992) pointed out that while therapeutic levels of most antipsychotics lead to substantial D2receptor blockade, clozapine alone has a low occupancy of D2 sites while blocking most D4 receptors. This is certainly cosistent with the interpretation that clozapine's efficacy is due to D4receptor blockade. It is less clear whether the strong correlation between therapeutic availability and D2 blockade for the other antipsychotics indicates the D2 site to be important in this respect; it may be that therapeutic levels of these other drugs are governed more by the threshold for the appearance of (D2-mediated) extrapyramidal symptoms than by antipsychotic efficacy. One further confounding factor is that the increased affinity of clozapine for the D4 receptor is not consistently reported; Mills et al. (1993) found a & of 53 nM in their expression system, some !%foldhigher than that reported by Van To1 et al. (1991). Recently, Seeman et al. (1993) attempted to determine D4 receptor density in human brain. Their approach involves the binding of ['Hlemonopride (to determine D2, D3, and D4 sites) from which the binding of ['Hlraclopride (selective for D2 and D3 receptors) was subtracted. If their interpretation is correct then striatal D4 receptors may be increased in the striatum in schizophrenia, since the binding of ['Hlemonopride, but not ['HI raclopride, was apparently increased. This contrasts with the increase in specific [3H]raclopride binding observed in schizophrenia by another group (Ruiz et al., 1992). Seeman et al. (1993) used these findings to provide an explanation for the differences between various research groups in the results of PET determination of DTlike receptor density in viva Thus, Wong et al. (1986), by using ["C]methylspiperone, identified elevated D4 sites in addition to DP and D3 receptors, while Farde et al. (1987), by using ["C] raclopride, only labeled the (unchanged) D2 and D3 sites. However, two attempts to replicate the study of Seeman et al. (1993) have provided no support for an elevation of D4 receptors in schizophrenia. D,-like receptors were determined in the putamen using a more conventional washed tissue preparation; by displacing ['Hlemonopride binding with raclopride (Reynolds and Mason, 1994), or by comparing the binding of [lZ5I]-epidepride(a new high affinity D2/D3 ligand) with that of [3H] emonapride (Reynolds and Mason, 1995), no D4component of D2-likedopamine receptors could be identified in either control subjects or schizophrenic patients. Other recent studies have complicated the issue still further; a polymorphism of the D4receptor gene in the human population has been identified

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(Van To1 et al., 1992). However, this polymorphism structure, which reflects differences in the number and form of a repeat sequence in the gene and, hence, the protein, does not appear to relate to differences in clozapine response (Shaikh et al., 1993). Nevertheless, while we have yet to see whether the D4 protein is functionally important in the human brain, the development of D,-specific antagonists certainly provides an approach with potential for the treatment of schizophrenia.

VI. Acetylcholine

The consideration given to cholinergic systems in schizophrenia primarily relates to the effect of antimuscarinic drugs in relieving some of the acute extrapyramidal side effects of antipsychotic drug treatment (McEvoy, 1983). A few antipsychotics have a relatively high affinity for the muscarinic acetylcholine receptor in brain tissue; for clozapine and thioridazine this is reportedly higher than their D4 receptor affinity (Richelson and Nelson, 1984) and is considered to relate to the low incidence of extrapyramidal symptoms found with these drugs (Miller and Hiley, 1974). Muscarinic blockade can lead to dry mouth and constipation. Conversely, however, excessive salivation has been reported following treatment with classical antipsychotic drugs (cited in Marsden et aL, 1986); this side effect is a major problem in patients receiving clozapine (Fitton and Heel, 1990). Although this has led to the suggestion that clozapine may be a partial agonist at muscarinic sites, which may contribute to the antipsychotic efficacy of the drug (Tandon and Kane, 1993), studies from this laboratory have failed to find any direct evidence for an agonist effect of clozapine at any muscarinic receptor subtypes (G. P. Reynolds et al., unpublished). It is known that anticholinergics may induce psychotic behavior. This may relate to the reciprocity of dopaminergic and cholinergic systems in the brain, an observation that has given support to a cholinergic hypothesis of schizophrenia. The ubiquity of cholinergic systems in the limbic structures of the brain, along with their involvement in awide range of behavioral functions, certainly indicates a possible role for acetylcholine in psychosis. Tandon and Greden (1989) reviewed some of the evidence linking cholinergic dysfunction with schizophrenia and proposed that the negative (type 11) syndrome reflects a hyperfunction of the cholinergic system(s), albeit in association with other neurotransmitter abnormalities. However, little direct supportive evidence for this hypothesis has emerged, although the paucity of neurochemical investigations into cholinergic markers and receptors in schizophrenia leaves the hypothesis inadequately tested.

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VII. 5-Hydroxyhyptamine

Since the identification of 5-HT as a neurotransmitter in the early 1950s, there has been an interest in its possible importance in schizophrenia. The initial finding that the hallucinogenic drug LSD was an effective 5-HT antagonist led to the development of hypotheses based on deficiency of 5-HT or transmethylation of endogeous arnines to form natural psychotogens. These hypotheses fell out of favor with the emergence of the dopamine hypothesis. Nevertheless, an intermittant interest in 5-HT systems in schizophrenia and its drug treatment has remained, fueled more recently by the observations that some antipsychotics have antagonist action at 5-HT receptors and that other 5-HT receptor antagonists are effective in certain animal models of antipsychotic action. However, in contrast with the dopamine hypothesis, no correlation between antipsychotic efficacy and 5-HT receptor antagonism has been shown for these drugs. It is perhaps notable that some animal behaviors induced by amphetamine which have, in the past, been equated to psychosis are mediated by 5-HT receptor systems (Sloviter et al., 1980).

A. PRESWAPTIC 5-HYDROXYTRWTAMINEFUNCTION Some reports have emerged of abnormalities in 5-HT and its major metabolite 5-hydroxyindoleaceticacid (5-HIAA)in schizophrenia. Low concentrations of 5-HIAA have been observed in CSF from schizophrenic subjects, an effect also seen for the major dopamine metabolite HVA (Section VJ). This is not specific for schizophrenia; the association of low 5-HIAAconcentrations in CSF with “violent” suicide attempts is a replicated finding that has long provided evidence for an abnormality of 5-HT function in (some cases of) depression (&berg et a[., 1976). The apparent influence of suicide and depression on the findings for schizophrenia is not easily clarified. Furthermore, it is conceivable that the effect of increased ventricular volume, a well-established finding in schizophrenia, may be to reduce metabolite concentrations in CSF, as for HVA under Section V,I. Certainly, CSF levels of 5-HIAA are also found to be reduced in schizophrenic patients with increased ventricular size (Potkin et uZ., 1983), although these authors suggest that low 5-HIAA is an independent indicator of frontal cortical dysfunction. Postmortem studies have not provided consistent evidence for a deficit of brain 5-HT and/or 5-HIAA in schizophrenia. An early study from Joseph et al. (1979) provided no indication of such abnormalities in several brain

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regions, and while others have reported significant increases in 5-HT in the lentiform nuclei (Korpi et al., 1986), the finding was not specific in that it was also observed in nonschizophrenic suicides. Whatever change there might be in neurotransmitters and/or metabolites, it is not easy to distinguish an effect secondary to abnormalities in other transmitter systems from primary changes in innervation. The assessment of transmitter uptake sites, however, may provide a measure of the integrity of presynaptic terminals. 5-HT uptake sites have been measured in brain tissue in schizophrenia using ligands such as ['Hlimipramine, ['HI cyanoimipramine, or the more selective ['HI paroxetine. Two recent studies have shown deficits in 5-HT uptake site density in prefrontal cortex (Laruelle et al., 1993) and in autoradiographic binding to these sites in frontal and cingulate cortices (Joyce et al., 1993), but have not observed abnormalities in motor, temporal, or occipital cortices or hippocampus. These latter authors also observed an increase in binding to 5-HT uptake sites in the striatal regions of the caudate and accumbens nuclei and putamen. This finding is consistent with the report mentioned previously of increases of 5-HT in the putamen and pallidum, although again the results may be confounded by an association with suicide.

B. POSTSYNAPTIC 5-HT2RECEPTORS 5-HT receptors have also been studied in postmortem brain tissue from schizophrenic patients. The 5-HT2 subtype was first investigated using ['HILSD; the binding of this ligand was found to be reduced in frontal cortex (Bennett et al., 1979), but this was not replicated by Whittaker et al. (1981) or by using the more specific ligand, ['Hlketanserin (Reynolds et al., 198313). Subsequently, however, a decrease in 5-HT2 receptor density defined by ['HI ketanserin binding was identified in frontal cortical tissue (Mita et al., 1986; Laruelle et al., 1993). Autoradiographic study of these receptors using ['HILSD identified no consistent changes in the prefrontal cortex, but did identify an increase in the middle layers of the cingulate and temporal cortex and in the hippocampus Uoyce et al., 1993). These authors also reported increases in ['HI ketanserin binding to limbic striatal regions. It is important to consider the possible role of prior drug treatment in these reported abnormalities of 5-HT2 receptor density, particularly since several antipsychotic drugs have a high affinity for the receptor. Along with some other antipsychotics, clozapine has a higher affinity for these sites than for the D2receptors. Its administration leads to a rapid downregulation of 5-HT2sites (Reynolds et aL, 1983a), an effect also found for chlorproma-

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zine (Matsubara and Meltzer, 1989) which also has a higher affinity for these sites than for D2 receptors. Nevertheless, there is substantial interest in the potential of mixed 5-HT2/D2receptor antagonists in the treatment of schizophrenia. This approach has been given substantial support by the theoretical analysis of Meltzer and colleagues (1989) who demonstrate that “atypicality” in a selected range of antipsychotic drugs corresponds to high ratios of 5-HT2and D2receptor affinities. Despite some problems with this study, it has served to stimulate the study of 5-HT2antagonist action in the treatment of schizophrenia, and there are now several mixed 5-HT2/D2 receptor antagonists which have been recently introduced or are in development, each of which has a higher affinity for 5-HT2 than for D2 receptors (Reynolds and Czudek, 1995). 5-HT2receptors apparently interact with dopaminergic function, providing a potential mechanism for effects of 5-HT2 antagonists on psychotic and/or extrapyramidal symptoms. Electrophysiological (Sorensen et al., 1991) and biochemical (Schmidt et al., 1992) studies have shown 5-HT2 antagonists to reverse the effects of amphetamines on dopaminergic neurons, findings which have been used to suggest the potential of pure 5-HT2 antagonists as antipsychotic agents. Recently, Casey (1993) investigated the effects in nonhuman primates of a range of mixed D2/5-HT2 antagonists including risperidone and clozapine. He observed that all drugs, with the sole exception of clozapine, induced an acute dystonia that was independent of the 5-HT2 affinity and concluded that in primates 5-HT2 antagonism does not contribute to diminishing extrapyramidal symptoms.

C. OTHER5-HT RECEPTORS Over the past 15 years the number of 5-HT receptor subtypes identified has increased from 3 to approximately 14. Several of these putative recep tors, in addition to the 5-HT2receptor, have been implicated either directly in the neurochemical pathology of schizophrenia or as possible sites of action of antipsychotic drugs. One of these is the 5-HT3receptor (Tricklebank, 1989). 5-HT3receptors do not occur in high densities in the cerebrum, although more are found in the hippocampus and amygdala than most other brain regions (Kilpatrick et aL, 1987). Clozapine, but few other antipsychotics, binds to this site with affinity similar to that for D2 (Watling et al., 1990). Ondansetron, the first selective 5-HT3antagonist available in the clinic, has been proposed to be of potential value for many psychiatric problems including schizophrenia. Thus, for example, ondansetron reportedly has inhibitory effects on limbic dopaminergic hyperfunction, an attractive indication of potential antipsychotic efficacy (Costal1 et al., 1987). How-

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ever, others have been unable to reproduce ondansetron's effects on dopamine-mediated hyperactivity (Greenshaw, 1993). No abnormality of 5-HT3 receptors has been reported in the brain in schizophrenia. A further 5-HT receptor that has been reported to be abnormal in density in the brain in schizophrenia is the 5-HTIAsite. Hashimoto et al. (1991) reported an increase above control values in 5-HTlAreceptors (defined by saturable binding of ['HI 8-hydroxy-DPAT, a 5-HTIAagonist) to frontal cortex membranes from a small series of schizophrenic subjects. This finding has received support from an autoradiographic study (Joyce et al., 1993) showing significant increases in binding to 5-HTIAsites in the outer cortical layers of prefrontal and anterior cingulate cortices. Recent evidence has emerged showing human cortical pyramidal neurons to be enriched with 5-HT1, receptors (Bowen et al., 1992). Thus, these receptors might provide a marker for cortical glutamatergic neurons, indicating that the above findings are consistent with some, but not all, evidence for increases in frontal cortical glutamatergic systems (see below). However, an alternative explanation might be found in the effects of stress on 5-HTIA receptors that may be upregulated in response to a variety of stressful stimuli (e.g., Mendelson and McEwen, 1991). Although most antipsychotic drugs have minimal affinity for the 5-HTIA site, this is not true for clozapine, which is able to bind to these receptors at concentrations that would be consistent with at least partial occupancy at normal drug dosages (Mason and Reynolds, 1992). This provides a potential receptor mechanism for the modulation of cortical glutamatergic neurons, raising the possibility that an interaction with 5-HTIAreceptors may contribute to the mechanism whereby clozapine exerts its unique action. Two of the many receptors recently identified by molecular biological cloning techniques are the 5-HTs and 5-HT7 subtypes. Several antipsychotic (and other psychoactive) drugs, notably clozapine, have high affinities for these receptors, which have been suggested to mediate some of the clinical effects of the antipsychotics (Roth et al., 1994). Of particular interest is 5-HT7which is expressed in regions of the brain that include limbic structures. These receptors have yet to be studied in schizophrenia, although the 5-HT7 receptor has been identified by ligand binding in mammalian brain tissue (Branchek et al., 1994).

WII. Nomdrenergic Systems

Originally proposed 20 years ago, the suggestion that an abnormality of noradrenaline transmission underlies schizophrenia was based on the

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fact that noradrenergic systems were thought to be involved in central reward pathways (Stein and Wise, 1971). Thus, a noradrenergic deficit could lead to loss of drive and anhedonia, which is a model appropriate for the syndrome of primarily negative symptoms. Although this involvement of noradrenaline in reward is not longer considered accurate, some support for a role for noradrenaline in schizophrenia has emerged from postmortem studies reporting increased concentrations in the nucleus accumbens (Farley et aL, 1978). Initial studies reporting deficits of the noradrenergic marker enzyme dopamine P-hydroxylase (Wise and Stein, 1973) remain unconfirmed (Cross et al., 1978). Hornykiewicz (1982) developed the noradrenaline hypothesis further; however; others found that the evidence he presented for a primary role of noradrenergic dysfunction in schizophrenia was unconvincing (Iversen et al., 1983). Nevertheless, there is circumstantial evidence supporting the involvement of noradrenaline in schizophrenic symptomatology. This is based on observed abnormalities in plasma and CSF concentrations of noradrenaline and metabolites (e.g., van Kammen et al., 1990), although they may well be an autonomic response to symptoms or effects of medication, rather than reflections of a primary abnormality. Interestingly, however, clozapine is able to increase plasma noradrenaline, probably via its effects on presynaptic a2receptors (Richelson and Nelson, 1984), and it has been suggested that this might provide a mechanism for the clinical efficacy of the drug (Pickar et al., 1992). Noradrenaline interacts with dopaminergic systems in various regions of the brain. Tassin (1992) reviewed the evidence for this interaction in the cortex and pointed out a reciprocal control between dopaminergic D, receptors and a-adrenoceptors. Many antipsychotic drugs are antagonists at a I adrenoceptors, and although the a, blockade shared by several of these drugs (Richelson and Nelson, 1984) is considered to be responsible for some of the unpleasant side effects, Tassin (1992) and Baldessarini et al. (1992) concluded that aI blockade may play a role in antipsychotic response, particularly in diminishing positive symptoms.

IX. GABA

y-Aminobutyric acid (GABA) has long been implicated in the neurochemical pathology of schizophrenia. A “GABA hypothesis” first emerged from observations of the reciprocal nature of the actions of GABA and dopamine in the basal ganglia (Roberts, 1972). Studies of brain tissue taken at postmortem provided little evidence for a dysfunction of GABA in these

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brain regions, although our understanding of the structures involved in schizophrenia would encourage the search for GABAergic abnormalities elsewhere. Concentrations of GABA have been reported to be diminished in the nucleus accumbens and amygdala in some patients (Perry et al., 1979; Spokes et al., 1980), although this has not been confirmed in all studies (Cross et al., 1979). More recent work has identified neurochemical changes related to GABAergic neurotransmission in parts of the brain associated with the neuropathology of schizophrenia. Deficits of (presumably GABAergic) interneurons have been found in the prefrontal and cingulate cortices (Benes et al., 1991), and increased densities of GABAA receptor binding have recently been reported in the cingulate cortex in schizophrenia (Benes et al., 1992), an effect thought to relate to a compensatory upregulation of postsynaptic receptor sites and consistent with an earlier study which found an increase in the prefrontal cortex (Hanada et al., 1987). Regions of the medial temporal lobe, the part of the brain in schizophrenia in which neuronal deficits or disorder have been most frequently reported (see Section 111), have demonstrated deficits in indicators of GABAergic neurons. GABA itself is reportedly diminished in the posterior hippocampus (Toru et al., 1988) in schizophrenia, while the hippocampus (Reynolds et al., 1990) and other temporal lobe structures (Simpson et al., 1989) do show deficits in another marker for GABAergic nerve terminals. Decreased densities in the binding of [3H]nipecotic acid to GABA uptake sites are found in hippocampal brain tissue in schizophrenia, and the more profound deficit occurring in the left hemisphere correlates with the increase in dopamine concentrations in the amygdala which is also found on this side of the brain (Reynolds et al., 1990). This correlation between the loss of hippocampal GABA neurons and the increase in dopamine concentrations in the left amygdala implies that the neurons lost in schizophrenia could result in a hyperactivity of dopaminergic neurons (Reynolds et al., 1990).Such an interpretation is highly speculative but provides a much needed mechanism whereby dopamine antagonist drugs might ameliorate some of the consequences of neuronal deficits in schizophrenia. Losses of cholecystokinin and somatostatin in limbic brain tissue in some schizophrenic patients (Roberts et al., 1983) are also consistent with losses of subgroups of GABAergic neurons in which these neuropeptides are known to be colocalized (Somogyi et al., 1984). There have been some studies of GABA receptors in brain tissue from schizophrenic subjects, in addition to the studies which showed increases in cortical receptors mentioned previously. A recent investigation reported an increase above control values in the binding of a single concentration of radiolabeled flunitrazepam to part of the hippocampus (Kiuchi et al.,

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1989); we have been unable to confirm this, finding no indication of an upregulatory increase in either affinity or density of hippocampal benzodiazepine receptors (Reynolds and Stroud, 1993). The investigation of GABA receptors is, like many neurochemical measurements, confounded by the effects of antipsychotic drug treatment. Such drugs can increase GABA receptors in the basal ganglia (Gale, 1980); this and other changes in GABAergic parameters in regions of the brain important in the control of motor function have been proposed to relate to the tardive dyskinesia side effect induced by chronic treatment with antipsychotic drugs (Gunne et al., 1984; Anderson et al., 1989).

X. Neuropeptide Systems

Many members of the growing band of neuroactive peptides have been implicated in schizophrenia. Sometimes this has reflected developments in understanding the neurochemical anatomy, behavioral neurophysiology and neuropharmacology of the neuropeptides, although often naive hypotheses are generated with minimal supporting evidence. This review concentrates on a select few peptides that have been closely investigated in schizophrenia and in which some substantial interest remains. It is notable that in several cases this interest has developed from the observation of an interaction between the peptide and dopamine systems in the mammalian brain.

A. NEUROTENSIN Neurotensin is a tridecapeptide that is found in several dopaminergic terminal regions, including mesolimbic structures, which partly reflects its coexistence with dopamine in some mesolimbic and/or mesocortical neurons (Seroogy et al., 1988). Diminished concentrations of neurotensin have often been reported in CSF from some diagnostic subgroups of drugfree patients (e.g., Lindstrom et al., 1988; Nemeroff et al., 1989), although abnormalities in brain tissue are not always consistently found in schizophrenia, perhaps due to the effects of antipsychotic drugs (Nemeroff et al., 1983). The peptide is increased following chronic antipsychotic drug administration, demonstrating a potentially important dichotomy: while haloperidol induces neurotensin increases in both caudate and accumbens nuclei, clozapine only affects the nucleus accumbens (Davis and Nemeroff, 1988).These results, along with other evidence for mutual regulatory effects

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between dopamine and neurotensin, have led to consideration of the peptide as having antipsychotic properties, an approach pursued with some success by Nemeroff and colleagues.

B. CHOLEC~STOKININ (CCK) CCK is another neuropeptide found in some mesolimbic dopamine neurons (H6kEelt et al., 1980), although the great majority of CCK immunoreactive neurons also contain neurotransmitter GABA. CCK has been reported to be diminished in certain limbic brain structures in schizophrenia (Roberts, et al., 1983); this has been interpreted as reflecting losses of GABAergic neurons (Reynolds, 1989), as discussed under Section IX, although Kleinman et al. (1983) were unable to identify any abnormality in CCK in a variety of brain regions. Nevertheless, the involvement of CCK with dopaminergic systems has stimulated interest in the possible role of the peptide in antipsychotic action. It has been suggested that CCK, and the CCK agonist ceruletide, are able to inhibit limbic dopaminergic activity, with behavioral effects following intracerebral injection in rats that resemble those of antipsychotic drugs (Van Ree et al., 1983). While there have been some positive effects reported in open trials of CCK or ceruletide in schizophrenia, better controlled studies have been disappointing (Montgomery and Green, 1988).

C. SOMATOSTATIN This neuropeptide interacts with many neurotransmitter systems, including dopamine, although like CCKit is found primarily to be colocalized in cortical GABAergic neurons. Postmortem neurochemistry has identified deficits in schizophrenia in the hippocampus (Roberts et al., 1983) and frontal cortex (Nemeroff et al., 1983); as with CCK, these findings may be interpreted as reflecting GABAergic neuronal deficits. CSF studies have been inconclusive, with both increases and decreases in the peptide having been reported (cited by Bissette et al., 1986), although CSF somatostatin may be diminished following antipsychotic drug treatment.

D. OPIOID PEPTIDES Bissette et al. (1986) pointed out that the original interpretations of the behavioral effects of b-endorphin as either catatonia or neuroleptic-like

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catalepsy led to two mutually exclusive hypotheses for the potential involvement of opioid peptides in psychosis: that they were psychotogenic or that they were antipsychotic. In retrospect, we can see that neither hypothesis is soundly based. Each approach has had its proponents, although interest has certainly waned over the past decade. The opioid psychotogenicity hypothesis has been tested by administration of antagonists such as naloxone; this drug, however, has not been consistently shown to be antipsychotic and, when reported to be of value in certain patients, its effect is usually fairly transient. It is likely that the akinetic effect of @endorphin relates to the role of opioid peptides in the motor functions of the basal ganglia. The concentrations of striatal met-enkephalin are increased by chronic antipsychotic drug treatment (Hong et d.,1978), providing some support for opioid peptides having a role as endogenous antipsychotics. But clinical trials have proven disappointing, although it is hardly surprising that short-term injection of peptides, such as @-endorphin,that are unlikely to reach the brain in any substantial quantity fail to provide positive results. The decrease in metenkephalin observed in the caudate nucleus of a chronic paranoid subgroup of schizophrenics (Kleinman et al., 1983) cannot thus be explained in terms of drug effects. An “opioid” peptide that has been studied as a potential antipsychotic agent is des-tyryendorphin (DTE). De Wied and colleagues have long pursued this approach which is based on the behavioral effects of this endorphin fragment in animal models predictive of antipsychotic efficacy. DTE has no opiate-like activity but is proposed to act at sites that allow endogenous endorphins to modulate mesolimbic dopamine systems (Van Ree and De Wied, 1982). Clinically, the promise shown in open studies failed to be confirmed by controlled investigations (reviewed by Verhoeven et d.,1988), although these authors remain convinced of the potential of DTE for the treatment of a subgroup of schizophrenic patients.

Xi. Glutamate Systems

Glutamate has recently received increasing interest in terms of its proposed role in schizophrenia (in this chapter “glutamatergic” will be taken as encompassing all excitatory amino acid systems in which aspartate may well also have a transmitter function). Several lines of evidence support this approach. One is the psychotomimetic effect of phencyclidine (PCP), a drug of abuse that is considered to produce a better human (and presumably animal) model of schizophrenia than amphetamine since it induces negative symptoms in addition to an acute psychosis of primarily positive

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symptoms. These behavioral effects of PCP are considered, primarily, to be due to its ability to block the ion channel associated with the glutamatergic N-methyl-D-aspartate (NMDA) receptor complex (Javitt and Zukin, 1991). PCP and other noncompetitive NMDA receptor antagonists, such as dizocilpine (MKSOl), bind to the open conformation of the NMDA receptor and act as negative allosteric modulators to close the cation channel and thereby noncompetitively antagonize the effect of glutamate. Drugs that bind to the PCP receptor therefore inhibit the functioning of normal NMDA receptor-mediated glutamatergic neurotransmission. Further hypotheses implicating glutamatergic dysfunction in schizophrenia have developed from an understanding of the close interrelationship between glutamate and dopamine systems. Carlsson and others have constructed elegant proposals for a dysfunction of this relationship in schizophrenia (Carlsson and Carlsson, 1990; Kim et al., 1980). Although necessary for an understanding of the antipsychotic action of dopamine antagonists in the context of a glutamatergic neuropathology of schizophrenia, such proposals do not explain the (primarily negative) symptoms that are less responsive to such treatment. No antipsychotic currently in use is thought to have direct effects on glutamatergic transmission at normal clinical dosages, although an effect of clozapine at the NMDA receptor site has been proposed (Schmidt et aL, 1987). Nevertheless, glutamate receptors, and particularly the NMDA subtype, offer a novel approach to the development of potential treatments for schizophrenia. These receptors have facilitatory effects on dopaminergic transmission in subcortical regions, since they can stimulate dopamine release. In the frontal cortex the action of glutamate on dopamine systems is less clear; some in vivo studies suggest NMDA receptors to diminish dopamine utilization (Ham et al., 1990), although release of dopamine from the frontal cortex in vitro is stimulated by both NMDA and non-NMDA glutamate receptors (Jones et al., 1993). Possible differences between cortical and subcortical glutamatergic control of dopamine systems present an interesting dichotomy which parallels some proposals relating to the dopamine hypothesis of schizophrenia; while it is generally accepted that a dopaminergic hyperactivity relates particularly well to positive symptoms, it has been suggested that dopamine hypofunction may underlie negative symptomatology in which frontal cortical dysfunction has been implicated. However, no neurochemical evidence from postmortem studies has offered any direct support for this hypothesis (Reynolds, 1989). A. PRESYNAPTIC GLUTAMATERGIC MARKERS There is now substantial evidence that schizophrenia might involve a dysfunction of glutamatergic transmission. First proposed by Kim et al.

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(1980), on the basis of (now unconfirmed) abnormalities of glutamate concentration in CSF, this has gained support from several postmortem neurochemical studies, particularly in the frontal cortex. Dsyfunction of this region, indicated by structural and functional imaging studies, has been associated with negative symptomatology in schizophrenia. Present neurochemical evidence supports hypotheses of either increased or decreased glutamatergic function in schizophrenia, although the PCP model would be more consistent with the latter. Ligand binding of [3H]~-aspartate to glutamate uptake sites is reportedly increased in frontal cortex (Deakin et al., 1989), as are concentrations of glutamate itself (Reynolds, 1991), although the latter measure, to which transmitter concentration only partially contribute, may be a poor indication of presynaptic glutamate. These findings have been interpreted as indicating a relative increase in innervation of the frontal cortex in schizophrenia. However, we have been unable to confirm the increase in uptake sites, finding deficits in binding to these sites in the caudate and accumbens nuclei, structures receiving glutamatergic projections from cortical regions (Reynolds and Cutts, 1994). The reports of increases in 5-HTlAreceptors provide another indication of increased density of glutamatergic neurons; the corticocortical neurons primarily express these 5-HT receptors in human cortex (Bowen et al., 1992). The limitations of the study of 5-HTIAreceptors in schizophrenia were discussed under Section VII,C. Neurochemistry of autopsy tissue has also provided some indication of glutamatergic abnormalites, primarily deficits, in the temporal lobe. Despite not finding a significant deficit of ['H)~-aspartatebinding in several temporal lobe structures, Deakin et al. (1989) did suggest that a decrease in this measure of glutamatergic neurons in left polar temporal cortex was related to increased left amygdalar dopamine. In a preliminary study, we have also observed diminished temporal cortical glutamate uptake sites in the left hemisphere in schizophrenia (Reynolds et al., 1993). The major findings, however, have been in the determination of glutamate receptors. RECEPTORS B. GLUTAMATE Kenvin et al. (1988,1990) have shown diminished binding to the kainatesensitive subtype of glutamate receptors in the hippocampus in schizophrenia, an effect most apparent in the left hemisphere and consistent with other neurochemical indications of left temporal lobe dysfunction discussed previously. This observation was supported by a reduction in mRNA for hippocampal kainate receptors (Harrison et al., 1991) which these authors interpreted as a dysfunction in gene expression, although alterna-

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tively the data may be consistent with a loss of neurons on which the receptors are found. A further report was unable to identify asymmetric deficits of hippocampal kainate receptors, but did observe an increase in binding to these sites in the orbital frontal cortex (Deakin et al., 1989), providing some support for the results from an earlier study (Nishikawa et al., 1983) in which the medial frontal cortex, but not the orbital cortex, demonstrated an increase in binding to kainate receptors. The NMDA receptor subtype has been less well studied, perhaps due to the absence, until recently, of adequate competitive antagonist radioligands. Kerwin et al. (1990) found NMDA-sensitive glutamate binding to the hippocampus to be fully preserved in schizophrenia in the presence of the losses in kainate receptors mentioned previously. ['HI MK801 binding to the NMDA-linked ion channel has been reported to be increased in the putamen in schizophrenia (Kornhuber et al., 1989),which is an interesting observation in the light of deficits in striatal glutamatergic innervation described previously. As mentioned previously, it is this NMDA site that mediates the psychotogenic effects of PCP and other drugs that block the associated ion channel. Like other psychotogenic drugs, these noncompetitive NMDA receptor antagonists can bring about a decrease in prepulse inhibition of the startle reflex (Mansbach, 1991). This behavioral effect is not influenced by dopaminergic D2 antagonists since it is not reversed by haloperidol (Geyer et aL, 1990). These results serve to strengthen the notion that manipulation of the NMDA receptor complex might be a valid target for antipsychotic drug development, particularly in treatmentresistant patients. The NMDA receptor complex has several sites open to pharmacological influence in addition to the transmitter binding site and the ion channel. For example, glutamate action at this site has an essential requirement for glycine; thus, glycine has a positive modulatory effect on the glutamate/NMDA receptor. Glycine itself, and the glycine prodrug milacemide, have been investigated as potential antipsychotics, although the results have been inconsistent. Noncompetitive antagonism of the glutamate/NMDA receptor may be brought about by antagonist action at the glycine binding site. One compound with this effect is HA966, which can inhibit the behavioral and biochemical effects of amphetamine and PCP in activating the mesolimbic dopamine system without affecitng dopamine function in nonstimulated animals (Hutson et al., 1991). Although this is a long way from a clinical antipsychotic response, it does provide an exciting stimulus to the development of novel antagonists at the glycine/NMDA receptor as potential drugs for the treatment of schizophrenia.

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Originally confused with the PCP binding site on the NMDA receptor complex, the haloperidol-sensitive u recognition site has attracted interest as being of possible importance in some aspects of antipsychotic action. Haloperidol and several newly developed antipsychotic drugs (but not clozapine) have a high affinity for this u receptor (Largent et al., 1988). However, several u ligands have been found not to be effective in the clinical treatment of schizophrenia, making it unlikely that action at u sites can make an important contribution to antipsychotic efficacy. It is unclear what role u receptors play in brain function, nor has the endogenous agonist for the receptor been identified. Nevertheless, there have been investigations of u receptors in the brain in schizophrenia, resulting in the identification of decreases in receptor density (Weissman et al., 1991; Simpson et al., 1992). This finding, however, has been interpreted as an effect of drug treatment; patients who had received haloperidol prior to death demonstrated a downregulation of u site density (Reynolds et al., 1991). XI!. Conclusions

A. A NEUROCHEMICAL PATHOLOGY OF SCHIZOPHRENIA This chapter has attempted to focus on the various changes in indicators of neurotransmitter function that are reported in schizophrenia. Some of these changes will eventually turn out to be artifactual, irreproducible, or simply erroneous findings. One obvious and well-recognized problem contributing to such artifacts is the effects of drug treatment, which nevertheless are not always fully defined. Ignoring artifact and error, changes in transmitter systems can be divided into two groups: those that directly reflect an abnormal neuropathology (neuronal deficits, abnormal neuronal connectivity, etc.) and those that may be secondary, perhaps compensatory, responses to these initial abnormalities. Candidates for the former group include deficits of GABAergic neurons in the frontal cortex and hippocampus, while glutamatergic neurons in the medial temporal lobe or projecting from the cortex to subcortical structures may also prove to be diminished in the disease. Several of the changes observed in neuropeptides and in receptors may reflect deficits of neurons containing the peptide or on which the receptors are sited. Although neither GABA- nor glutamatergic systems are directly influenced by antipsychotic drugs, secondary effects of these neuropathologies on dopamine systems provide potential (and

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necessary) mechansims for understanding the clinical efficacy of dopamine antagonists in treating schizophrenia. Such interactions have been tentatively proposed for deficits in hippocampal GABAergic neurons (Reynolds et aZ., 1990) and for a dysfunctional corticostriatal glutamate pathway (Carlsson and Carlsson, 1990). Further research is particularly needed in two related directions. One is to accept that there is a lateralization of function within the human brain and to pursue the observation of neurochemical asymmetry that is also seen in a range of behavioral, physiological,and neuroanatomical measures in schizophrenia. Another is to recognize the disease as heterogeneous and to avoid some of the limitations this has inevitably imposed on research by undertaking neurochemical studies of syndromes within the disease. Although this requires the collection of larger series of subjects for study, the work of Liddle (1987) has given us valuable clues in relation to the regions of the brain to be investigated in the search for transmitter correlates of the neural abnormalities associated with the three syndromes. Having established that there are abnormalities of neurotransmitter systems in schizophrenia, probably reflecting neuronal deficits in the brain, we should consider their origins. There is much evidence in support of schizophrenia as a disorder of development, a finding that is potentially compatible with both environmental and genetic etiologies. If one accepts that the primary pathology is in frontal cortical and/or hippocampal neurons containing GABA and/or glutamate, then it would seem that a search for linkage between hereditary schizophrenia and molecular variants of, for example, a dopamine receptor, is unlikely to be particularly fruitful. Thus, future research must draw on basic studies in neuronal development to identify the mechanisms that go awry which produce the subtle deficits and disorder in neuronal systems which, in turn, are reflected by the abnormalities in transmitter systems described here.

B. PROSPECTS FOR DRUGTREATMENT The ubiquity of GABA and glutamate within the brain indicates the difficulty of developing drug treatment that addresses regional-specific neuronal deficits. Nevertheless, there are experimental indications of the potential offered by drugs acting on the glutamate/NMDA receptor (Section X1,B). This, and other approaches to the future treatment of schizophrenia have been reviewed elsewhere (Reynolds and Czudek, 1995). Molecular biological studies may also contribute to the future development of more specific drug treatments addressing amino acid transmitter systems. Thus, the ion channel receptor complexes that make up the GABAAand gluta-

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mate/NMDA receptors are composed of several subunits which may vary subtly in structure and, hence, in agonist and antagonist selectivity. Since the relative composition of these receptors varies between different brain regions, a potential means of introducing regional selectivity is apparent. This has been utilized in the case of drugs acting on GABAergic systems; the benzodiazepine partial agonists show some regional selectivityreflecting differences in receptor structure and are being proposed for the treatment of schizophrenia (Delini-Stula et d.,1992). However, for the foreseeable future the dopamine antagonists and subtle variants on the same theme (e.g, 5-HT2/D2 antagonists) will remain central to antipsychotic drug treatment. The deficiencies associated with these drugs were mentioned under Section IV as are the clues provided by clozapine in developing approaches to treat otherwise drug-resistant patients. Research to identify the neuronal and neurotransmitter abnormalities that distinguish this substantial subgroup would be a major advance toward providing a rational treatment for these patients.

References

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PHYSIOLOGY OF BERGMANN GLlAL CELLS

Thomas Muller and Helmut Kettenmann Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 345, 69 120 Heidelberg, Germany and Max-Delbrirck Center for Molecular Medicine, Robert-Rossle Strasse 10, 13 129 Berlin-Buch, Germany

I. Introduction 11. Morphology 111. Gap Junctions IV. Ion Channels A. K+ Currents

B. Kt Uptake V. Neurotransmitter Receptors A. Glutamate Receptors B. GABA Receptors C. Functional Importance of Glutamate and GABA Receptors in Bergmann Glial Cells VI. Ion Exchangers VII. Transmitter Uptake WII. Summary References

Until recently little was known about the physiological and pharmacological properties of the different types of glial cells. Intracellular recording techniques yielded stable, negative membrane potentials of about -80 mV (Orkand et al., 1966) but, unlike neurons, glia were not considered to generate active responses. Recent investigations (for review see Barres et al., 1990b; Bevan, 1990) have challenged this view and it is now clear that glial cells have the potential to express many membrane components and cellular responses that were formerly thought to be typical of neurons. These findings have triggered a number of speculations on glial cell function, suggesting a more dynamic role of the glial cells in the functioning of the nervous system. Much of our recent knowledge originates from studies on glial cells grown in primary cell culture. This approach offers many advantages, such as working with relatively pure, defined populations of cells. A major drawINTERNATIONAL REVIEW O F NEUROBIOLOGY, VOL. 38

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back, however, is the difficulty to extrapolate such data to in situ situations in which the cellular environment and external influences are more complex. Since glial cell properties in situ may differ from those in culture, the question remained unanswered whether glial cells in situ express the same wide range of transmitter receptors and voltagegated ion channels as those in culture. This chapter focuses on the physiological properties of a particular type of glial cell, the Bergmann glial cell in the cerebellum. This radial glial cell of the astrocytic lineage can be studied in situ employing the patch-clamp technique in the cerebellar slice preparation. It is evident that glial cell physiology in a tissue is more complex and dynamic than previously anticipated.

II. Morphology

Bergmann glial cells, also named Golgi epithelial cells, are confined to the cerebellum. While they belong to the astrocytic lineage, they do not represent the only form of astrocytes in the cerebellar cortex. Two more subclasses of astrocytes are present, namely the lamellar or velate astrocytes and the smooth astrocytes (Chan-Palay and Palay, 1972). Moreover, the cerebellar cortex contains oligodendrocytes and microglia cells (ChanPalay and Palay, 1972). Bergmann glial cells were noted as fibrous structures in the cerebellar cortex of cat and dog and in the human case of atrophy (Bergmann, 1857). According to Bergmann, the radial striae in the molecular layer are fine fibers passing vertically through the cortex and forming a limiting membrane at the surface by the fusion of conical swellings at the fiber tips. In the following decades the Bergmann glial cell morphology has been thoroughly investigated (Golgi, 1885;Ram6n y Cajal, 1909;Schroder, 1929) and is best summarized by Palay and Chan-Palay (1974). In the immature brain Bergmann glial cells possess a quite distinct morphology which may relate to their functional importance during the development of the cerebellum. During the first postnatal days, Bergmann glial cells are a necessary requirement for the proper development of the cerebellum. They form radial structures extending from the external granular cell layer to the inner granular cell layer on which granule cells migrate to their final destination (for review see Hatten and Mason, 1986; Rakic, 1971). This distinct functional property is reflected in a distinct morphology. Young Bergmann glial cells possess not only processes extending through the molecular layer to the pia mater but also an additional process extending into the inner granular cell layer, sometimes even pene-

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trating into the white mater. These processes appear smooth due to the lack of the fine elaborated appendages which appear at later stages of the development (Fig. 1 ) . This property is maintained during the migratory period of the granular cells up to Postnatal Day 15 as studied in the mouse. From this time on the descending process is retracted and is no longer present in the more mature cerebellum. In addition, fine appendages are formed by highly branched fine processes which are located in synaptic areas where parallel fibers and climbing fibers contact Purkinje cells. These processes are only a few tenths of a micrometer wide and are closely associated with pre- and postsynaptic terminals (Baude et at., 1994). Moreover, the cell somata of Bergmann glial cells translocate from deeper locations, where they are not confined to a single layer, to a very restricted layer, namely the Purkinje cell layer. In the adult stage, Bergmann glial cells give rise to at least two, but commonly about six processes which bifurcate and ascend in an almost parallel fashion through the molecular layer ending at the pial surface with the formation of endfeet at the glia limitans (Fig. 1 ) . The processes are “studded with a multiude of lamellae and granular, moderately branching appendages. These appendages and lamellae . . . form a sponge-like fabric in which the neuronal elements are lodged” (Ramon y Cajal, 1909). These processes form intimate interactions with the dendritic tree and cell body of the Purkinje cells. None of the other nerve cells of the cerebellum show such a specific interaction with a particular type of glia cell. This isolating role of neuroglial processes was suggested by Ram6n y Cajal (1909) and elaborated by Peters and Palay (1965) and Palay (1966). Bergmann glial cells and their typical orientated structure are part of the radial glial system which is found in many areas of the developing brain. In contrast to most radial glial cells present in early development, Bergmann glial cells are still present in the mature brain. This implies that the cells acquire functions for the mature brain in addition to their role during development.

111. Gap Junctions

Chemical transmission is the general pathway by which the neuronal elements interact and communicate. Between glial cells, gap junctions are common, forming more direct pathways for intercellular communication. However, these mechanisms are not exclusive; gap junctions have been found between at least some populations of neurons, and chemical responsiveness is increasingly appreciated in glia (see Section V) .

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@I -170mV

15ms FIG.1. Bergmann glial cell in a cerebellar slice: morphological and ekctrophysiologicdl features at different developmental stages. (A) On the left, a Lucifer yellow-injected Bergmann glial cell from P6 is shown in the fluorescence micrograph. In the middle, the morphological features were reconstructed by computer based on a series of photographs taken at different focal planes. On the right, a process from that cell (see box in the middle) is shown at higher magnification. (B) Same as for that described in A, ;I Bergmann glial cell from P20 is depicted. Note the numerous elaborated appendages on the processes of the P20 cell compared to those of the P6 cell. In the background a few weakly coupled cell bodies of adjacent Bergmann glial cells are visible. The bar on the left denotes 50 p m and the bar on the right 25 pm. (C) Membrane currents were recorded with the patch-clamp technique from a Bergmann glial cell from P6. The membrane was clamped from a holding potential of -70 mV to increasing depolarizing potentials (-50, -30, - 10, 10, and 30 mV) and hyperpolarizing potentials (-170, -150, - 130, -110, and -90). Note the presence of voltage-gated currents, i.e., the delayed rectifying outward and the inactivating inward rectifier K +current. (D) Using the same voltage clamp protocol as that described in B, Bergmann glial cells from Postnatal Day 20 responded to de- and hyperpolarizing voltages by large time- and voltage-independent currents (with modifications from Muller et al., 1994).

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Evidence for gap junctional coupling between Bergmann glial cells has been documented by a variety of different techniques ranging from immunohistochemistry, electron microscopy, electrophysiology, to dye coupling (Palay and Chan-Palay, 1974; Yamamoto et al., 1990; Muller et al., 1995). In the adult brain, the major connexin, the molecular element forming the gap junction channels, is connexin43 and is expressed by astrocytes (for review see Dermietzel and Spray, 1993). Bergmann glial cells as a specialized form of astrocytes also express connexin43. Gap junctions formed by this connexin are nonrectifylng as demonstrated for astrocytes in culture (Kettenmann and Ransom, 1988; Dermietzel et al., 1991). During the development of the brain the expression of connexin43 is increased. In the cerebellum of mice connexin43 appears after the end of the granule cell migration around Postnatal Day 15 (Milller et al., 1994). This is consistent with the finding that Lucifer yellow injection revealed no dye coupling among Bergmann glial cells before Postnatal Day 15. Similarly, electrical coupling was only observed at mice after Postnatal Day 15. In contrast, Bergmann glial cells at Day 20 are always electrically and dye coupled. Recent studies suggest that the degree of coupling among glial cells is not a steady property but is subject to high plasticity, regulated by intracellular signaling mechanisms and membrane receptors (De Vries and Schwartz, 1989; Giaume et aZ., 1991). Gap junction permeability between glial cells was studied by simultaneously recording currents from a pair of Bergmann glial cells and monitoring the current passage between the two. Halothane readily uncouples Bergmann glial cells. This process is reversible and is consistent with the properties of gap junctions in general. Another wellknown feature which regulates gap junction permeability is the level of intracellular pH. Cytosolic alkalization, experimentally achieved by applying NH,Cl, increases the coupling permeability between pairs of glial cells. In turn, acidification, experimentally achieved by removing NH,Cl, causes a decrease in gapjunction permeability. Another common agent to regulate gap junction permeability is the level of intracellular Ca2+.This is particularly interesting in the light of the finding that glutamate receptor activation in Bergmann glial cells increases cytosolic Ca2' (also see Section V). NonNmethyl-D-aspartate (NMDA) receptor activation using the specific ligand kainate causes a significant block of the junctional conductance in the presence of extracellular Ca2+.Omitting Ca2' from the bath leads only to a slight reduction of the junctional conductance (Mtkller et al., 1995). Since glutamate receptors are in close vicinity to synaptic areas, and may be activated during synaptic transmission, glutamatergic synaptic activity will regulate the gap junction permeability in Bergmann glial cells via a Ca2+mediated mechanism. This blockade is reversible and mainly depends on

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the velocity of intracellular Ca2+regulation. A more prolonged effect of glutamate on connexin43 levels was reported by Hossain et al. (1994) demonstrating that the level of connexin43 is reduced after intracerebral kainate treatment. This also implies that the expression of the junctional protein may be controlled by synaptic activity. Ultrastructurally identified gap junctions were found on the ascending process (Palay and Chan-Palay, 1974; Yamamoto et al., 1990; Mtiller et al., 1995). However, it has not been established whether the gap junctions are between neighboring Bergmann glial cells or whether they connect processes originating from the same cell. Moreover, Yamamoto et al. (1990) demonstrated that gap junctions are also localized on glial processes ensheathing Purkinje cell somata using gap junction-specific antibodies. Gap junctions among glial cells may serve different functions. They can be important mediators for exchanging low molecular substances between cells such as cyclic nucleotides. Another important property is the participation of gap junctions in controlling extracellular K+ homeostasis. Neuronal activity leads to an accumulation of extracellular K+. Glial cells are important in the uptake and redistribution of excess K+. Gap junctions may serve to interconnect glial pathways for the removal of Kt. They might provide a direct pathway to the side of K+ disposal into the perivascular compartments (Gardner-Medwin et aL, 1981; Newman, 1986). Another equally plausible notion is that coupling increases the volume of the buffer sink, so that focal activity of neurons, and thus focal increases of K+, are distributed to more than one glial compartment. Thus, the coupled Bergmann glial cell compartments could share the excess K+ load. Recent evidence suggest that gapjunctions are also important in signaling between glial cells. Increases in cytosolic Ca4+can be activated by a variety of stimuli including glutamate receptor activation or mechanical stress (Cornell-Bell et al., 1990; Mtiller et al., 1992; Charles et al., 1993). These Ca2+waves can propagate between glial cells via gap junctions. Such Ca2+waves have been demonstrated for astrocytes in culture and from slice preparations (Finkbeiner, 1992). In Bergmann glial cells Ca2+waves have not yet been demonstrated.

IV. ion Channels

In the classical view glial cells are considered as the unexcitable cells of the central nervous system exhibiting passive membrane properties and negative stable resting potential at about -80 mV. In an elegant series of studies Kuffler, Nicholls, and Orkand demonstrated that the membrane of glial cells in the leech is dominated by passive Kt currents (Nicholls and

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Kuffler, 1965; Orkand et aL, 1966). A number of recent studies performed mainly in cell culture demonstrated that glial cells have the capability of expressing a number of voltagegated channels including delayed rectifiers, Na+ channels, and Ca2+channels. Bergmann glial cells of the adult brain seem to be similar to the classical glial cells in that their membrane is dominated by a passive Kt conductance (Fig. 1). While these passive currents are only slightly affected by the classical K+ channel blockers, such as barium, 4Ap, and TFA, the currents are markedly reduced when the cytosolic CaZt concentration is increased. Such an increase occurs after kainate/a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor activation or can be experimentally introduced by applying the Ca2+ionophore ionomycin (Muller et aL, 1992).

A. K'CURRENTS

In the immature cerebellum the membrane properties of Bergmann glial cells are quite different. At this stage the passive Kt currents are less pronounced and voltage-gated channels can be detected (Fig. 1).A delayed rectifying K+ channel can be activated with membrane depolarization and this channel is blocked by barium, 4Ap, and TEA. Hyperpolarization activates an inwardly rectifying K+ channel which inactivates at more negative potentials. Thus, this channel has the classic properties of an inward rectifier. It is blocked by 4AP and barium, while TEA is not effective. In the first postnatal days (Postnatal Days 3-7 of mice) the membrane is dominated by these voltagegated channels. At about Postnatal Day 15 voltage-gated channels disappear and later the membrane of Bergmann glial cells is dominated by the passive Kt conductances.

B. K'UPTAKE

K+ channels in glia cells fulfill the important task of redistributing excess K' released by neuronal activity. In the cerebellum Kt levels rise by 1-3 mM around the neuronal soma and dendrites during physiological spike activity. It was indeed possible to detect Kt elevations with single action potentials (Hounsgaard and Nicholson, 1982). As expected, only neuronal depolarization led to the accumulation of Kt, while a hyperpolarization was not effective. As described under Section 11, the cell body of the Purkinje cell including all its dendrites and its initial segment of the axon is ensheathed by lamellar processes from Bergmann glial cells. This close morphological interaction is responsible for Bergmann

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glial cell depolarization on neuronal stimulation (Hounsgaard and Nicholson, 1982). Using the patch-clamp technique and simultaneously recording from Purkinje cells and Bergmann glial cells it could be confirmed that Purkinje cell activity induces K+ currents in the Bergmann glial cell. On depolarization of the Purkinje cell an inward current is activated in the Bergmann glial cell. In contrast, no glial current response was observed on hyperpolarization of the Purkinje cell. The uptake of KC can be reversibly blocked by barium. The glial K+ current correlates with the development of the morphological interactions between Bergmann glial and Purkinje cells. While on early postnatal days the glial processes are slender and unbranched and serve as pathways for the migration of granular cells, on later days Bergmann glial processes intermingle with Purkinje cell dendrites. Concommitant with this development K+ currents in Bergmann glial cells due to Purkinje cell stimulation are minute early in development and increase in size with further development. The Muller cell of the retina is an excellent example of a radial glial cell which serves an important function in the clearance of K'. In an elegant series of studies on isolated amphibian Muller cells Newmann and coworkers (1984, 1987) demonstrated that these cells can take up K+ at their soma and release it at the endfoot region. The Muller cell endfoot is specialized for releasing K+ since its K' conductance is higher compared to that of other regions of the cell. In turn, inwardly rectifying K+ channels are found in nonendfoot regions and these channels, due to their properties, are optimized for K' uptake. Recordings from the Muller cell endfoot in situ demonstrated that this structure is an electrically isolated unit in the mouse, while current can more easily spread from (or to) somatic regions in the guinea pig or (even better) in amphibians (Reichelt et aL, 1993; Newman, 1984, 1987). This implies that with recordings from the soma one may easily overlook voltage-gated channels in more distant regions such as the endfoot or the fine appendages. Bergmann glial cells also show endfoot formation and it is likely that the lack of voltage-gated channels in mature Bergmann glial cells may be due to the lack of the present technique to detect such channels. One future possibility would be to perform single-channel recordings from subcellular regions of Bergmann glial cells. K+ channels may not only serve for the spread of voltage signals or for ion redistribution but also may serve for control of basic cellular functions such as a control of proliferation. For T lymphocytes and Schwann cells it has been demonstrated that the expression of delayed rectifylng Kt channels is important for mitosis (DeCoursey et al., 1984; Wilson and Chiu, 1990).

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V. Neurotransmitter Receptors

In the past years it has become evident that gial cells can express a large variety of transmitter receptors. These receptors have mainly been characterized in cell cultures. (For review see Murphy, 1993; Kimelberg et al., 1993.) These receptors include those for the major inhibitory and excitatory neurotransmitter in the central nerval system of mammals, the glutamate, and y-aminobutyric acid (GABA) receptors, respectively (Blankenfeld and Kettenmann, 1992).

A. GLUTAMATE RECEPTORS Glutamate receptors comprise a family of transmitter receptors which can be activated by the common ligand glutamate. Specific ligands can be distinguished between subtypes, namely NMDA, kainate, and AMPA (Monaghan et aL, 1989). 1. Kainate/AMF'A Receptors

Bergmann glial cells respond with an inward current to kainate which is a common agonist for kainate and AMPA receptors (Fig. 2). Indeed, molecular biological studies using in situ hybridization have demonstrated that Bergmann glial cells express subunits of the AMPA/kainate receptor. Bergmann glial cells in the cerebellum are labeled very strongly by GluRA and GluR, probes, but no GluRB or G l u h mRNA could be detected (Blackstone et al., 1992; Martin et al., 1993; Petralia and Wenthold, 1992, Baude et al., 1994).In the recombinant AMPA receptors the voltage independence and the low divalent-action permeability have been traced to the presence of the GluRB subunit, whereas channels lacking the GluR, subunit are characterized by double rectifylng current voltage relationship and a high

NM~A

Glutamate k p A

-iV----60 s

:f

Kainite

FIG.2. Comparison of NMDA (lo-' M ) , glutamate (lo-' M ) , and kainate (lo-' M ) evoked current responses from a Bergmann glial cell from a 2May otd mouse (with modifications from Moller et al., 1992, 1993).

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Ca2+permeability (Hollmann et aL, 1989; Verdoorn et aZ., 1991). In patchclamp recordings from cultured fusiform glial cells of the cerebellum and from Bergmann glial cells in brain slices it could be demonstrated that the kainate/AMPA receptors on Bergmann glia are highly Ca2+permeable and have a double rectifylng current voltage curve (Burnashev et aL, 1992; Milller et aL, 1992). To record membrane currents and changes in intracellular Ca2+concentration simultaneously, Bergmann glial cells were filled with the Ca2'-sensitive fluorescent dye fura-2 by dialysis of the cell during the electrophysiological recordings via the patch pipette (Mtlller et al., 1992). Application of kainate induced an increase in the intracellular Ca2' concentration. This increase was reversibly blocked in Ca2+-freebath solution, indicating an influx of Ca2+from the extracellular space, whereas the kainate-induced inward current was still observed. In contrast, application of 6-cyano-7nitroquinoxaline-2,3ione (CNQX), a blocker of non-NMDA receptors, blocks both the Ca2+entry and the membrane current (Fig. 3). Since Bergmann glial cells lack the presence of voltagegated Ca2+ channels, these experiments clearly demonstrate the permeability of kainate/AMPA receptors on Bergmann glial cells not only for Kt and Na+, but also, as predicted by the recombinant and in situ hybridization results, for Ca2+. These characteristics could be observed in Bergmann glial cells of cerebellular slices of mice from Postnatal Day 5, when the migration of the granule cells on Bergmann glial cell processes starts, up to Postnatal Day 25, when the migration is finished and Bergmann glial cells form intimate contacts to the Purkinje cell synapses.

FIG.3 Figure 3 shows the kainate-induced changes in intracellular Ca". A Bergmann glial cell in a cerebellar slice was filled with the Ca2+ indicator fura-2 by dialysis via the patch pipette. The cytoplasmic [Cap+]levels were measured as the fluorescence ratio (&,,saa) and are color coded as indicated on the calibration bar in the middle of the figure (colors on the bottom indicate low Ca'+ concentrations). The color-coded pictures illustrate the increase in Cap+concentration during application of kainate (lo->M, middle picture) compared to the control before (left picture) and after (right picture). In the presence of kainate, the tips of the processes disappear due to a saturation of the Cap' imaging system. From a series of Cap' images, continuous traces of intracellular Ca2+levels were constructed by averaging the changes restricted to the cell processes (left). The pointer indicates the time when the color-coded pictures were taken. (A) Effect of low extracellular calcium: in the top row application of kainate led to an increase in the fluorescence ratio, indicative of an increase in Ca2+concentration (&qu,JBo) (left trace). In Capt-free bathing solution, the kainate-induced change in Cap' concentration is blocked (middle row). (Bottom) a control experiement was repeated in normal Ca''concentration. (B) Effect of CNQX: Same as that described in A, the effect of the kainate receptor antagonist CNQX (45 p M ) was analyzed (with modifications from Muller d al., 1992).

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35 1

2. NMDARecqtors In the cerebellum, NMDA receptors play an important role in neuronal differentiation (Rabacchi et al., 1992; Komuro and Rakic, 1993) and excitatory synaptic transmission (Garthwaite and Brodbelt, 1989; Silver et al., 1992; D'Angelo et al., 1993). Although the conventional NMDA receptor seems not to be expressed by Bergmann glial cells (Moriyoshi et al., 1991) electrophysiological experiments demonstrated an intrinsic response of Bergmann glial cells to NMDA (Muller et al., 1993; Fig. 2). In some experiments it could be demonstrated that NMDA increases the membrane conductance in Bergmann glial cells indicating the expression of NMDA receptors in these cells. Similarities with neuronal NMDA receptors include that the NMDA-induced current is mimicked by the NMDA receptor agonist homocysteate and blocked by the NMDA receptor antagonist ketamine, but not by the non-NMDA receptor antagonist CNQX. Despite of some similarities, most of the properties of the Bergmann glial NMDA response are different from those described in neurons: the current-voltage relationship is linear and the response is not modulated by Mg''. In addition, no changes in cytosolic Ca2+after application of NMDA were observed. Although NMDA receptor expression by Purkinje cells is still controversial, NMDA receptors have been shown to be involved in synapse elimination at Purkinje cells during cerebellar development (Rabacchi et al., 1992). It is conceivable that this process may be mediated by NMDA receptors located on Bergmann glial cells. In addition, recent experiments demonstrate changes in NMDA receptor properties at cerebellar granule cells at various developmental stages (Farrant et al., 1994). Since Bergmann glial cells are in close contact with migrating granule cells it is quite possible that glial NMDA receptors may be involved in this neuron-glia interaction. B. GABA RECEPTORS In the cerebellum GABA is the main neurotransmitter mediating synaptic inhibition (Fagg and Foster, 1983). GABA activates two types of membrane receptors, a GABA-gated anion channel (the GABAA receptor) and a G protein-coupled GABABreceptor (Bowery et al., 1987). The ligandgated GABAA receptor is the target of action for a variety of psychoactive compounds as barbiturates, benzodiazepines, neurosteroids, and ethanol (Olsen and Tobin, 1990; Wafford et al., 1990). Electrophysiological experiments on Bergmann glial cells in acute slices of mouse cerebellum demonstrate that application of GABA leads to a pronounced conductance increase. The involvement of GABAAreceptor is inferred by the observation that the reversal potential of the response

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under these experimental conditions is close to the C1- equilibrium potential. Moreover, the GABA response can be mimicked by the GABAA receptor agonist muscimol and blocked by the antagonists bicuculline and picrotoxin. The GABA response in Bergmann glial cells can be modulated by the barbiturate pentobarbital as described for GABAA receptors. The activation of single channels is further substantiated by the observation that during GABA application a pronounced current noise increase can be recorded. GABA receptors in glial cells are heterogeneous with respect to their benzodiazepine pharmacology. In cultured astrocytes from rat cortex the inverse benzodiazepine agonist DMCM augments GABA responses in contrast to neurons and oligodendrocytes (for review see Blankenfeld and Kettenmann, 1992). Since Bergmann glial cells are of the astrocytic type, one would also expect an augmentation of the GABA response by DMCM. In contrast, recordings from Bergmann glial cells demonstrate that these cells are benzodiazepine insensitive. Neither the inverse agonist Ro15-4513 nor normal benzodiazepine agonists, such as diazepam, are able to modulate the GABA response (Fig. 4).Thus, GABA receptors in situ on Bergmann glial cells are distinct from those described in cultured cortical astrocytes, oligodendrocytes, or neurons. This is substantiated by identifymg the Bergmann glial GABAA receptor subunits using subunit-specificantibodies. Bergmann glial cells are positive for the a2,as,and S subunit and lack the expression of a y subunit (Fig. 5). The y subunit, however, is particularly important for benzodiazepine sensitivity of the receptor (Pritchett et al., 1989). Both the electrophysiological and the immunocytochemical studies indicate that GABA receptors in Bergmann glial cells do not bind and are not modulated by benzodiazepines. The expression of the GABA receptors changes markedly during development. Staining with the antibodies against the aq,a3,and 6 subunits is first observed around Postnatal Day 5 in mice and becomes most prominent at Postnatal Days 7-10 and decreases gradually thereafter. At Postnatal Day 20 staining of the Bergmann glial cell has disappeared except for a faint a2subunit reactivity. Since other groups demonstrated the weak expression for the a2and y subunit in the adult rat Bergmann glial cell (Laurie et al., 1992; Wisden et al., 1992), it seems likely that a subunit of the GABAA receptor is replaced by the y subunit later in development. This is substantiated by electrophysiological recordings. In young Bergmann glial cells large GABA-mediated currents can be recorded, while in the more mature cerebellum only very small responses can be evoked in Bergmann glial cells. In this context is it noteworthy that the diazepam-binding inhibitor, a peptide that is binding to benzodiazepine recognition sites associated with GABA, receptors or mitochondria1 benzodiazepine receptors, is also

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A

"-7 Bicuculline G

A

Y

-

GABA

B

1 GAG + Pentobarbital

C

-

li

L v 15s

-

GABA

GABA + Diazepam

I

R015-4513

15s

FIG.4. Pharmacological properties of the GABA response. Membrane currents were recorded with the patchdamp technique from individual Bergmann glial cells in the cerebellar slice. Drugs were tested as indicated by bar (with modifications from Mlrller et aL, 1994). (A) The GABA response (lo-%M) was significantlyreduced by the competitive GABA, blocker M). (B) Potentiation of the GABA response by pentobarbital. A 2-min bicuculline preincubation with lo-' M pentobarbital resulted in a clear increase of the GABA-induced M) at the holding potential of -70 mV. (C and D) Lack of effects of the current M) on benzodiazepine agonist diazepam (5X10-4 M) and inverse agonist Ro15-4513 M) currents. Note the current noise increase during GABAapplication GABA-induced (5X in C.

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FIG.5 . Colocalization of the aPsubunit with GFAP in Bergmann glial cell processes. Video images from confocal laser microscopy displaying double-immunofluorescence staining of Bergmann glial cell processes with antibodies to GFAP (left) and to the GABA, receptor cu, subunit (right). Notice the punctate distribution of the a2subunit immunoreactivity. Arrowheads point to apsubunit immunoreactive puncta localized in a GFAP-positive appendage which stems from a large process; arrows show a2subunit staining in several fine processes immunoreactive for GFAP. Scale bar denotes 10 p n .

found on adult Bergmann glial cells (Costa and Guidotti, 1991;VidnyAnszky et al., 1994). While the classic action of GABAAreceptors is the activation of a C1conductance, a second mechanism has been described in Bergmann glial cells which is mediated by GABAAreceptors. In both Bergmann glial cells from young and older animals, GABA transiently blocks the resting K+ conductance. This blockade is longer lasting than the activation of the C1channel. The GABA-mediated K+ channel blockade is due to the activation of GABAAreceptors since the response can be mimicked by muscimol and is blocked by bicuculline. However, so far the link between GABAAreceptor activation and the decrease in the resting K+ conductance is unresolved. C. FUNCTIONAL IMPORTANCE OF GLUTAMATE AND GABA RECEPTORSIN BERCMA"GLIAL CELLS The expression of GABAAreceptors on Bergmann glial cells is remarkably linked with the period of granule cells migration. Since GABA is

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thought to modulate maturation and differentiation of brain cells during development (Chronwall and Wolff, 1980; Lauder et al., 1986; see Meier et al., 1991 for review), several hypotheses may be formulated concerning the putative functional roles of these channels. First, it is possible that GABA modulates the maturation of Bergmann glial cells by means of GABAA receptors. Second, it is conceivable that activation of GABAA receptors influences the migration of granule cells. Third, since an upregulation in the expression of ionic currents is reported for astrocytes in contact with neurons (Barres et al., 1990b), the presence of the granule cells on the Bergmann glial cell processes may influence the expression of these channels. With regard to the source of GABA in early development, cerebellar interneurons have been reported to be immunoreactive for GABA as early as P1 (basket cells) and P7 (stellate cells) (Aoki et al., 1989),while migrating granule cells do not express detectable levels of GABA. VI. IonExchangers

Astrocytes like other cells, have numerous ion transport systems. There are channels for sodium, chloride, and calcium (for review see Barres et aL, 1990b; Bevan, 1990). There are also chloride/bicarbonate exchangers, Na+/H+ exchangers, and uptake systems for numerous transmitters that involve cotransport of sodium (Kimelberg et al., 1993). In contrast to other astrocytes, little is known about ion exchangers on Bergmann glial cells. Ortega et al. (1991) demonstrated by means of ion flux measurements that the dissipation of Na+ after kainate application is clearly due to the activation of Na+-dependent exchange systems which lead to the release of Na+ ions and the incorporation of Ca2+and H+ ions. They have been able to show that the kainate-induced Ca2+influx is partially mediated by the activation of an Nat/Ca2+ exchanger (see also Section V,A,1) . This transporter is, under resting conditions, responsible for intracellular Ca2+homeostasis. Due to the elevation of cytosolic Na+ after glutamate receptor activation, the cycle is reversed: Nat is expelled and Ca2+ enters the cell. Similarly, the Nat/Ht exchanger, under resting conditions responsible for acid extrusion, is activated and leads to an influx of Ht. The activation has been shown to occur with a delay (Saktor and Kinsella, 1988). VII. Transmitter Uptake

It is now well established that astrocytes in culture and i n situ possess Na+-dependent, high-afiinity uptake carriers for glutamate, GABA, taurine, and other amino acids (Hosli et aL, 1986).

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. Na' Fit;. 6. Possible modulation of synaptic efficiency by Bergmann glial cells. The scheme represents the synaptic regions consisting of the pre- and postsynaptic membrane of the Purkinje cell and the Bergmann glial ensheathment (shaded areas). It hypothetically illustrates the events following presynaptic glutamate release. To illustrate the sequence of events an area of neuron-glia interaction is marked by the square and is shown in A-D. (A) In the resting state the Bergmann glial cell is dominated by a large resting Kt permeability. (B) Neuronal activity of the Purkinje cell inputs leads to the vesicular release of glutamate into the synaptic cleft. Glutamate activates both the kainate/AMPA receptors on the Purkinje cell and the Bergmann glia. In the neuron, a postsynaptic potential is generated, while in the Bergmann glial cell intracellular Ca'+ and Na+ 1r:vels are increased by an influx through the kainate/AMPA receptor. The resulting depolarization of the glial membrane results in a K+ efflux; this, however, is strongly reduced, since the (Ca*+-sensitive)K+ channels are blocked by the Ca2+influx. Moreover, the K+ channel blockade augments the extent of the glial depolarization. (C) The rise in the intracellular Na+ concentrations and the membrane depolarization, in concert, strongly hampers the activity of the glial glutamate uptake system. Thus, glutamate in the cleft is not rapidly removed. It depends on the activity of the Na+/ Kt -ATPase and the Ca2' extrusion mechanism to restore the optimal working conditions for the glutamate uptake system. (D) When the Bergmann glial Na' and Ca2+levels are at resting levels again, the glutamate uptake system can efficiently remove the excess glutamate from the cleft. The time course of glutamate removal thus depends on a complex system involving carriers, receptors, and ion channels of the glial cell. A modulation of any of these components will ultimately alter the efficacy of synaptic transmission.

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In sztu localization of glutamate visualized by gold immunocytochemistry demonstrated that such an uptake mechanism must also be present in Bergmann glial cells (Ottersen, 1990) An increase in extracellular K+ led to a loss of glutamate immunoreactivity from mossy fiber terminals and parallel fiber terminals. In contrast, Bergmann glial cell bodies and processes show an increased staining after Kc-induced depolarization. In addition, Ortega et al. (1991) showed that incorporation of glutamate and aspartate into cultured Bergmann glial cells was significant, whereas the incorporation of GABA was poor. The uptake of glutamate by Bergmann glial cells is a major means of terminating the transmitter action at climbing fiber and parallel fiber terminals. An increase in intracellular Na' ions mediated by the opening of the kainate receptor could cause an inhibition of the glial glutamate uptake, as suggested by Ortega et al. (1991). This would delay the removal of glutamate from the synaptic region and would then lead to a potentiation of the excitatory postsynaptic potential at the Purkinje cells.

WII. Summary

While Bergmann glial cells play an important role in the development of the cerebellum they were thought to serve as passive insulators of the Purkinje cell dendritic tree and its synaptic connections. New results challenge this view and demonstrate that Bergmann glial cells are equipped with a large repertoire of receptors allowing them to sense the activity of synapses. These receptors have distinct biophysical and pharmacological features activating second-messenger pathways in the Bergmann glial cells. It is evident that the synapse has to be viewed as consisting of three elements, the presynaptic and postsynpatic region and the glial ensheathment. All three elements of this synaptic complex may undergo plastic changes as a prerequisite for central nervous system plasticity. Glial cells could interfere with synaptic transmission by communicating with neurons via the extracellular space, e.g., by modulating ion concentrations or transmitter levels in the cleft (Fig. 6 ) .

References

Aoki, E., Semba, R., and Kashiwamata, S. (1989). Bruin Res. 502, 245-251. Barres, B. A., Chun, L. Y. Y., and Corey, D. P. (1990b). Annu. Rev. Neurosci. 13, 441-474.

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INDEX

ethanol interactions binding site pharmacology, 122 characterization, 13-15 chronic administration effects, 15-19 subunit composition, 20-21 subunit mRNA levels, 22-24 [3H]zolpidem binding, 24 function, 1-3 GABA binding, molecular pharmacology, 117-119 loreclezole binding site, pharmacology, 122-123 neurosteroid interactions characterization, 24-26 receptor regulation in vim,26-28 phosphorylation by protein kinase A, 103-104 by protein kinase C, 104-105 picrotoxin binding sites, pharmacology, 121-122 posterior pituitary nerve terminal, 228-232 in schizophrenia, 325 structure, 1-3 subunit genes alternative splicing, 100-101 cDNAs, cloning, 96-100 chromosome assignment, 101-102 structure, 102 subunits antisera immunoprecipitation, 107-109 composition alterations, 32-33 composition after chronic ethanol exposure, 20-21 dendrogram of primary amino acid sequences, 99 immunolocalization, 109-1 1 I mRNA levels after chronic ethanol exposure, 22-24 in situ hybridization, 106-107 stoichiometry, 111-113 Western blot analysis, 109

A

Acetylcholine in schizophrenia, 319 striatal, genetic effects, 78-83 Alternative splicing, GAB& receptor subunits, 100-101 Amino acid sequences dopamine transporter, 146-150 GABAAreceptor subunit family, human, 99 glutamate transporters, 159-160 Na+/CI-dependent transporters, 146-150 y-Aminobutyric acid binding, molecular pharmacology, 117-119 receptors in Bergmann glial cells, 351-354, 354-355 in schizophrenia, 324-326 striatal, genetic effects, 83-84 y-Aminobutyric acid, GABAAreceptor subtype anesthetic binding sites, pharmacology, 123 barbiturate interactions characterization, 9-10 chronic administration effects, 10-13 benzodiazepine binding sites, pharmacology a subunits, 114-115 p subunits, 115 characterization, 113-114 6 subunits, 116-117 y subunits, 115-116 p subunits, 11 7 benzodiazepine interactions characterization, 6-7 chronic administration effects, 7-9 desensitization, 4-6 developmental alterations, 28-32 drug binding sites, 113-125

361

362

INDEX

zinc binding sites, pharmacology 123-123 Anesthetics, binding sites on GABA, receptor, pharamcology, 123 Annelids, monoamine catabolism, 276-279 Antisera, GABA, receptor subunit-specific, immunoprecipitation, 107-109 Anxiolytic/anticonvulsantcompounds, effects on GABAAreceptors, 125 Aromatic L-amino acid decarboxylase, aminergic neurotransmitter synthesis, 268-269 Arthropods, monoamine catabolism, 282-285

B Barbiturates, GABA, receptor interactions binding sites, pharmacology, 120-121 characterization, 9-10 chronic administration effects, 10-1 3 Basal ganglia, genetics behavioral responses to dopamine receptor agonists, 50-52 dopamine receptor antagonists, 54-57 ethanol administration, 62 methylxanthines, 60-61 morphine, 58-60 phencyclidine, 61 scopolamines, 60 functional architecture dopamine levels, 67-69 dopamine neurons, midbrain number, 62-67 neuropeptides, 70 neurotransmitter transporters, 77-78 opiate receptors, 84-85 serotonin levels, 67-69 striatal cholinergic system, 77-78 striatal GABA system, 83-84 wv mutants, 85-86 Benzodiazepine-GABA, receptor interactions binding site pharmacology a subunits, 114-115 /3 subunits, 115 characterization, 113-1 14 6 subunits, 116-117 y subunits, 115-116 p subunits, 117

characterization, 6-7 chronic administration effects, 7-9 Benzodiazepine receptors function, 1-3 structure, 1-3 Bergmann glial cells gap junctions, 343-346 glutamate receptors, 349-350 ion channels, 346-348 ion exchangers, 355 Kf currents, 347 K+ uptake, 347-348 morphology, 342-343 transmitter uptake, 355-357 Betaine transporter amino acid sequences, 146-150 characteristics, 153 Biogenic amines, see Monoamine neurotransmitters Bipolar cells, retinal, presynaptic excitability Ca2+channels, 235-237 neurotransmitters, 237-239 Birds, monoamine catabolism, 286-288 t-Butylbicyclophosphorothionate,see Picrotoxin

C Calcium channels, presynaptic ciliary ganglion, 232-234 motor nerves, 205-208 posterior pituitary, 217-218 squid synapse, 212-214 summary, 241 Chelicerates, monoamine catabolism, 282 Cholecystokinin, in schizophrenia, 327 Cholinergic system in schizophrenia, 319 striatal, genetic effects, 78-83 Ciliary ganglion nerve terminals, presynaptic excitability Cap' channels, 232-234 K+ channel, 234 neurotransmitters, 234-235 Cloning effect on noradrenergic systems, 324 GABA, receptor subunit cDNAs, 96-100 glutamate transporters, 157-158

INDEX Na'/Cl-dependent neurotransmitter transporters, 145, 151 Clozapine a n i t y of 5-HT2 receptors, 321-322 D, antagonist actions, 316 efficacy in schizophrenia, 31 1-312 Coelenterates, monoamine catabolism, 274-276 Complementary DNA GABAA receptor subunit, cloning, 96-100 glutamate transporters, isolation, 157-158 Creatine transporter characteristics, 155 cloned, predicted amino acid sequence, 146-150 Crustaceans, monoamine catabolism, 282-284

D Desensitization, GABA,/benzodiazepine receptors, 4-6 Development, GABA, receptor expression, 28-32 DNA, complementary, see Complementary DNA Dopamine in acoelomates, 259-261 catabolism in animals, schematic, 272 in deuterostomes, 263-264 presynaptic function in schizophrenia, 3 12-31 4 in protostomes, 262-263 in pseudocoelomates, 261 steady-state levels and turnover in inbred mouse strains, 67-69 structure, 254 Dopamine-P-hydroxylase, aminergic neurotransmitter synthesis, 269 Dopamine hypothesis, in schizophrenia research, 310 Dopamine receptors agonists, response of inbred strains to, 50-51, 51-54 antagonists, response of inbred strains to, 54-57 basal ganglia, genetic factors affecting, 70-77 binding, genetic aspects, 70-77

363

murine genes, chromosomal location, 58 in schizophrenia D1 subtype, 316-317 D2 subtype, 314-316 D3 subtype, 317-318 D4 subtype, 317-318 Dopamine transporter amino acid sequence, 146-150 characteristics, 152 DTE (destyr-yendorphin), antipsychotic efficacy, 328

E Echinoderms, monoamine catabolism, 285 Electrophysiological recordings, presynaptic mechanisms ciliary ganglion Ca2+channels, 232-234 K+ channels, 234 motor nerve terminals Caz+channels, 205-208 channel locations, 209-21 1 K+ channels, 204-205 presynaptic transmitters, 208-209 posterior pituitary Ca2+channels, 217-218 K+ channels, 219-226 Na+ channels, 226-228 neurotransmitters, 228-232 retinal bipolar cells Ca2+channels, 235-237 neurotransmitters, 237-239 squid synapse Ca2+channels, 212-214 channel locations, 216 K+ channels, 215-216 presynaptic transmitters, 216-217 Endorphins, basal ganglia, genetic differences, 84-85 Enflurane, effects on GAB& receptor, 123 Enkephalins, basal ganglia, genetic differences, 84-85 Epithelial cells, Golgi, see Bergmann glial cells Ethanol binding sites on GABAAreceptor, pharmacology, 122

364 effects on basal ganglia-related behavior, 62 GABA, receptor interactions characterization, 13-15 chronic administration effects, 15-19 subunit composition, 20-21 subunit mRNA levels, 22-24 [SH]zolpidem binding, 24

INDEX Glycine transporters characteristics, 154 heterogeneity, 189-190 localization, 187-188 predicted amino acid sequences, 146-150 Golgi epithelial cells, see Bergmann glial cells

H G

GABA hypothesis, in schizophrenia, 324 GABA transporter characteristics, 152, 153 cloned, predicted amino acid sequence, 146-150 ionic requirements, 181-183 localization, 187- 188 predicted amino acid sequences, 146-150 purification and isolation, 143-144 regulation, 176-180 structure, 165-169 Gap junctions, Bergmann glial cells, 343-346 Gas chromatography/mass spectrometry, neurotransmitter transporters, 257 Genes GABA, receptor subunits chromosome assignment, 101-102 structure, 102 unc-17, vesicular acetylcholine transporter, 165 wv, and basal ganglia organization, 85-86 Giant synapse, see Squid synapse Glial cells, Bergmann, see Bergmann glial cells Glutamate receptors Bergmann glial cells, 349-350, 354-355 schizophrenia, 330-331 Glutamatergic markers, in schizophrenia, 324-326 Glutamate transporters amino acid sequences, 159-160 characteristics, 162 cloning, 157-158 ionic requirements, 183-184 structure, 170

Halothane, effects on GABA, receptor, 123 High-performance liquid chromatography, with electrochemical detection, for neurotransmitter transporters, 257 Histofluorescence techniques, for neurotransmitter transporters, 258 Homovanillic acid, CSF levels in schizophrenia, 312-313 Hybridization, in situ, GABA, receptor subunits, 106-107 5-Hydroxytryptamine in acoelomates, 261 catabolism in animals, schematic, 273 in deuterostomes, 263-264 in inbred mouse strains, steady-state levels and turnover, 67-69 in protostomes, 262-263 in pseudocoelomates, 261 in schizophrenia presynaptic function, 320-321 receptor subtypes, 321-323 structure, 254 5Hydroxytryptamine transporter amino acid sequence, 146-150 characteristics, 152

I Immunolocalization, GABAAreceptor subunits, 109-1 1 1 Immunoprecipitation, GABA, receptor subunit-specific antisera, 107-109 Inbred strains dopamine receptor binding, 70-77 for genetics studies, 44-46 recombinant, and quantitative trait loci, 46-49 Insects, monoamine catabolism, 284-285 Invertebrates, monoamine catabolism annelids, 276-279

INDEX arthropods, 282-285 chordates, 285 coelenterates, 274-276 echinoderms, 285 molluscs, 279-282 platyhelminthes, 276 pseudocoelomates, 276 routes of, 271-274 Ion channels, see also specific channel in Bergmann glial cells, 346-348 Ion exchangers, in Bergmann glial cells, 355 Isoflurane, effects on GABAA receptor, 123

K Kainate/AMPA receptors, Bergmann glial cells. 349-350

Lanthanum, effects on GABAA receptors, 124 Loreclezole, binding sites on GABAA receptor, 122-123

M Mammals, monoamine catabolism, 286-288 Messenger RNA, GABAAreceptor subunits, chronic ethanol exposure effects, 22-24 N-Methyl-o-aspartate receptors, in Bergmann glial cells, 351 Methylxanthines, effects on basal gangliarelated behavior, 60-61 Molluscs, monoamine catabolism, 279-282 Monoamine neurotransmitters analysis methods, 256-259 catabolism annelids, 276-279 arthropods, 282-285 birds, 286-288 chordates, 285 coelenterates, 274-276 echinoderms, 285 lower vertebrates, 277, 285-286 mammals, 286-288 molluscs, 279-282 platylhelminthes, 276

365

pseudocoelomates, 276 teleost fish, 285-286 distribution acoelomates, 259-261 deuterostomes, 263-264 protostomes, 262-263 pseudocoelomates, 261 release, 269-270 reuptake, 269-270 storage, 269-270 synthesis dopamine-P-hydroxylase in, 269 tryptophan hydroxylase in, 267-268 tyrosine hydroxylase in, 266-267 Monoamine oxidase, activity measurement techniques, 258-259 Monoamine vesicular transporters characteristics, 161-162 cloning, 161 heterogeneity, 190 membrane topology, 163, 166 sequence homology, 163, 166 Morphine, effects on basal ganglia-related behaviors, 58-60 Morphology, Bergmann glial cells, 342-343 Motor nerves, presynaptic excitability Ca" channels, 205-208 channel locations, 209-211 K' channels, 204-205 presynaptic transmitters, 208-209

N Nerve terminals, presynaptic excitability ciliary ganglion Ca" channels, 232-234 K' channel, 234 neurotransmitters, 234-235 motor nerves CaZt channels, 205-208 channel locations, 209-21 1 K' channels, 204-205 presynaptic transmitters, 208-209 posterior pituitary Ca2' channels, 217-218 Nat channels, 226-228 neurotransmitters, 228-232 retinal bipolar cells Ca2' channels, 235-237 neurotransmitters, 237-239

366 squid synapse Ca2+channels, 212-214 channel locations, 216 K+ channels, 215-216 presynaptic transmitters, 216-21 7 Neuropeptides, basal ganglia, genetic aspects, 70 Neurotensin, in schizophrenia, 326-327 Neurotransmitters ciliary ganglion newe terminals, 234-235 monoamine, see Monoamine neurotransmitters posterior pituitary nerve terminals, 228-232 presynaptic motor nerve terminals, 208-209 squid giant synapse, 216-217 retinal bipolar cells, 237-239 uptake by Bergmann glial cells, 355-357 Neurotransmitter transporters classification, 143-144 genetics, 77-78 glutamate transporters amino acid sequences, 159-160 characteristics, 162 cloning, 157-158 ionic requirements, 183-184 structure, 170 H+dependent vesicular, 158- 165 characteristics, 162, 164 cloning, 161, 165 comparative amino acid sequences, 159-160 ionic requirements, 284 membrane topology, 163, 166 sequence homology, 163, 166 structure, 170 heterogeneity of, 189-190 localization distribution, 187 glial vs. neuronal, 187-188 subcellular, 188-189 Na+/Cl-dependent, 143-157 characteristics, 152-155 cloning, 143-145, 151 comparative amino acid sequences, 146-150 ionic requirements, 180-183

INDEX membrane topology, 156 sequence homology, 156 structure, 165-169 regulation, 170-180 in disease states, 171 by hypertonicity, 1’79 by monoamine receptors, 179-180 second-messenger systems, 171-179 research focus, 142-143 research history, 141-142 transmitter efflux, 184-186 Nonsteroidal anti-inflammatory drugs, effects on GABAA receptors, 125 Noradrenaline in deuterostomes, 263-264 in protostomes, 262-263 in schizophrenia, 323-324 structure, 254 Norddrenergic systems, in schizophrenia, 323-324 Norepinephrine transporter characteristics, 152 cloned, predicted amino acid sequence, 146-150 0

p-Octopamine in protostomes, 262-263 in pseudocoelomates, 261 structure, 254 Opiate receptors, basal ganglia, genetic differences, 84-85 Opioid peptides, in schizophrenia, 327-328

P Pentobarbital, chronic administration effects on GABA, receptors, 10-13 Pesticide compounds, effects on GABAA receptors, 124 Phencyclidine, effects on basal gangliarelated behavior, 61 Phenotypic variance, classical analysis, 46 Picrotoxin, GABAAreceptor binding sites, pharmacology, 121-122 Pituitary, posterior, see Posterior pituitary Platylhelminthes, monoamine catabolism, 276

INDEX

Posterior pituitary, presynaptic excitability Ca2+channels, 217-218 Na+ channels, 226-228 neurotransmitters, 228-232 Potassium channels in Bergmann glial cells, 347-348 in nerve terminals ciliary ganglion, 234 motor nerves, 204-205 posterior pituitary, 219-226 squid synapse, 215-216 summary, 241 Proline transporter characteristics, 154 cloned, predicted amino acid sequence, 146-150 Propofol, effects on G U A Areceptor, 123 Protein kinase A, GABAAreceptor phosphorylation, 103- 104 Protein kinase C, GABAAreceptor phosphorylation, 104-105 Pseudocoelomates, monoamine catabolism, 276

Q Quantitative trait loci, recombinant inbred strains for, 46-49

s Schizophrenia cholinergic systems, 319 dopamine hypothesis, 310 dopamine receptors D, subtype, 316-317 D2subtype, 314-316 Ds subtype, 317-319 D4subtype, 317-319 drug treatment prospects, 333-334 etiological factors, 307-308 GABA receptors, 324-326 glutamate receptors, 330-331 Shydroxyuyptamine receptors 5-HTlAsubtype, 323 5-HT2 subtype, postsynaptic, 321-322 5-HTs subtype, 322-323 neurochemical pathology, 332-333 neuropathology, 308-309 neuropeptide systems, 326-327

367

nondopamine neurotransmitter systems, 310-31 2 noradrenergic systems, 323-324 presynaptic dopamine function, 312-314 presynaptic glutamatergic markers, 329-330 presynaptic 5-HT function, 320-321 sigma receptors, 332 types I and 11, 306 Scopolamines, effects o n basal gangliarelated behavior, 60 Selective breeding technique, 49 Sigma receptors, in schizophrenia, 332 Sodium channels, presynaptic, in posterior pituitary, 226-228 Sodium/chloridedependent transporters characteristics, 152-155 cloning, 143-145, 151 comparative amino acid sequences, 146- 150 heterogeneity, 189-190 ionic requirements, 180-183 membrane topology, 156 sequence homology, 156 structure, 165-169 Somatostatin in inbred mouse strains, 70 in schizophrenia, 327 Squid synapse, presynaptic excitability Ca2' channels, 212-214 channel locations, 216 Kt channels, 215-216 presynaptic transmitters, 216-217 Stoichiometry, GABAAreceptor subunits, 111-113

T Taurine transporter characteristics, 154-1 55 cloned, predicted amino acid sequence, 146-1 50 Teleost fish, monoamine catabolism, 285-286 Tetrahydrodeoxycorticosterone-GABAA receptor interactions characterization, 24-26 receptor regulation in uiuo, 26-28

368 Transporters, see Neurotransmitter transporters Tryptophan hydroxylase, aminergic neurotransmitter synthesis, 267-268 Tyrosine hydroxylase aminergic neurotransmitter synthesis, 266-267 inbred mouse strains, midbrain and striatal activity, 62-67

INDEX

mammals, 286-288 teleost fish, 285-286 terrestrial tetrapods, 286 W

Western blotting, GABAA receptor subunits, 109

Z V Vertebrates, monoamine catabolism birds, 286-288

Zinc, binding sites on GABAAreceptor, pharmacology, 123- 123 Zolpidem binding, chronic ethanol exposure effects, 24

CONTENTS OF RECENT VOLUMES

Batrachotoxin: A Window on the Allosteric Nature of the Voltage-Sensitive Sodium Channel Georgp B. Brown

Volume 28

Biology and Structure of Scrapie Prions Michael P. McKinlty and Stan@ B. Prusiner

Neurotoxin-Binding Site on the Acetylchcline Receptor Thomos L. Lentz and Paul T. Wilson

Different Kinds of Acetylcholine Release from the Motor Nerve S. Thessleff

Calcium and Sedative-Hypnotic Drug Actions Peter L. CarIen and Peter H. Wu

Neuroendocrine-Ontogenetic Mechanism of Aging: Toward an Integrated Theory ofAging V. M. Dilman, S. Y. Revskloy, and A. G. Golube0

Pathobiology of Neuronal Storage Disease Steven U. Walklty

The Interpeduncular Nucleus Barbara J. M w l q

Thalamic Amnesia: Clinical and Experimental Aspects Stephen G. Waxman

Biological Aspects of Depression: A Review of the Etiology and Mechanisms of Action and Clinical Assessment of Antidepressants S. I. Ankier and B. E. Leonard

Critical Notes on the Specificity of Drugs in the Study of Metabolism and Functions of Brain Monoamines S. Garattini and T. Mennini

Does Receptor-Linked Phosphoinositide Metabolism 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 andJ M. Wojtowin

Retinal Transplants and Optic Nerve Bridges: Possible Strategies for Visual Recovery as a Result of Trauma or Disease James E. Turner, Jerry R Blair, Magdalene Seiler, Robert Aramant, Thomas W. Laedtke, E. Thomas Chappell, and Lauren Clarkson

Immunology and Molecular Biology of the Cholinesterases: Current Results and Prospects Stephen Brimijoin and Zoltan Rakonnay

Schizophrenia: Instability in Norepinephrine, Serotonin, and y-Aminobutjric Acid Systems Joel Gelernter and Daniel P. van Kammen

INDEX

INDEX

Volume

29 Volume 30

Molecular Genetics of Duchenne and Becker Muscular Dystrophy Ronald G. Worton and Arthur H. M. Burghes

Biochemistry of Nicotinic Acetylcholine Receptors in the Vertebrate Brain Jakob Schmidt 369

370

CONTENTS OF RECENT VOLUMES

The Neurobiology of NAcetylaspartylglutamate Randy D. Blakely and Joseph T. Cuyle

Neuropeptide-Processing, Converting, and -Inactivating Enzymes in Human Cerebrospinal Fluid Lars Terenius and Fred Nyberg

Nerve Blood Flow and Oxygen Delivery in Normal, Diabetic, and Ischemic N e u r o p athy Phillip A. Low, Tmena D. Lagerlund, and Philip G. McManis INDEX

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. Vandmolf

O n the Contribution of Mathematical Models to the Understanding of Neurotransmitter Release H. Parnas, I. Parnas, and L. A. Segel Single-Channel Studies of Glutamate Receptors M . S. P. Sansom and P. N. R Ushmood

INDEX

Volume

Volume 32

31

Animal Models of Parkinsonism Using Selective Neurotoxins: Clinical and Basic Implications MichaelJ. Zigmond and Edward M. Stricker Regulation of Choline Acetyltransferase Paul M. Salvatma and J a m s E. Vaughn Neurobiology of Zinc and Zinc-Containing Neurons ChristopherJ. Fredmickson Dopamine Receptor Subtypes and Arousal Ennio Ongini and Vincenzo G. Long0 Regulation of Brain Atrial Natriuretic Peptide and Angiotensin Receptors: Quantitative Autoradiographic Studies Juan M. Saavedra, Eero Castrin, Jorge S. Gutkind, and Adil J, Naulrali 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

Coinjection of h o p u s Oocytes with cDNA Produced and Native m R N h A Molecular Biological Approach to the Tissue-Specific Processing of Human Cholinesterases Shbmo Seidman and Hennona Soreq Potential Neurotrophic Factors in the Mammalian Central Nervous System: Functional Significance in the Developing and Aging Brain Dalia M. Araujo, Jean-Guy Chabot, and &i Quirion Myasthenia Gravis: Prototype of the Antireceptor Autoimmune Diseases Simone Schonbeck, Susanne Chreslel, and Reinhard Hohlfeld Presynaptic Effects of Toxins Alan I,. Haruqr Mechanisms of Chemosensory Transduction in Taste Cells Myks H. Akabas Quinoxalinediones as Excitatory Amino Acid Antagonists in the Vertebrate Central Nervous System Stephen N. Davies and Graham L. Collinp'dge

CONTENTS OF RECENT VOLUMES Acquired Immune Deficiency Syndrome and the Developing Nervous System Douglas E. Brenneman, Susan K. McCune, and Illuna Gates

371

Release, and Presynaptic Modulation by Autoreceptors and Adrenoceptors Ignaz Wessler INDEX

INDEX

Volume 33

Volume

35

Olfaction S. G. Shirley

Biochemical Correlates of Long-Term Potentiation in Hippocampal Synapses S a t m Otani and Yehakel Ben-An

Neuropharmacologic and Behavioral Actions of Clonidine: Interactions with Central Neurotransmitters JenyJ Buccafusco

Molecular Aspects of Photoreceptor Adaptation in Vertebrate Retina S a t m Kawamura

Development of the Leech Nervous System Gunther S. Stent, William B. Kristan, Jr., Steven A. Tmence, Kathleen A. French, and David A. Weisblal GABA, Receptors Control the Excitability of Neuronal Populations Armin Stelrer Cellular and Molecular Physiology of Alcohol Actions in the Nervous System Forrest F. Weight INDEX

Volume 34

Neurotransmitters as Neurotrophic Factors: A New Set of Functions Joan P. Schwartz Heterogeneity and Regulation of Nicotinic Acetylcholine Receptors RonaldJ Lukas and Merouane Bencheriif Activity-Dependent Development of the Vertebrate Nervous System R Douglas Fields and Phillip G. Nelson A Role for Glial Cells in Activity-Dependent Central Nervous Plasticity? Review and Hypothesis Christian M. Muller Acetylcholine at Motor Nerves: Storage,

The Neurobiology and Genetics of Infantile Autism Linda J. Lotspeich and Roland D. Ciaranello Humoral Regulation of Sleep Leuente Kaph, Ferenc Owl, Jr., and James M . Krueger Striatal Dopamine in Reward and Attention: A System for Understanding the Symptomatology of Acute Schizophrenia and Mania Robert Miller Acetylcholine Transport, Storage, and Release Stanley M. Parsons, Chris Prior, and Ian G. Marshall Molecular Neurobiology of Dopaminergic Receptors David R Sibley, Fredm'ckJ. M o n s m , Jr., and Yong S h a INDEX

Volume 36 Ca2+, &Methyl-maspartate Receptors, and AIDS-Related Neuronal Injury Stuart A. Lipton Processing of Alzheimer A@Amyloid Precursor Protein: Cell Biology, Regulation, and Role in Alzheimer Disease Sam Gandy and Paul Greengard

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CONTENTS OF RECENT VOLUMES

Molecular Neurobiology of the GABA, Receptor Susan M. J Dunn, A h n N. Bateson, and Ian L. Martin The Pharmacology and Function of Central GABABReceptors David D. Molt and Dave11 V. Lewis The Role of the Amygdala in Emotional Learning Michael Davis Excitotoxicity and Neurological Disorders: Involvement of Membrane Phospholipids Akhluq A . Farooqui and L l q d A. Horrocks Injury-Related Behavior and Neuronal Plas ticity: An Evolutionary Perspective on Sensitization, Hyperalgesia, and Analgesia Edgar T. Walters

Exploration and Selection in the Early Acquisition of Skill Esther Thelen and Danieh Curbetta Population Activity in the Control of Movement Apostolos P. Georgopoulos Section 111: Functional Segregation and Integration in the Brain Reentry and the Problem of Cortical Integration Giulio Tononi Coherence as an Organizing Principle of Cortical Functions Wolf Singer Temporal Mechanisms in Perception Ernst P w e l Section N Memory and Models

INDEX

Selection versus Instruction: Use of Computer Models to Compare Brain Theories George N . Reeke, J .

Volume 37

Memory and Forgetting: Long-Term and Gradual Changes in Memory Storage Lany R Squire

Section I: Selectionist Ideas and Neurobiology Selectionist and Instructionist Ideas in Neuroscience Olaf s p m s Population Thinking and Neuronal Selection: Metaphors or Concepts? Ernst Mayr Selection and the Origin of Information Manfred Eigen Section 11: Development and Neuronal Populations Morphoreguldtory Molecules and Selectional Dynamics during Development Kathryn L. Crossin

Implicit Knowledge: New Perspectives on Unconscious Processes Daniel L. Schacter Section V Psychophysics, Psychoanalysis, and Neuropsychology Phantom Limbs, Neglect Syndromes, Repressed Memories, and Freudian Psychology 1’. S. Ramachandran Neural Darwinism and a Conceptual Crisis in Psychoanalysis Arnold H. Modell A New Vision of the Mind Oliver Sacks INDEX

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